InteractiveFly: GeneBrief

G protein-coupled receptor kinase 2: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Heterotrimeric G protein-coupled signaling is a form of intracellular communication that is mediated by the family of G protein-coupled receptors (also known as heptahelical/7TM receptors). In the continued presence of ligand, G protein-coupled receptors become less efficient in transducing signals, a process called desensitization. Desensitization of G protein-coupled receptors is thought to occur in several steps: binding of G protein-coupled receptor kinases (GRKs) to receptors, receptor phosphorylation, kinase dissociation, and finally binding of beta-arrestins to phosphorylated receptors.

The Drosophila G protein receptor-coupled kinase 2 (Gprk2) gene encodes a member of the G protein-coupled receptor kinase family (Cassill, 1991; Schneider, 1997). The Gprk2 protein has a high level of sequence identity to members of the mammalian GRK4 subfamily (GRK4, GRK5, and GRK6). Expression of the Gprk2 protein in the mushroom bodies of the brain is similar to the expression of genes involved in cAMP-mediated signaling pathways, such as dunce (dnc) and rutabaga (rut). The rutabaga locus encodes an adenylate cyclase and mutation of this gene results in a decrease in cAMP synthesis, whereas mutation in dnc, coding for cyclic AMP phosphodiesterase (an enzyme that degrades cAMP), causes an increase in cAMP synthesis.

Because of the potential role of the Gprk2 gene in G protein signaling it was hypothesized that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger. To examine this hypothesis, tests were performed for genetic and biochemical interactions between dunce and Gprk2 mutants; dunce is able to suppress the sterility defect of a gprk2 mutant. Similarly, gprk2 mutation is able to rescue the viability and sterility defects of dunce mutants. Results of the biochemical analysis of cAMP levels in the dunce;gprk2 mutant combinations further strengthen the genetic findings. These results suggest that Gprk2 is involved in a receptor-mediated signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

The genetic interaction between dunce and Gprk2 was examined through the effects of these two genes on oogenesis. A mutant allele of Gprk2, called gprk2⁶⁹³⁶, has decreased fertility as a result of reduced levels of egg laying and hatching, and developing egg chambers display defects in the formation of anterior structures. Similarly, many alleles of dunce are sterile, with an ovary phenotype that resembles gprk2⁶⁹³⁶. Introduction of a single copy of a hypomorphic or null allele of dunce into the gprk2⁶⁹³⁶ background suppresses all of these defects to a significant degree. Suppression is also observed when a single copy of gprk2⁶⁹³⁶ is introduced into a dunce background. Like rutabaga mutants (rutabaga encodes a calcium/calmodulin-dependent adenylate cyclase), gprk2⁶⁹³⁶ has reduced levels of cAMP. Ovaries from gprk2⁶⁹³⁶ females contain about one third the normal amount of cAMP. In addition, in every mutant combination where fertility is increased, cAMP levels are closer to wild type levels. These results suggest that Gprk2 is functioning in a cAMP-signaling pathway and that the underlying basis of the interaction between Gprk2 and dunce is a normalization of cAMP levels (Lannutti, 2001).

Homozygous gprk2⁶⁹³⁶ females produce eggs with dorsal appendages that are malformed or truncated, and nurse dumping is often incomplete. The most severely affected egg chambers are not laid. Of the eggs that are laid, many have a flaccid appearance and there is a significant reduction in the rate of hatching. The embryos that fail to hatch show a range of defects including twisted gastrulation, fusion of adjacent segments, and a perforated cuticle (Schneider, 1997). Although some embryos do hatch, the number of animals that survive to adulthood is very low. The defects seen in gprk2⁶⁹³⁶ egg chambers are primarily the result of a lack of expression in the germline. Egg chambers from gprk2⁶⁹³⁶ germline clones display all of the defects seen in gprk2⁶⁹³⁶ homozygotes and they hatch at a similarly reduced rate. The gprk2⁶⁹³⁶ mutant is the only allele of Gprk2 that has been identified and there are no deficiencies that uncover this locus. However, expression of wild type Gprk2 under the control of either a heat shock or germline-specific promoter is sufficient to rescue the sterility defect of gprk2⁶⁹³⁶ (Lannutti, 2001).

Because of the possibility that dunce may be acting in a common pathway with Gprk2, the dunce ovarian phenotype was reexamined. Females homozygous for hypomorphic (dnc²) and null (dnc^M14 ) alleles have been shown to lay few or no eggs. This has been attributed to defects in the chorion and vitelline membrane. In addition to these defects, nurse cell dumping is incomplete in egg chambers from dnc²homozygous females. This incomplete cytoplasmic transfer may cause or contribute to the formation of misshapen dorsal appendages. Incomplete cytoplasmic dumping is also apparent in dnc^M14 homozygotes and dnc²/dnc^M14 transheterozygotes. In both of these genotypes, egg chambers have severely truncated dorsal appendages or the dorsal appendages fail to form altogether. The percentage of egg chambers that fail to complete cytoplasmic dumping does not increase significantly in the stronger allelic combinations, probably because many egg chambers fail to develop to that stage (Lannutti, 2001).

In addition to the dumping and dorsal appendage defects, ovaries from dunce mutant mothers contain degenerating egg chambers. In dnc² homozygotes, 70% of stages 10B and 11 egg chambers are degenerating, as determined by the presence of condensed, DAPI-bright nuclei. In dnc²/dnc^M14 and dnc^M14 homozygotes, egg chamber degeneration is detected in earlier stages (beginning at stage 6) and occurs more frequently. All three aspects of the dunce ovary phenotype, the incomplete cytoplasmic dumping, the malformed dorsal appendages, and the degeneration of egg chambers, resemble those of the gprk2⁶⁹³⁶ mutant (Lannutti, 2001).

Mutations in three genes, chickadee, quail, and singed, cause a block in nurse cell dumping and disrupt dorsal appendage formation. These three genes encode proteins whose mammalian homologs (profilin, villin, and fascin, respectively) are involved in actin bundling. In all three mutants, there is a failure in the formation of the cytoplasmic fibers that tether the nurse cell nuclei during cytoplasmic dumping. Oocytes from homozygous gprk2⁶⁹³⁶ mothers have dorsal appendages that are malformed, and nurse cell dumping is often incomplete. Immunofluorescence analysis of gprk2⁶⁹³⁶ egg chambers that are stained with DAPI reveal that some nuclei of nurse cells have a defect in tethering during cytoplasmic dumping. In stage 10B egg chambers from wild type mothers, cytoplasmic actin filaments extend from the nucleus to the plasma membrane. These filaments anchor the nuclei during dumping, thus preventing their movement into the ring canals. In contrast, in 31% of stage 10B egg chambers from gprk2⁶⁹³⁶ nuclei, stretching has been observed in the direction of cytoplasmic dumping and extending through the ring canals. More than two nuclei affected in a single egg chamber are never observed, and the nurse cells that dump directly into the oocyte are usually not affected. Phalloidin staining in gprk2⁶⁹³⁶ egg chambers demonstrates that cytoplasmic actin fibers do form in the mutant. However, these fibers often appear more clumped and irregular than those in wild type egg chambers. A tethering defect is never observed in the dnc² and dnc^M14 alleles (Lannutti, 2001).

There is a marked increase in fertility in allelic combinations of dunce and gprk2. This increase suggests that there should be a corresponding improvement in the ovary morphology of combinations of both mutants. To test this, a quantitative analysis of the ovaries from the different mutant combinations was performed. The feature of the dunce phenotype that is most readily quantified is egg chamber degeneration; this defect can be assayed by the presence of DAPI-bright nuclei. In dnc² homozygotes, DAPI-bright, nurse cell nuclei were observed in 39% of stages 9 and 10 egg chambers. This defect is more severe in dnc²/dnc^M14 transheterozygotes and dnc^M14/dnc^M14 homozygotes. In these genotypes 52% (dnc²/dnc^M14) and 58% (dnc^M14/dnc^M14) of stages 9 and 10 egg chambers display a degenerating phenotype. These numbers are an underestimate of the level of degeneration in dnc²/dnc^M14 and dnc^M14/dnc^M14 allelic combinations because egg chambers often degenerate earlier in oogenesis. As a result, ovaries from dnc²/dnc^M14 and dnc^M14/dnc^M14 females produce few egg chambers past stage 11. One copy of gprk2⁶⁹³⁶, which suppresses the sterility of the two dunce alleles, also suppresses degeneration in the dnc²/dnc², dnc²/dnc^M14, and dnc^M14/dnc^M14 mutants. Although degeneration is dramatically reduced in all three genotypes, rescue is not complete. These results support the suppression of sterility that is observed in dunce;gprk2⁶⁹³⁶ allelic combinations (Lannutti, 2001).

Mutations in the dunce gene cause an increase in embryonic and adult cAMP levels (two- to fivefold, depending on the allele) and most dunce alleles are sterile. In contrast, mutations in rutabaga cause a slight decrease in cAMP levels but are completely fertile. Double mutants of rutabaga and dunce have intermediate levels of cAMP and fertility is partially restored. Similarly, one copy of gprk2⁶⁹³⁶ partially restores fertility in dunce mutants. By analogy to the dunce-rutabaga interaction, the simplest explanation for the mutual suppression of gprk2⁶⁹³⁶ and dunce is that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are lower than those in wild type flies. In agreement with this suggestion, it was found that cAMP levels in the ovaries of gprk2⁶⁹³⁶ homozygotes are almost threefold lower than those in wild type females. Furthermore, the suppression of the gprk2⁶⁹³⁶ phenotype by dunce (and vice versa) is reflected in the cAMP levels of the dunce, gprk2⁶⁹³⁶ allelic combinations. Introducing a single copy of the dnc² or dnc^M14 mutant into gprk2⁶⁹³⁶ homozygotes resulted in a wild-type cAMP content, threefold higher than that in gprk2⁶⁹³⁶ homozygotes. The gprk2⁶⁹³⁶ mutant also suppresses the elevated levels of cAMP seen in mutant dunce alleles. In short, in every mutant combination in which an increase in fertility is observed, the cAMP content in the ovaries changes in the expected direction. Taken together, these results strongly support the hypothesis that Gprk2 is involved in a signaling pathway that utilizes cAMP as a second messenger (Lannutti, 2001).

Mutations in rutabaga result in a decrease in cAMP synthesis. By analogy, it is possible that there is a decrease in the synthesis of cAMP in the gprk2⁶⁹³⁶ mutant. This could be explained if the Gprk2 protein regulates receptors that inhibit adenylate cyclase through a Galphai protein. When the receptor becomes activated it would first decrease cAMP levels by inhibiting adenylate cyclase. Then, when the receptor becomes desensitized through the action of Gprk2, the inhibition of adenylate cyclase would be relieved and cAMP levels would increase. This model would explain the mutual suppression between gprk2⁶⁹³⁶ and the dunce alleles. In the gprk2⁶⁹³⁶ mutant, receptor activity (and, consequently, Galphai activity) would be prolonged and adenylate cyclase activity would be inhibited to a greater extent, thus leading to reduced levels of cAMP. Decreasing the dose of dunce would decrease the amount of phosphodiesterase activity. Thus the cAMP that is produced would be degraded more slowly, resulting in a greater accumulation than that in gprk2⁶⁹³⁶ homozygotes (Lannutti, 2001).

Similarly, in dunce mutants, cAMP levels are elevated as the result of a lack of cAMP metabolism. Decreasing the dose of Gprk2 would alleviate this problem, to some degree, by causing a reduction in the amount of cAMP that is synthesized. One Galphai (G-oalpha 65A) gene has been characterized in Drosophila and analysis of the Drosophila genome sequence suggests that there are no others. The Galphai protein is expressed in the follicle cells and is present in granules in the oocyte. There are no reported loss-of-function mutants of this gene. However, a dominant gain-of-function line has been generated using the UAS/GAL4 expression system. This mutant has altered signaling in the nervous system, as shown by its increase in sensitivity to cocaine (Li, 2000). If this simple model of Gprk2 function is correct, it would suggest that overexpression of activated Galphai would decrease fertility. An alternative mechanism is that the Gprk2 protein desensitizes receptors that couple to other G protein subunits that inhibit adenylate cyclases. Mammalian Galphao and certain combinations of betagamma have been shown to inhibit type I adenylate cyclases (rutabaga is a type I cyclase). Galphao, beta, and gamma subunits have all been identified in Drosophila and searches of the Drosophila genome suggest that there are six Galpha genes (including Galphas, Galphai, Galphao, Galphaq, and Galpha12/13), two beta-subunit genes, and two gamma-subunit genes). The expression of most of the alpha subunits in the ovaries has not been described. The exceptions are Galphai and Galphao, whose mRNA is expressed at high levels in the nurse cells, although there is no detectable protein expression. The expression of beta and gamma genes in the ovaries has not been reported (Lannutti, 2001).

It is interesting that gprk2⁶⁹³⁶ and dunce mutants have similar ovary phenotypes, even though they have opposite effects on cAMP levels in the ovaries. It appears that cAMP levels must be maintained at an optimum level; both an increase and a decrease cause defects in dorsal appendage formation, cytoplasmic dumping, and probably other maternal and zygotic functions. This effect has also been documented in assays of learning in the fly. Both rutabaga and dunce mutants cause defects in learning and memory, although they cause opposite changes in cAMP levels. Apparently, the level of cAMP must be tightly regulated for proper functioning of cAMP-dependent pathways (Lannutti, 2001 and references therein).

Another parallel with the dunce, rutabaga studies is the observation that the degree of suppression does not always correlate with the level of cAMP in the ovaries. In earlier studies it has been shown that a dunce, rutabaga double mutant (homozygous for both alleles) is still defective in learning, although cAMP levels are rescued. Similarly, rescue of fertility is not complete, even in combinations that produced cAMP levels very close to those of wild type. For example, the ovaries from dnc²/+;gprk2⁶⁹³⁶ /gprk2⁶⁹³⁶ females have cAMP levels that are not statistically different from wild type levels. However, the fertility of these females is still statistically less than that in wild type. There are several possible explanations for this effect. (1) It has been suggested that the kinetics of cAMP metabolism are as important as the level of cAMP per se. Similarly, the subcellular distribution of cAMP may play an equally important role in fertility. (2) The Gprk2 and dunce genes may have functions that are not dependent on one another. For example, because there are many heptahelical receptors in Drosophila and only two known GRKs, it is likely that Gprk2 protein phosphorylates many different receptor types. If some of these receptors act through second messengers other than cAMP, then they would not be rescued by a decrease in the dosage of dunce. Similarly, Dunce is the major source of phosphodiesterase activity in Drosophila. Therefore, a decrease in phosphodiesterase activity could disrupt signaling from Gprk2-independent receptors. (3) Gprk2 and Dunce could carry out functions that are not directly related to production or metabolism of second messengers. For example, mammalian GRKs have been shown to interact with cytoskeletal proteins, suggesting that they have a potential scaffolding or docking function (Lannutti, 2001 and references therein).

In germline clones of dnc^M14, eggs are laid but fail to hatch, suggesting that dunce activity is required in the somatic cells for egg laying and in the germline for hatching. This appears to contradict the data suggesting a germline requirement for dunce in egg laying. However, there are several possible explanations for this apparent discrepancy. (1) The analysis of egg laying from females with dnc^M14germline clones could not be quantitative because the ovarioles do not all contain clones. Therefore, while the data demonstrate that somatic expression of dunce is necessary for egg laying, it does not exclude a role in the germline as well. (2) By the same argument, a role for Gprk2 activity in the somatic cells has not been ruled out. Although Gprk2 expression in the follicle cells cannot be detected, it is possible that a low level of somatic expression plays a role in egg laying. (3) Although the Gprk2 gene plays a role in regulating the level of cAMP in the ovaries, it has not been directly shown that Gprk2 and dunce are acting in the same molecular pathway. Perhaps dunce and Gprk2 act in different signaling pathways that are active in somatic and germline cells, respectively, and the interaction of the two pathways is necessary for normal levels of egg laying (Lannutti, 2001).

Cytoplasmic dumping in nurse cells involves both tubulin-based microtubules and actin-based microfilaments. The submembranous cytoskeleton provides the contractile forces for dumping, and cytoplasmic actin fibers tether the nurse cell nuclei so that they do not become lodged in the ring canals. In the gprk2⁶⁹³⁶ mutant, a small number of nurse cell nuclei fail to remain tethered and, instead, begin to extend into the ring canals. This phenotype resembles the mutants quail, singed, and chickadee. The proteins encoded by these genes (homologs of villin, fascin, and profilin, respectively) are structural components of the cytoplasmic actin fibers, and in the mutants the fibers fail to form. In strong alleles of these loci the 'dumpless' phenotype is completely penetrant. In contrast, blocked ring canals are observed in the gprk2⁶⁹³⁶ mutant at a low frequency, although incomplete cytoplasmic dumping is much more common. Furthermore, the cytoplasmic actin fibers do form in gprk2⁶⁹³⁶, even in nurse cells whose nuclei are extending through ring canals. The clumped appearance of these fibers in these nurse cells suggests that they are not arranged in a normal fashion. At this time, whether the relationship between the cytoplasmic actin fibers and the lack of nuclear tethering is causative or correlative cannot be determined. The localization of the Gprk2 protein to a submembranous region suggests that Gprk2 may play a role in the interaction between the cytoplasmic filaments and the membrane, rather than a structural role in the filaments themselves (Lannutti, 2001).

Mammalian GRKs have also been shown to interact with the cytoskeleton. GRK5 (the closest mammalian homolog to Gprk2) phosphorylates ß-tubulin and microtubules, although the functional significance of this interaction is unknown (Carman, 1998). In addition, GRK5 binds to actin monomers and filaments, and stabilizes polymerized actin filaments in vitro. Actin binding, in turn, decreases the activity of GRK5 toward receptors (Freeman, 1998; Pitcher, 1998). The sequence of the actin-binding region in GRK5 is conserved in Gprk2, suggesting that Gprk2 could also bind to actin at this site. The low penetrance of the tethering defect in the gprk2⁶⁹³⁶ mutant suggests that the lack of cytoplasmic dumping cannot be completely explained by blockage of the ring canals. In addition, the dunce mutants have a similar dumping defect, although no nurse cell nuclei extending through the ring canals has been seen in these mutants. The formation of cytoplasmic fibers and nuclear breakdown that accompany normal cytoplasmic dumping fail to occur in germline clones that lack expression of Drosophila Caspase protein-1 (DCP-1), suggesting that the events that occur during cytoplasmic dumping are part of an apoptotic pathway. It has been hypothesized that DCP-1 activity leads to two parallel pathways: one resulting in the formation of cytoplasmic actin bundles and the other resulting in nuclear envelope permeabilization, elevation of cytoplasmic Ca2+, and contraction of the submembranous actin network. Because egg chambers from gprk2⁶⁹³⁶ and dunce mutants form cytoplasmic fibers and undergo contraction, it suggests that these genes are not involved in initiation of apoptosis. It is possible that Dunce and Gprk2 could affect cytoplasmic dumping through signaling processes that regulate Ca2+ levels during the apoptotic process (Lannutti, 2001 and references therein).

The rescue of the tethering defect, by reducing the dosage of dunce, suggests that this aspect of the gprk2⁶⁹³⁶ phenotype is also sensitive to the level or subcellular distribution of cAMP. It might be expected that a similar phenotype would be seen in mutant alleles of DCO, the gene encoding the catalytic subunit of PKA. DCO is an essential gene; however, combinations of certain weak alleles are semilethal and sterile. These egg chambers have an earlier defect than observed in gprk2⁶⁹³⁶. Mutant DCO egg chambers have unstable ring canals and, beginning at stage 7, nurse cells begin to fuse, forming binucleate cells. Therefore, their egg chamber morphology is partially disrupted by stage 10B, when cytoplasmic dumping begins. Nevertheless, the nurse cell nuclei appear to remain anchored in the center of the cell. Certain allelic combinations of chickadee also form bi- and multinucleate nurse cells, suggesting that cAMP signaling and the cytoskeleton both play critical roles in maintaining membrane integrity. Binucleate cells have never been observed in the gprk2⁶⁹³⁶ mutant. However, it is likely that there are receptors that contribute to cAMP signaling that are not regulated by Gprk2. These receptors could regulate PKA in the absence of Gprk2 activity (Lannutti, 2001 and references therein).

In summary, analysis of the interaction between gprk2⁶⁹³⁶ and dunce mutants demonstrates that the regulation of cAMP synthesis and metabolism is critical for development. The gprk2⁶⁹³⁶ mutant has not only reduced levels of cAMP but also decreased egg laying and hatching. These defects were all suppressed by weak and null alleles of dunce. Similarly, the increased levels of cAMP and sterility of dunce are suppressed by the gprk2⁶⁹³⁶ mutant. Further analysis of the developmental functions of gprk2⁶⁹³⁶ in vivo will continue to complement the biochemical characterization of this protein (Lannutti, 2001).

REGULATION

Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease

Alpha-synuclein is phosphorylated at serine 129 (Ser129) in intracellular protein aggregates called Lewy bodies. These inclusion bodies are the characteristic pathologic lesions of Parkinson disease. This study defines the role of phosphorylation of Ser129 in alpha-synuclein toxicity and inclusion formation using a Drosophila model of Parkinson disease. Mutation of Ser129 to alanine to prevent phosphorylation completely suppresses dopaminergic neuronal loss produced by expression of human alpha-synuclein. In contrast, altering Ser129 to the negatively charged residue aspartate, to mimic phosphorylation, significantly enhances alpha-synuclein toxicity. The G protein-coupled receptor kinase 2 (Gprk2) phosphorylates Ser129 in vivo and enhances alpha-synuclein toxicity. Blocking phosphorylation at Ser129 substantially increases aggregate formation. Thus Ser129 phosphorylation status is crucial in mediating alpha-synuclein neurotoxicity and inclusion formation. Because increased number of inclusion bodies correlates with reduced toxicity, inclusion bodies may protect neurons from alpha-synuclein toxicity (Chen, 2005).

The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila

Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. Gprk2 function is required for transducing the Smo signal, and when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary (Molnar, 2007).

Smo is the key transducer of a conserved signaling pathway regulating many developmental processes in vertebrates and invertebrates. The transmembrane protein Patched (Ptc) is the receptor for the ligand Hedgehog (Hh) and represses Smo activity in the absence of ligand. The binding of Hh to Ptc relieves this repression and allows Smo to signal to a protein complex that includes the transcription factor Ci/Gli. Smo controls the activation of Ci in the presence of the Hh ligand in part by preventing Ci proteolytic processing into a transcriptional repressor. In the Drosophila wing disc, the epithelium giving rise to the wing and thorax of the fly, Smo signaling controls the expression of several genes in anterior cells close to the anterior-posterior (A/P) compartment boundary and promotes the growth and patterning of the wing (Molnar, 2007).

The cytoplasmic tail of Drosophila Smo is a target for phosphorylation by protein kinase A and casein kinase I, and it has been shown that Smo phosphorylation by these kinases is essential for its activity and membrane accumulation. However, most of these phosphorylated residues are not conserved in its vertebrate counterparts. Recently, the G protein-coupled receptor kinase 2 (Grk2) has been shown to phosphorylate mammalian Smo (Chen, 2004). G protein-coupled receptor kinases (GRKs) selectively phosphorylate the ligand-activated form of G protein-coupled receptors (Lefkowitz, 2005). This phosphorylation promotes uncoupling from G proteins and also the recruitment of β-arrestins, which target the receptor for clathrin-mediated endocytosis. In addition, GRKs and β-arrestins also participate in signal propagation by recruiting additional proteins to the receptor complex. There are two Drosophila GRKs, GPRK1 and GPRK2. GPRK1 modulates the amplitude of the visual response acting as a Rhodopsin kinase, whereas GPRK2 regulates the level of cAMP during Drosophila oogenesis (Schneider, 2005; Lannutti, 2001). Phosphorylation of mammalian Smo by GRK2 promotes its endocytosis in clathrin-coated pits in a process dependent on β-arrestin2 (Chen, 2004). However, whether this form of Smo internalization is part of a desensitization mechanism, as is the case for different G protein-coupled receptors (Lefkowitz, 2005), or if it participates in Hh signaling is still not known. To address the participation of GRKs during Smo signaling in Drosophila, the function was analyzed of Gprk2 during imaginal wing disc development. It was found that Gprk2 activity is required for Smo activation. Thus, the reduction of Gprk2 expression by interference RNA, or its elimination by a genetic mutation, causes the accumulation of Smo in wing disc anterior cells exposed to Hh. The accumulation of Smo is, however, correlated with reduced activity, because Smo high-level targets are not correctly activated and flies expressing Gprk2-RNAi display Hh loss-of-function phenotypes. Interestingly, the reduction in Gprk2 expression is able to antagonize the activity of Smo mutant forms that mimic its phosphorylation by protein kinase A and casein kinase 1, suggesting that additional phosphorylation by Gprk2 is a necessary step to obtain the correct activation of Smo to promote the expression of its targets requiring high levels of signaling (Molnar, 2007).

The expression of Gprk2 mRNA in the wing disc is generalized but appears increased in a stripe of cells located close to the A/P compartment boundary. To better characterize this pattern, the P-lacZ insertion Gprk2⁰⁶⁹³⁶, which is localized in the 5' untranslated region of the gene, was used. Interestingly, β-gal expression is restricted to the A/P compartment boundary of the wing disc during the third larval instar. The cells expressing β-gal were further identified by using a combination of region-specific markers such as Engrailed (En), Patched (Ptc), Blistered (Bs), and Caupolican (Caup). This analysis places the stripe of maximal expression of Gprk2 to anterior cells abutting the A/P boundary. These cells express Ptc and En in the anterior compartment and are localized in the region exposed to high-level Hh signaling. In fact, Hh signaling regulates the expression of Gprk2 in these anterior cells, because β-gal expression in Gprk2⁰⁶⁹³⁶ discs is expanded to the entire anterior compartment when hh is ectopically expressed, and it is repressed when the activity of the pathway is reduced by ectopic expression of Ptc). The regulation of Gprk2 accumulation in anterior cells by Hh suggests that Hh signaling and Gprk2 might be functionally related (Molnar, 2007).

All available Gprk2 alleles are P element insertions in the 5' region of the gene. These alleles are homozygous viable, and the mutant wings do not display any visible phenotype. Stronger loss-of-function conditions of the gene were generated by (1) expressing Gprk2 interference RNA (Gprk2i) under the control of yeast upstream activator sequences (UAS; UAS-Gprk2i) and (2) constructing a synthetic deletion of the gene. In wing discs of the combination Gal4-638/UAS-Gprk2i, a reduction of Gprk2 mRNA levels of 66% was found. The corresponding adult wings show a range of striking phenotypes similar to loss of Hh function, displaying a reduction of the L3/L4 intervein, fusion of the L3 and L4 veins, and in a lower percentage of wings, the loss of the L3 and L4 veins. These veins and the L3/L4 intervein correspond to the territory specified by Hh signaling. In fact, reduction of Gprk2 levels results in wings very similar to those with a moderate loss of Hh signaling, generated either by ectopic expression of Ptc or by expression or hh-interference RNA. This phenotype is very different from that observed upon increased activity of the pathway. The Gal4-638 line is expressed in the entire wing, and to distinguish between the effects of lowering Gprk2 levels in cells producing or responding to Hh, three other Gal4 lines were used expressed in either the anterior (Gal4-Ci and Gal4-ptc) or the posterior (Gal4-hh) compartments. It was found that the expression of Gprk2i only in anterior cells recapitulates the reduction of the L3/L4 intervein observed in Gal4-638/UAS-Gprk2i wings. Thus, the combinations Gal4-Ci/UAS-Gprk2i and Gal4-ptc/UAS-Gprk2i show a reduction or elimination of the L3/L4 intervein, whereas the wings of the Gal4-hh/UAS-Gprk2i combination display a normal pattern of veins. The phenotypes observed upon a reduction of Gprk2 unambiguously indicate that Gprk2 function is necessary for the transduction of the Hh signal. Furthermore, when the expression of Gprk2 is reduced in flies expressing lower levels of the ligand Hh, the resulting wings have stronger hh loss-of-function phenotypes, and a previously unrecognized phenotypic class indistinguishable to those of wings formed by smo mutant cells is now observed (Molnar, 2007).

To directly monitor the activity of the Hh pathway, the expression of several Hh targets was studied in Gal4-638/UAS-Gprk2i discs. The expression of En and Ptc in anterior cells is always impaired when Gprk2 levels are reduced. These two genes correspond to Hh targets activated by a high level of signaling. The expression of Knot (Kn) is also reduced in Gal4-638/UAS-Gprk2i discs, and the stripe of maximal accumulation of Ci is also modified in Gal4-638/UAS-Gprk2i discs compared with wild-type ones. The expression of other genes regulated directly or indirectly by Hh signaling was studied in Gal4-638/UAS-Gprk2i discs. The expression of the Notch ligand Delta (Dl) is very weak or absent in the primordia of the veins L3 and L4, where it accumulates at high levels in normal discs. Similarly, the expression of Bs in the L3/L4 intervein is reduced or absent in Gal4-638/UAS-Gprk2i discs. The expression of the low-level Hh signaling targets caup and decapentaplegic (dpp) is also modified in Gal4-638/UAS-Gprk2i discs. Caup expression in the presumptive L3 vein is generally expanded toward the A/P compartment boundary in Gal4-638/UAS-Gprk2i discs, most likely because En, a repressor of Caup in anterior cells, is not expressed upon a reduction of Gprk2 levels. The domain of Caup expression in the L3 vein is reduced or lost compared with wild-type discs in only a small fraction of discs (7%). The expression of dpp is detected in Gal4-638/UAS-Gprk2i discs at lower levels but in a domain broader than the characteristic of normal discs. Taken together, these data suggest that Gprk2 plays a positive role in the Hh signaling pathway. The lowering of Gprk2 levels reduces very efficiently high-level Hh signaling and much less efficiently low-level Hh signaling. Thus, a complete elimination of Hh signaling is observed only when Gprk2 levels are reduced in wing discs with lower hh. Finally, the expression of spalt, a target of the Dpp/BMP4 pathway, is almost normal upon Gprk2 reduction, indicating specificity of Gprk2 function toward Hh signaling (Molnar, 2007).

To confirm the specificity of the Gprk2 RNAi, the expression of two Hh-targets, En and Ptc, was analyzed in wing disc cells homozygous for a deficiency that removes all of the Gprk2 coding region. In both cases it was found that anterior Gprk2 ^- clones eliminate, in a cell-autonomous manner, the anterior expression of En. Gprk2 ^- clones located in the posterior compartment did not affect the expression of En, confirming that Gprk2 activity is required in cells receiving Hh (Molnar, 2007).

To further analyze where Gprk2 function is required in the Hh signaling pathway, the expression of En was studied in clones of cells ectopically expressing hh or both hh and Gprk2i. It was found that clones expressing Gprk2i located in the domain of En expression in the anterior compartment cell-autonomously suppress the expression of En. The expression of En is induced by Hh signaling in hh-expressing clones, both within the clone and in the surrounding cells. However, in the hh+Gprk2i-expressing clones, the expression of En is induced only in wild-type anterior cells that do not express Gprk2i. These observations confirm that Gprk2 activity is required for transducing the Hh signal in Hh-receiving cells and not for Hh secretion (Molnar, 2007).

Experiments in mammalian cells in culture have shown that beta-arrestin2 and GRK2 mediate internalization of active Smo (Chen, 2004). Consequently, the expression and subcellular localization of Smo was studied in wing discs where Gprk2 activity is reduced. In wild-type discs, smo RNA is expressed in all cells, but Smo protein accumulates associated to cell membranes only in the posterior compartment and in some anterior cells exposed to Hh. Intriguingly, the reduction in Gprk2 levels in the entire wing blade eliminates the distinction in Smo accumulation between anterior and posterior cells, and Smo is detected at similar levels in both compartments. When the levels of Gprk2 are reduced only in the dorsal compartment or in clones of Gprk2 ^- homozygous cells, the changes in Smo expression in anterior cells are more evident. Thus, it was observed that Smo accumulates at high levels associated to cell membranes in a broader anterior domain of cells within the range of Hh. The extension of Smo accumulation in anterior cells might be due to an extension of the Hh diffusion range because Ptc is not expressed in Gprk2 mutant cells. This is a previously unrecognized instance in which Smo accumulation and signaling can be uncoupled, because it was thought that, at least in Drosophila, Smo membrane accumulation leads to signaling. The same effects are observed when S2 cells were used. Thus, Smo is expressed in S2 cells in intracellular vesicles at low levels. Upon Hh treatment, Smo translocates close to the plasma membrane in these cells. In cells that have been treated for 4 days with Gprk2 dsRNA (causing a reduction of Gprk2 mRNA levels of 77%) the levels of Smo are higher independently of Hh (Molnar, 2007).

To further analyze the relationship between Smo and Gprk2 functions, Gprk2i was expressed in the same wing with different N-terminal (smoDeltaN; extracellular) and C-terminal (smoDeltaC2 and smoDeltaC4; intracellular) deletions of Smo. The expression of Smo proteins bearing either N-terminal or C-terminal deletions fails to rescue Smo mutants, but their overexpression does not interfere significantly with Smo signaling. A strong synergic genetic interaction was found when Smo C-terminal deletions were coexpressed with Gprk2i. Thus, wings expressing C-terminal deletions of Smo with reduced Gprk2 levels display a strong hh loss-of-function phenotype that is comparable to the elimination of smo. Gprk2i combined with UAS-smoDeltaN resulted in additive phenotypes. It is suggested that the reduction of Gprk2 uncovers a dominant-negative effect of SmoDeltaC proteins, reducing the efficiency of Smo signaling. The basis for this dominant negative effect could be the inclusion of a form of Smo, SmoDeltaC, unable to be phosphorylated by Gprk2, in the Smo complexes that have been postulated to mediate Smo activity. Therefore, it is proposed that Gprk2 function, acting through the C-terminal tail of Smo, is involved in an activation step promoting Smo interaction with the Costal2/Fused/Su(fu) complex to prevent Ci processing into a repressor form and to accumulate Ci in an activating form. Based on the effects of mammalian GRK2 and beta2-arrestin on Smo (Chen, 2004, Meloni, 2006), it is possible that Gprk2-mediated activation of Smo involves the recycling of Smo from the cell membrane to an intracellular signaling compartment (Molnar, 2007).

The interaction between SmoDeltaC and Gprk2 indicates a critical role of the Smo intracellular C-terminal domain for its relationship with Gprk2 function. Interestingly, the Smo intracellular C-terminal domain is where all of the consensus phosphorylation sites by casein kinase 1 and protein kinase A are located, as well as other serine and threonine residues in the vicinity of acidic residues that are similar to mammalian GRK2 phosphorylation consensus. A form of Smo was expressed in the wing disc that mimics its phosphorylation by these kinases (Smo^SD123), and whether this Smo-activated form is sensitive to Gprk2 levels was analyzed. The expression of Smo^SD123 in the wing disc causes overgrowth of the anterior compartment and defects in the L3 and L2 veins. In the corresponding wing discs, the accumulation of Smo and the expression of its targets En and Ptc are expanded to occupy the entire anterior compartment. When Gprk2 levels are reduced in discs expressing Smo^SD123, Smo accumulation is still observed in all anterior cells. In contrast, the expression of both En and Ptc is now restricted to their normal domains adjacent to the A/P compartment boundary. The overgrowth phenotype characteristic of Gal4-638/+; UAS-Smo^SD123 discs is not rescued by the reduction of Gprk2 expression, suggesting that the low-level Hh target dpp is still expressed through the anterior compartment. These data suggest that to generate the high levels of Smo activity required to activate the expression of its targets En and Ptc, the Smo^SD123 protein has to be phosphorylated by Gprk2 (Molnar, 2007).

In conclusion, Drosophila Gprk2 is critically required to generate high levels of Hh signaling in the wing disc. The genetic interactions between Gprk2 and Smo proteins bearing C-terminal deletions or Smo phosphomimic variants suggest that Smo is a target of Gprk2. The modifications in Smo protein accumulation detected in wing discs and S2 cells with reduced Gprk2 expression suggests that a likely step affected by Gprk2 is the activation of Smo by a phosphorylation step that could prime Smo for internalization to a signaling compartment. GRK2 has recently been shown to play a positive role in Shh transduction in mammalian cells (Meloni, 2006). Taken together, these findings and the data indicate that Smo phosphorylation by GRK homologues constitute a conserved component of the Smo signal transduction cascade (Molnar, 2007).

Spike amplitude of single-unit responses in antennal sensillae is controlled by the Drosophila circadian clock

Circadian changes in membrane potential and spontaneous firing frequency have been observed in microbial systems, invertebrates, and mammals. Oscillators in olfactory sensory neurons (OSNs) from Drosophila are both necessary and sufficient to sustain rhythms in electroanntenogram (EAG) responses, suggesting that odorant receptors (ORs) and/or OR-dependent processes are under clock control. This study measured single-unit responses in different antennal sensillae from wild-type, clock mutant, odorant-receptor mutant, and G protein-coupled receptor kinase 2 (Gprk2) mutant flies to examine the cellular and molecular mechanisms that drive rhythms in olfaction. Spontaneous spike amplitude, but not spontaneous or odor-induced firing frequency, is under clock control in ab1 and ab3 basiconic sensillae and T2 trichoid sensillae. Mutants lacking odorant receptors in dendrites display constant low spike amplitudes, and the reduction or increase of levels of GPRK2 in OSNs results in constant low or constant high spontaneous spike amplitudes, respectively. It is concluded that spike amplitude is controlled by circadian clocks in basiconic and trichoid sensillae and requires GPRK2 expression and the presence of functional ORs in dendrites. These results argue that rhythms in GPRK2 levels control OR localization and OR-dependent ion channel activity and/or composition to mediate rhythms in spontaneous spike amplitude (Krishnan, 2008).

One hypothesis to explain rhythms in the amplitude of spontaneous spikes and electroanntenogram rhythms (EAGs) is that ion channel activity and/or composition is under circadian control. Drosophila ORs have been found to form heteromeric odor-gated and cyclic-nucleotide-activated cation channels. It has been demonstrated that ORs accumulate in OSN dendrites in a circadian fashion, where OR abundance peaks near the middle of the night and is low during the day (Tanoue, 2008). These rhythms are dependent on the levels of GPRK2 and coincide with rhythms in the amplitude of both EAGs and spontaneous spikes (Tanoue, 2008). Taken together, these results suggest a model whereby GPRK2 controls the abundance and/or activity of OR-dependent odor-gated cation channels in OSN dendrites, which in turn alter membrane conductance to generate rhythms in the amplitude of spontaneous spikes and EAG responses. The possibility cannot be excluded that the clock modulates other molecular or cellular targets to generate rhythms in EAG and spike amplitude such as other ion channels expressed in OSNs, the composition of sensillar lymph, and/or the size and shape of OSNs (Krishnan, 2008).

Though a clear ecological explanation for rhythms in spike amplitude is not understood, one could hypothesize that the circadian clock could tune the olfactory system to a higher gain level (higher signal-to-noise ratio) by modulating spike amplitude irrespective of the stimulus preferentially in the subjective night. For instance, rhythms in OSN spike amplitude might produce increased activity in downstream neurons during the night and decreased activity during the day in response to the same stimulus. Recent evidence demonstrates that locomotor activity of paired male and female Drosophila is increased during the subjective night and is dependant on an intact olfactory system (Fujii, 2007). In addition, behavioral responses to odors in Drosophila are lower during the day than at night and are controlled by the circadian clock (Zhou, 2005). These phenomena may represent behavioral consequences of this electrophysiological rhythm that could provide an advantage in courtship, food acquisition, or predator avoidance. These data are consistent with circadian-clock-dependent rhythms in mating activity (Sakai, 2001). The phase of the peak in spontaneous spikes in trichoids could translate to heightened behavioral activity associated with mating during the subjective night. Thus, the results suggest that spike amplitude, in addition to firing frequency, can also encode meaningful information in the peripheral OSNs, which is transmitted to higher processing centers of CNS that mediate behavioral responses to odors (Krishnan, 2008).

G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila.

G protein-coupled receptor kinase 2 (Gprk2/GRK2) plays a conserved role in modulating Hedgehog (Hh) pathway activity, but its mechanism of action remains unknown. This study provides evidence that Gprk2 promotes high-level Hh signaling by regulating Smoothened (Smo) conformation through both kinase-dependent and kinase-independent mechanisms. Gprk2 promotes Smo activation by phosphorylating Smo C-terminal tail (C-tail) at Ser741/Thr742, which is facilitated by PKA and CK1 phosphorylation at adjacent Ser residues. In addition, Gprk2 forms a dimer/oligomer and binds Smo C-tail in a kinase activity-independent manner to stabilize the active Smo conformation, and promotes dimerization/oligomerization of Smo C-tail. Gprk2 expression is induced by Hh signaling, and Gprk2/Smo interaction is facilitated by PKA/CK1-mediated phosphorylation of Smo C-tail. Thus, Gprk2 forms a positive feedback loop and acts downstream from PKA and CK1 to facilitate high-level Hh signaling by promoting the active state of Smo through direct phosphorylation and molecular scaffolding (Chen, 2010).

A genetic modifier screen for novel Hh signaling components identified Gprk2 as a positive regulator of Smo. Gprk2 was shown to be required for high but not low levels of Hh signaling activity. Evidence was provided that Gprk2 is a Smo kinase and that Gprk2 promotes maximal Smo activity by phosphorylating S₇₄₁/T₇₄₂ in Smo C-tail. Furthermore, a kinase-independent function of Gprk2 in Hh signaling was uncovered. Gprk2 forms a dimer/oligomer and binds Smo C-tail to promote the active state of Smo. Thus, this study reveals a novel mechanism for regulating a GPCR-like protein by GRK (Chen, 2010).

Previous studies suggest that Drosophila Gprk2 and mammalian GRK2 are involved in Smo phosphorylation because their knockdown in cultured cells either increased Smo mobility on SDS-PAGE or decreased metabolic labeling of Smo by γ-³²p-ATP. However, these studies did not distinguish whether Gprk2/GRK2 phosphorylates Smo directly or indirectly through regulating other kinases. Neither did they reveal any biological relevance of Gprk2/GRK2-mediated phosphorylation in Hh signaling, since the relevant phosphorylation sites on Smo were not identified. In an in vitro kinase assay using purified substrates and a recombinant GRK, this study found that Smo is phosphorylated by GRK at S₇₄₁/T₇₄₂ and S₁₀₁₃/S₁₀₁₅. A mutagenesis study demonstrated that phosphorylation at S₇₄₁/T₇₄₂ is required for optimal Smo activation. Indeed, a previous study showed that Smo is phosphorylated at S₇₄₁/T₇₄₂ in cultured cells in the presence of Hh. In further agreement with the functional significance of S₇₄₁/T₇₄₂ phosphorylation, conserved S/T residues are found at the corresponding location in other insect Smo proteins (FlyBase) (Chen, 2010).

Interestingly, the in vitro kinase assay revealed that phosphorylation of S₇₄₁/T₇₄₂ by Gprk2 is regulated by PKA/CK1 phosphorylation at adjacent Ser residues, including S₇₄₀, S₇₄₃, and S₇₄₆. Previous studies in mammalian systems suggest that GRKs tend to phosphorylate S/T residues embedded in an acidic environment. Phosphorylation at S₇₄₀, S₇₄₃, and S₇₄₆ improves the acidic environment for S₇₄₁/T₇₄₂, which may account for the observed stimulation of S₇₄₁/T₇₄₂ phosphorylation by PKA/CK1. Indeed, mutating S₇₄₀, S₇₄₃, and S₇₄₆ to Ala abolished PKA/CK1-mediated stimulation of S₇₄₁/T₇₄₂ phosphorylation, whereas converting these residues to acidic residues mimicked PKA/CK1-mediated stimulation. As Hh induces Smo phosphorylation by PKA and CK1, phosphorylation at S₇₄₁/T₇₄₂ by Gprk2 is likely to be stimulated by Hh in vivo (Chen, 2010).

Although phosphomimetic mutation at S₇₄₁/T₇₄₂ promotes Smo activity, it does not bypass the requirement for Gprk2 for optimal Smo activation because SmoSDGPSD failed to induce ectopic en expression in Gprk2 mutant discs. This implies that Gprk2 promotes Hh signaling through a mechanism in parallel to S₇₄₁/T₇₄₂ phosphorylation. It is possible that Gprk2 might act at an additional step downstream from Smo activation by phosphorylating intracellular Hh signaling components, or at the level of Smo activation by phosphorylating Smo at additional sites that have been missed by the in vitro kinase assay. However, the finding that the constitutively active form of Smo lacking the autoinhibitory domain (SAID: SmoΔ661-818) is insensitive to Gprk2 inactivation suggests that Gprk2 acts mainly at the level of Smo, although the possibility cannot be ruled out that Gprk2 may also play a minor role downstream from Smo. Interestingly, it was found that the kinase-dead form of Gprk2 (Gprk2^KM) can rescue the activity defect of SmoSDGPSD in Gprk2 mutants, demonstrating that Gprk2 also regulates Smo in a phosphorylation-independent manner. The observation that Gprk2^KM does not rescue the activity defect of SmoSD123 in Gprk2 mutants suggests that the phosphorylation-dependent and phosphorylation-independent mechanisms act in parallel rather than redundantly to promote Smo activation. Furthermore, evidence was obtained that Gprk2 interacts with the SAID independently of its kinase activity. Therefore, it is proposed that Gprk2 promotes Smo activation by counteracting Smo autoinhibition through binding to and phosphorylating the SAID (Chen, 2010).

At least two paralleled mechanisms have been attributed to Smo activation by Hh: (1) Smo cell surface accumulation, and (2) conformation change in Smo C-tail. Intriguingly, it was found that loss of Gprk2 resulted in increased rather than decreased Smo levels in cells that are not exposed to Hh or are exposed to low levels of Hh. However, unlike Hh stimulation, which preferentially stabilizes Smo on the cell surface, Gprk2 inactivation appears to stabilize Smo both inside the cell and on the cell surface. Furthermore, in the presence of high levels of Hh where Smo is accumulated at high levels on the cell surface, Gprk2 inactivation does not cause any discernible changes in either the level or subcellular distribution of Smo. Thus, the reduced Smo activity in Gprk2 mutant cells exposed to high levels of Hh is unlikely to be due to a change in Smo level or subcellular localization (Chen, 2010).

It is not clear what role Gprk2-mediated down-regulation of Smo levels might play in Hh signaling, although this may reflect an ancient mechanism by which GRK family kinases 'desensitize' GPCRs. In this regard, Gprk2-mediated down-regulation could serve as a mechanism to restrict the basal level of Hh signaling activity or to terminate or tune down Hh signaling activity once the Hh signal is withdrawn. However, this negative role of Gprk2 could be masked by its positive role. The mechanism by which Gprk2 down-regulates Smo levels remains unclear, although the kinase activity of Gprk2 appears to be required. Gprk2 could phosphorylate Smo and/or other proteins to promote Smo internalization and degradation. High levels of Hh could counteract Gprk2-mediated down-regulation of Smo by preventing Gprk2-meidated Smo internalization or by promoting Smo recycling (Chen, 2010).

FRET analysis provided strong evidence that Gprk2 is required for Smo to adopt and/or maintain its active conformation in response to Hh stimulation. A previous study suggested that Hh induces a conformational switch in Smo C-tail that is mediated by PKA and CK1 phosphorylation. In the absence of Hh, the Smo C-tail adopts a closed conformation in which the tail folds back, resulting in a close proximity between the C terminus and the third intracellular loop. The closed conformation is maintained, at least in part, through intramolecular electrostatic interactions between the multiple Arg clusters in the SAID and multiple acidic clusters near the C terminus. Hh-induced phosphorylation at PKA and CK1 sties disrupts the intramolecular electrostatic interactions, resulting in unfolding of the C-tail, which is reflected by a decreased intramolecular FRET (FRET^L3C). In addition, phosphorylation promotes dimerization of two C-tails within a Smo homodimer, leading to increased proximity of the two C termini, as reflected by an increased C-terminal FRET (FRET^C). Multiple intermediate conformational states may exist, depending on the levels of Smo phosphorylation, as increasing the number of phosphomimetic mutations progressively decreased FRET^L3C and gradually increased FRET^C. It was found that both an Hh-induced decrease in FRET^L3C and an Hh-induced increase in FRET^C were compromised by loss of Gprk2, suggesting that Gprk2 is critical for Smo to adopt and/or maintain the fully open conformation (Chen, 2010).

How does Gprk2 regulate Smo conformation? Genetic and FRET analyses demonstrated that Gprk2 promotes high levels of Hh signaling activity and regulates Smo conformation through both phosphorylation-dependent and phosphorylation-independent mechanisms. Furthermore, this study found that Gprk2 self-associates, binds the SAID, and promotes self-association of Smo C-tail. Interestingly, both Gprk2/SAID interaction and S₇₄₁/T₇₄₂ phosphorylation by Gprk2 are enhanced by PKA/CK1 phosphorylation. Taken together, the following model is proposed to account for the regulation of Smo conformation by Gprk2. In response to Hh, PKA/CK1-mediated phosphorylation of Smo C-tail promotes its unfolding and dimerization; however, in the absence of Gprk2, the open conformational state of Smo is unstable and may exist in equilibrium with the closed and/or partially open conformational states. Phosphorylation of Smo by PKA/CK1 promotes the binding of Gprk2 to the SAID and phosphorylation at S₇₄₁/T₇₄₂, both of which may stabilize Smo in the fully open and active conformation by preventing refolding of Smo C-tail and by 'cross-linking' the two C-tails within a Smo dimer via dimerization of Gprk2. In essence, Gprk2 may function as a 'molecular clamp' to promote the clustering of Smo C-tails. It is also possible that Gprk2 could cross-link two or more Smo dimers to form high-order oligomers, which might be essential for high levels of Hh signaling activity. This study thus reveals an unanticipated complexity in the regulation of Smo conformational states, and provides the first evidence that Smo conformation states are regulated by not only phosphorylation and intramolecular interactions, but also intermolecular interactions. It is possible that the closed conformation state of Smo is also regulated by intermolecular interactions in addition to intramolecular interactions. For example, it has been shown that Fu can directly bind the Smo C terminus in the absence of Hh, and this interaction may help stabilize the closed conformation of Smo C-tail. Indeed, disrupting Smo/Fu interaction led to increased basal activity of Smo (Chen, 2010).

Recent studies have emphasized the differences between vertebrate and Drosophila Hh signaling mechanisms. The sequence divergence between Drosophila and vertebrate Smo proteins and the lack of conserved PKA/CK1 phosphorylation sites in vertebrate Smo proteins have led to the proposal that vertebrate Smo proteins are activated through fundamentally distinct mechanisms. Nevertheless, a previous study revealed that Shh induces a conformational change in mSmo similar to that of dSmo, and forced clustering of mSmo also leads to pathway activation). GRK2 has been implicated as a positive regulator of the Shh pathway, and mSmo phosphorylation is affected by GRK2 silencing, although direct phosphorylation of mSmo by GRK2 has not been demonstrated. It is possible that GRK2 may substitute the role of PKA and CK1 and act as a major Smo kinase in vertebrates to promote the active Smo conformation. Alternatively, GRK2 may act in conjunction with other GRKs and/or yet-to-be-identified kinases to regulate Smo conformation, subcellular localization, and activity in vertebrates. The relatively weak phenotypes exhibited by GRK2 mutants are consistent with the latter possibility. This study also raised an interesting possibility that GRK2 may regulate mSmo not only by phosphorylation, but also by a kinase-independent mechanism such as a protein-protein interaction. Further investigation of the mechanism by which GRK2 and other kinases regulate mSmo will shed an important light on how vertebrate Smo activation is achieved (Chen, 2010).

Drosophila G-protein-coupled receptor kinase 2 regulates cAMP-dependent Hedgehog signaling

G-protein-coupled receptor kinases (GRKs) play a conserved role in Hedgehog (Hh) signaling. In several systems, GRKs are required for efficient Hh target gene expression. Their principal target appears to be Smoothened (Smo), the intracellular signal-generating component of the pathway and a member of the G-protein-coupled receptor (GPCR) protein family. In Drosophila, a GRK called Gprk2 is needed for internalization and downregulation of activated Smo, consistent with the typical role of these kinases in negatively regulating GPCRs. However, Hh target gene activation is strongly impaired in gprk2 mutant flies, indicating that Gprk2 must also positively regulate Hh signaling at some level. To investigate its function in signaling, several different readouts of Hh pathway activity were analyzed in animals or cells lacking Gprk2. Surprisingly, although target gene expression was impaired, Smo-dependent activation of downstream components of the signaling pathway was increased in the absence of Gprk2. This suggests that Gprk2 does indeed play a role in terminating Smo signaling. However, loss of Gprk2 resulted in a decrease in cellular cAMP concentrations to a level that was limiting for Hh target gene activation. Normal expression of target genes was restored in gprk2 mutants by stimulating cAMP production or activating the cAMP-dependent Protein kinase A (Pka). These results suggest that direct regulation of Smo by Gprk2 is not absolutely required for Hh target gene expression. Gprk2 is important for normal cAMP regulation, and thus has an indirect effect on the activity of Pka-regulated components of the Hh pathway, including Smo itself (Cheng, 2012).

Based on existing evidence, the role of GRKs in Hh signaling is complex. Elimination of Gprk2 in flies leads to increased accumulation of Smo at the cell surface, generally a sign of high-level signaling and consistent with the typical function of GRKs in negatively regulating GPCRs. However, activation of Hh target genes is lost, indicating that Gprk2 plays a positive role in the pathway. Based on analysis of how signaling downstream of Smo is affected in the absence of Gprk2, this study draws three main conclusions that provide insight into this apparent contradiction. First, Gprk2 does indeed act as a negative regulator of the Hh pathway by limiting accumulation of active Smo. Second, Gprk2 activity is required for normal regulation of cellular cAMP levels, and thus Pka activity. Third, much of the positive effect of Gprk2 on Hh pathway activation is indirect, through promotion of Pka-dependent Smo phosphorylation and activation (Cheng, 2012).

GRKs regulate homologous desensitization of GPCRs, thereby limiting their signaling activity. Consequently, GRK loss often leads to increased surface receptor levels and exaggerated signaling responses. Consistent with this, previous studies have shown that Gprk2 downregulates Smo subsequent to its activation. This study finds that Smo-dependent activation of the canonical Hh pathway is increased at a cellular level in the absence of Gprk2, in a manner roughly proportional to the increase in Smo levels. This is the first demonstration that Gprk2 does in fact restrict Smo activity. Taken together the evidence suggests that Smo undergoes Gprk2-dependent homologous desensitization; that in the absence of Gprk2, Smo activity is not dramatically impaired; and that the accumulation of active Smo leads to more total pathway activation. As a result, Ci¹⁵⁵ is stabilized and enters the nucleus in gprk2-mutant Hh-responding cells, as in wild-type cells. However, high-threshold target genes are not expressed (Cheng, 2012).

Some of the same features of Hh pathway misregulation are found in other pathway mutants. Loss of Fu leads to similar effects on Ci stabilization and target gene expression. In contrast to fu mutants, SuFu is strongly phosphorylated and Ci¹⁵⁵ accumulates in the nucleus in gprk2 mutants. Manipulation of SuFu and Gα activity clearly points to distinct underlying causes of the signaling defects in these mutants. In dally and lipophorin (lpp; Rfabg -- FlyBase) mutants, Ci¹⁵⁵ stabilization and nuclear import are uncoupled from target gene expression, as in the gprk2 mutants. Knockdown of lpp lipoprotein particle production in particular has some similar, if more severe, consequences as loss of gprk2 - ectopic Smo stabilization and nuclear accumulation of Ci¹⁵⁵ (although in cells not exposed to Hh), without activating target genes. Although the phenotypic similarities suggest that Gprk2 could work through the same mechanism, there are important differences that suggest this is unlikely. For example, in contrast to gprk2 mutants, high-threshold target genes are expressed in the absence of Dally and Lpp, although over a narrower range. Loss of lpp appears to impair the ability of Ptc to silence Smo in the absence of Hh, whereas Ptc regulates Smo normally in the absence of Gprk2. Furthermore, the genetic and biochemical evidence for Gprk2 acting directly on Smo is compelling. Instead of a direct mechanistic link, what these mutants may have in common is accumulation of a not-fully active form of Smo. In lpp and dally mutants, this is due to inappropriate Smo activation in the absence of Hh, whereas in gprk2 mutants it reflects the failure to downregulate Smo in Hh-responding cells (Cheng, 2012).

The reduction of cAMP levels caused by loss of Gprk2 is a general effect, occurring not only in adult animals but during development and in cultured cells as well. Importantly, the results clearly implicate this cAMP misregulation in the impairment of Hh target gene expression. The sensitivity of gprk2 mutants to alterations in cAMP levels or Pka activity indicates that these are limiting for target gene expression in the mutants. This is not the case in fu mutants, highlighting the specificity of the effect (Cheng, 2012).

An important outstanding issue is why cAMP levels decrease in gprk2 mutants. Given that Smo can signal through Gα_i, excessive Smo-Gα_i coupling (as observed for other GPCRs in GRK knockout mice) would be the most obvious explanation. However, the fact that elimination of Smo in Gprk2-depleted S2 cells does not restore cAMP levels to normal means that there must be other factors. This is consistent with the magnitude of the drop in cAMP levels observed in gprk2 mutant larvae, which would be difficult to explain solely in terms of Smo activity (Cheng, 2012).

An alternative explanation is that misregulation of G-protein-dependent signaling by GPCRs other than Smo in the gprk2 mutants affects cAMP levels and output of the Hh pathway. Individual GRKs can regulate many GPCRs and loss of a single GRK can have a major impact on cAMP production, as previously observed in GRK3 knockout mice. Furthermore, cross-talk between the Sonic Hh pathway and Gα-coupled GPCRs has been demonstrated in mammals. A more global misregulation of Gprk2-regulated GPCRs could thus explain the changes in cAMP levels and impairment of Hh signaling seen in the absence of this kinase (Cheng, 2012).

The gprk2 mutants reveal an important positive role for Pka in Hh signaling. Both genetic and biochemical analyses suggest that reduced Pka-dependent phosphorylation and activation of Smo contributes to the Hh signaling defect in gprk2 mutants. How can the seemingly contradictory observations that elimination of Gprk2 increases Smo-dependent canonical pathway activity while also reducing Smo activation? In the absence of Gprk2, activated Smo accumulates to ~3-fold higher levels than normal be reconciled. The increase in total canonical pathway activation can most easily be explained by the increase in Smo levels or duration of its activity. However, because of reduced Pka phosphorylation, individual Smo molecules may be unable to achieve the most active conformation required for full Ci activation (Cheng, 2012).

If the positive effect of Pka were mediated entirely through the three phosphorylation sites in Smo, one would expect Smo^SD123 to be at least as effective as Gα_s or Pka in rescuing target gene expression in the gprk2 mutants. That this seems not to be the case implies that a Pka target other than the three sites in Smo is required for maximal target gene activation. This is consistent with observations in other systems. Despite evidence for a positive role for Pka in vertebrate Hh signaling, the three sites in the Smo C-terminus are not conserved and there is no clear evidence that Pka phosphorylates vertebrate Smo proteins. Analyses in Drosophila embryos also point to the existence of a positively acting Pka target other than the three sites in Smo. This target is unlikely to be Ci, as Pka strongly inhibits Ci¹⁵⁵ both by promoting its cleavage and reducing its specific activity. In fact, the stimulatory effects of Pka on Hh target gene transcription in embryos appear to be mediated by promoter sequences distinct from known Ci binding sites (Cheng, 2012).

The conclusion that Gprk2 affects Hh signaling indirectly through Pka appears to contradict a recent study that found that direct binding and phosphorylation of Smo by Gprk2 are required for high-level signaling. In fact, both mechanisms may normally contribute to Smo activation. Mutation of the Gprk2 sites at serine-740 and -741 (GPS1) to alanine reduced the ability of overexpressed Smo^SD123 to activate ectopic en expression, pointing to the importance of phosphorylation at these sites for maximal Smo activity. It may be that other kinases can substitute for Gprk2 to phosphorylate these sites in some circumstances. It has been previously shown that the second Drosophila GRK, Gprk1, does contribute to Hh pathway regulation at permissive temperature in the absence of Gprk2, making it a potential candidate. However, unlike Gprk2, Gprk1 does not associate with Smo, nor has it been able to demonstrate direct regulation of Smo by this kinase, suggesting that its effects may also be indirect. It is also possible that both Gprk2 and Pka can phosphorylate the GPS1 sites, or that Pka acting through a distinct positively acting target renders Hh target genes easier to activate, bypassing the requirement for GPS1 phosphorylation. In any case, it is clear from the results that the Gprk2-dependent changes in Smo phosphorylation extend well beyond the four mapped Gprk2 sites. The full effects, both direct and indirect, of Gprk2 on Smo and their respective contributions to Smo activation will need to be carefully defined (Cheng, 2012).

DEVELOPMENTAL BIOLOGY

Alpha-synuclein phosphorylation controls neurotoxicity and inclusion formation in a Drosophila model of Parkinson disease

Alpha-synuclein is phosphorylated at serine 129 (Ser129) in intracellular protein aggregates called Lewy bodies. These inclusion bodies are the characteristic pathologic lesions of Parkinson disease. This study defines the role of phosphorylation of Ser129 in alpha-synuclein toxicity and inclusion formation using a Drosophila model of Parkinson disease. Mutation of Ser129 to alanine to prevent phosphorylation completely suppresses dopaminergic neuronal loss produced by expression of human alpha-synuclein. In contrast, altering Ser129 to the negatively charged residue aspartate, to mimic phosphorylation, significantly enhances alpha-synuclein toxicity. The G protein-coupled receptor kinase 2 (Gprk2) phosphorylates Ser129 in vivo and enhances alpha-synuclein toxicity. Blocking phosphorylation at Ser129 substantially increases aggregate formation. Thus Ser129 phosphorylation status is crucial in mediating alpha-synuclein neurotoxicity and inclusion formation. Because increased number of inclusion bodies correlates with reduced toxicity, inclusion bodies may protect neurons from alpha-synuclein toxicity (Chen, 2005).

The G protein-coupled receptor regulatory kinase GPRK2 participates in Hedgehog signaling in Drosophila

Signaling by Smoothened (Smo) plays fundamental roles during animal development and is deregulated in a variety of human cancers. Smo is a transmembrane protein with a heptahelical topology characteristic of G protein-coupled receptors. Despite such similarity, the mechanisms regulating Smo signaling are not fully understood. Gprk2, a Drosophila member of the G protein-coupled receptor kinases, plays a key role in the Smo signal transduction pathway. Lowering Gprk2 levels in the wing disc reduces the expression of Smo targets and causes a phenotype reminiscent of loss of Smo function. Gprk2 function is required for transducing the Smo signal, and when Gprk2 levels are lowered, Smo still accumulates at the cell membrane, but its activation is reduced. Interestingly, the expression of Gprk2 in the wing disc is regulated in part by Smo, generating a positive feedback loop that maintains high Smo activity close to the anterior-posterior compartment boundary (Molnar, 2007).

Smo is the key transducer of a conserved signaling pathway regulating many developmental processes in vertebrates and invertebrates. The transmembrane protein Patched (Ptc) is the receptor for the ligand Hedgehog (Hh) and represses Smo activity in the absence of ligand. The binding of Hh to Ptc relieves this repression and allows Smo to signal to a protein complex that includes the transcription factor Ci/Gli. Smo controls the activation of Ci in the presence of the Hh ligand in part by preventing Ci proteolytic processing into a transcriptional repressor. In the Drosophila wing disc, the epithelium giving rise to the wing and thorax of the fly, Smo signaling controls the expression of several genes in anterior cells close to the anterior-posterior (A/P) compartment boundary and promotes the growth and patterning of the wing (Molnar, 2007).

The cytoplasmic tail of Drosophila Smo is a target for phosphorylation by protein kinase A and casein kinase I, and it has been shown that Smo phosphorylation by these kinases is essential for its activity and membrane accumulation. However, most of these phosphorylated residues are not conserved in its vertebrate counterparts. Recently, the G protein-coupled receptor kinase 2 (Grk2) has been shown to phosphorylate mammalian Smo (Chen, 2004). G protein-coupled receptor kinases (GRKs) selectively phosphorylate the ligand-activated form of G protein-coupled receptors (Lefkowitz, 2005). This phosphorylation promotes uncoupling from G proteins and also the recruitment of β-arrestins, which target the receptor for clathrin-mediated endocytosis. In addition, GRKs and β-arrestins also participate in signal propagation by recruiting additional proteins to the receptor complex. There are two Drosophila GRKs, GPRK1 and GPRK2. GPRK1 modulates the amplitude of the visual response acting as a Rhodopsin kinase, whereas GPRK2 regulates the level of cAMP during Drosophila oogenesis (Schneider, 2005; Lannutti, 2001). Phosphorylation of mammalian Smo by GRK2 promotes its endocytosis in clathrin-coated pits in a process dependent on β-arrestin2 (Chen, 2004). However, whether this form of Smo internalization is part of a desensitization mechanism, as is the case for different G protein-coupled receptors (Lefkowitz, 2005), or if it participates in Hh signaling is still not known. To address the participation of GRKs during Smo signaling in Drosophila, the function was analyzed of Gprk2 during imaginal wing disc development. It was found that Gprk2 activity is required for Smo activation. Thus, the reduction of Gprk2 expression by interference RNA, or its elimination by a genetic mutation, causes the accumulation of Smo in wing disc anterior cells exposed to Hh. The accumulation of Smo is, however, correlated with reduced activity, because Smo high-level targets are not correctly activated and flies expressing Gprk2-RNAi display Hh loss-of-function phenotypes. Interestingly, the reduction in Gprk2 expression is able to antagonize the activity of Smo mutant forms that mimic its phosphorylation by protein kinase A and casein kinase 1, suggesting that additional phosphorylation by Gprk2 is a necessary step to obtain the correct activation of Smo to promote the expression of its targets requiring high levels of signaling (Molnar, 2007).

The expression of Gprk2 mRNA in the wing disc is generalized but appears increased in a stripe of cells located close to the A/P compartment boundary. To better characterize this pattern, the P-lacZ insertion Gprk2⁰⁶⁹³⁶, which is localized in the 5' untranslated region of the gene, was used. Interestingly, β-gal expression is restricted to the A/P compartment boundary of the wing disc during the third larval instar. The cells expressing β-gal were further identified by using a combination of region-specific markers such as Engrailed (En), Patched (Ptc), Blistered (Bs), and Caupolican (Caup). This analysis places the stripe of maximal expression of Gprk2 to anterior cells abutting the A/P boundary. These cells express Ptc and En in the anterior compartment and are localized in the region exposed to high-level Hh signaling. In fact, Hh signaling regulates the expression of Gprk2 in these anterior cells, because β-gal expression in Gprk2⁰⁶⁹³⁶ discs is expanded to the entire anterior compartment when hh is ectopically expressed, and it is repressed when the activity of the pathway is reduced by ectopic expression of Ptc). The regulation of Gprk2 accumulation in anterior cells by Hh suggests that Hh signaling and Gprk2 might be functionally related (Molnar, 2007).

All available Gprk2 alleles are P element insertions in the 5' region of the gene. These alleles are homozygous viable, and the mutant wings do not display any visible phenotype. Stronger loss-of-function conditions of the gene were generated by (1) expressing Gprk2 interference RNA (Gprk2i) under the control of yeast upstream activator sequences (UAS; UAS-Gprk2i) and (2) constructing a synthetic deletion of the gene. In wing discs of the combination Gal4-638/UAS-Gprk2i, a reduction of Gprk2 mRNA levels of 66% was found. The corresponding adult wings show a range of striking phenotypes similar to loss of Hh function, displaying a reduction of the L3/L4 intervein, fusion of the L3 and L4 veins, and in a lower percentage of wings, the loss of the L3 and L4 veins. These veins and the L3/L4 intervein correspond to the territory specified by Hh signaling. In fact, reduction of Gprk2 levels results in wings very similar to those with a moderate loss of Hh signaling, generated either by ectopic expression of Ptc or by expression or hh-interference RNA. This phenotype is very different from that observed upon increased activity of the pathway. The Gal4-638 line is expressed in the entire wing, and to distinguish between the effects of lowering Gprk2 levels in cells producing or responding to Hh, three other Gal4 lines were used expressed in either the anterior (Gal4-Ci and Gal4-ptc) or the posterior (Gal4-hh) compartments. It was found that the expression of Gprk2i only in anterior cells recapitulates the reduction of the L3/L4 intervein observed in Gal4-638/UAS-Gprk2i wings. Thus, the combinations Gal4-Ci/UAS-Gprk2i and Gal4-ptc/UAS-Gprk2i show a reduction or elimination of the L3/L4 intervein, whereas the wings of the Gal4-hh/UAS-Gprk2i combination display a normal pattern of veins. The phenotypes observed upon a reduction of Gprk2 unambiguously indicate that Gprk2 function is necessary for the transduction of the Hh signal. Furthermore, when the expression of Gprk2 is reduced in flies expressing lower levels of the ligand Hh, the resulting wings have stronger hh loss-of-function phenotypes, and a previously unrecognized phenotypic class indistinguishable to those of wings formed by smo mutant cells is now observed (Molnar, 2007).

To directly monitor the activity of the Hh pathway, the expression of several Hh targets was studied in Gal4-638/UAS-Gprk2i discs. The expression of En and Ptc in anterior cells is always impaired when Gprk2 levels are reduced. These two genes correspond to Hh targets activated by a high level of signaling. The expression of Knot (Kn) is also reduced in Gal4-638/UAS-Gprk2i discs, and the stripe of maximal accumulation of Ci is also modified in Gal4-638/UAS-Gprk2i discs compared with wild-type ones. The expression of other genes regulated directly or indirectly by Hh signaling was studied in Gal4-638/UAS-Gprk2i discs. The expression of the Notch ligand Delta (Dl) is very weak or absent in the primordia of the veins L3 and L4, where it accumulates at high levels in normal discs. Similarly, the expression of Bs in the L3/L4 intervein is reduced or absent in Gal4-638/UAS-Gprk2i discs. The expression of the low-level Hh signaling targets caup and decapentaplegic (dpp) is also modified in Gal4-638/UAS-Gprk2i discs. Caup expression in the presumptive L3 vein is generally expanded toward the A/P compartment boundary in Gal4-638/UAS-Gprk2i discs, most likely because En, a repressor of Caup in anterior cells, is not expressed upon a reduction of Gprk2 levels. The domain of Caup expression in the L3 vein is reduced or lost compared with wild-type discs in only a small fraction of discs (7%). The expression of dpp is detected in Gal4-638/UAS-Gprk2i discs at lower levels but in a domain broader than the characteristic of normal discs. Taken together, these data suggest that Gprk2 plays a positive role in the Hh signaling pathway. The lowering of Gprk2 levels reduces very efficiently high-level Hh signaling and much less efficiently low-level Hh signaling. Thus, a complete elimination of Hh signaling is observed only when Gprk2 levels are reduced in wing discs with lower hh. Finally, the expression of spalt, a target of the Dpp/BMP4 pathway, is almost normal upon Gprk2 reduction, indicating specificity of Gprk2 function toward Hh signaling (Molnar, 2007).

To confirm the specificity of the Gprk2 RNAi, the expression of two Hh-targets, En and Ptc, was analyzed in wing disc cells homozygous for a deficiency that removes all of the Gprk2 coding region. In both cases it was found that anterior Gprk2 ^- clones eliminate, in a cell-autonomous manner, the anterior expression of En. Gprk2 ^- clones located in the posterior compartment did not affect the expression of En, confirming that Gprk2 activity is required in cells receiving Hh (Molnar, 2007).

To further analyze where Gprk2 function is required in the Hh signaling pathway, the expression of En was studied in clones of cells ectopically expressing hh or both hh and Gprk2i. It was found that clones expressing Gprk2i located in the domain of En expression in the anterior compartment cell-autonomously suppress the expression of En. The expression of En is induced by Hh signaling in hh-expressing clones, both within the clone and in the surrounding cells. However, in the hh+Gprk2i-expressing clones, the expression of En is induced only in wild-type anterior cells that do not express Gprk2i. These observations confirm that Gprk2 activity is required for transducing the Hh signal in Hh-receiving cells and not for Hh secretion (Molnar, 2007).

Experiments in mammalian cells in culture have shown that beta-arrestin2 and GRK2 mediate internalization of active Smo (Chen, 2004). Consequently, the expression and subcellular localization of Smo was studied in wing discs where Gprk2 activity is reduced. In wild-type discs, smo RNA is expressed in all cells, but Smo protein accumulates associated to cell membranes only in the posterior compartment and in some anterior cells exposed to Hh. Intriguingly, the reduction in Gprk2 levels in the entire wing blade eliminates the distinction in Smo accumulation between anterior and posterior cells, and Smo is detected at similar levels in both compartments. When the levels of Gprk2 are reduced only in the dorsal compartment or in clones of Gprk2 ^- homozygous cells, the changes in Smo expression in anterior cells are more evident. Thus, it was observed that Smo accumulates at high levels associated to cell membranes in a broader anterior domain of cells within the range of Hh. The extension of Smo accumulation in anterior cells might be due to an extension of the Hh diffusion range because Ptc is not expressed in Gprk2 mutant cells. This is a previously unrecognized instance in which Smo accumulation and signaling can be uncoupled, because it was thought that, at least in Drosophila, Smo membrane accumulation leads to signaling. The same effects are observed when S2 cells were used. Thus, Smo is expressed in S2 cells in intracellular vesicles at low levels. Upon Hh treatment, Smo translocates close to the plasma membrane in these cells. In cells that have been treated for 4 days with Gprk2 dsRNA (causing a reduction of Gprk2 mRNA levels of 77%) the levels of Smo are higher independently of Hh (Molnar, 2007).

To further analyze the relationship between Smo and Gprk2 functions, Gprk2i was expressed in the same wing with different N-terminal (smoDeltaN; extracellular) and C-terminal (smoDeltaC2 and smoDeltaC4; intracellular) deletions of Smo. The expression of Smo proteins bearing either N-terminal or C-terminal deletions fails to rescue Smo mutants, but their overexpression does not interfere significantly with Smo signaling. A strong synergic genetic interaction was found when Smo C-terminal deletions were coexpressed with Gprk2i. Thus, wings expressing C-terminal deletions of Smo with reduced Gprk2 levels display a strong hh loss-of-function phenotype that is comparable to the elimination of smo. Gprk2i combined with UAS-smoDeltaN resulted in additive phenotypes. It is suggested that the reduction of Gprk2 uncovers a dominant-negative effect of SmoDeltaC proteins, reducing the efficiency of Smo signaling. The basis for this dominant negative effect could be the inclusion of a form of Smo, SmoDeltaC, unable to be phosphorylated by Gprk2, in the Smo complexes that have been postulated to mediate Smo activity. Therefore, it is proposed that Gprk2 function, acting through the C-terminal tail of Smo, is involved in an activation step promoting Smo interaction with the Costal2/Fused/Su(fu) complex to prevent Ci processing into a repressor form and to accumulate Ci in an activating form. Based on the effects of mammalian GRK2 and beta2-arrestin on Smo (Chen, 2004, Meloni, 2006), it is possible that Gprk2-mediated activation of Smo involves the recycling of Smo from the cell membrane to an intracellular signaling compartment (Molnar, 2007).

The interaction between SmoDeltaC and Gprk2 indicates a critical role of the Smo intracellular C-terminal domain for its relationship with Gprk2 function. Interestingly, the Smo intracellular C-terminal domain is where all of the consensus phosphorylation sites by casein kinase 1 and protein kinase A are located, as well as other serine and threonine residues in the vicinity of acidic residues that are similar to mammalian GRK2 phosphorylation consensus. A form of Smo was expressed in the wing disc that mimics its phosphorylation by these kinases (Smo^SD123), and whether this Smo-activated form is sensitive to Gprk2 levels was analyzed. The expression of Smo^SD123 in the wing disc causes overgrowth of the anterior compartment and defects in the L3 and L2 veins. In the corresponding wing discs, the accumulation of Smo and the expression of its targets En and Ptc are expanded to occupy the entire anterior compartment. When Gprk2 levels are reduced in discs expressing Smo^SD123, Smo accumulation is still observed in all anterior cells. In contrast, the expression of both En and Ptc is now restricted to their normal domains adjacent to the A/P compartment boundary. The overgrowth phenotype characteristic of Gal4-638/+; UAS-Smo^SD123 discs is not rescued by the reduction of Gprk2 expression, suggesting that the low-level Hh target dpp is still expressed through the anterior compartment. These data suggest that to generate the high levels of Smo activity required to activate the expression of its targets En and Ptc, the Smo^SD123 protein has to be phosphorylated by Gprk2 (Molnar, 2007).

In conclusion, Drosophila Gprk2 is critically required to generate high levels of Hh signaling in the wing disc. The genetic interactions between Gprk2 and Smo proteins bearing C-terminal deletions or Smo phosphomimic variants suggest that Smo is a target of Gprk2. The modifications in Smo protein accumulation detected in wing discs and S2 cells with reduced Gprk2 expression suggests that a likely step affected by Gprk2 is the activation of Smo by a phosphorylation step that could prime Smo for internalization to a signaling compartment. GRK2 has recently been shown to play a positive role in Shh transduction in mammalian cells (Meloni, 2006). Taken together, these findings and the data indicate that Smo phosphorylation by GRK homologues constitute a conserved component of the Smo signal transduction cascade (Molnar, 2007).

Spike amplitude of single-unit responses in antennal sensillae is controlled by the Drosophila circadian clock

Circadian changes in membrane potential and spontaneous firing frequency have been observed in microbial systems, invertebrates, and mammals. Oscillators in olfactory sensory neurons (OSNs) from Drosophila are both necessary and sufficient to sustain rhythms in electroanntenogram (EAG) responses, suggesting that odorant receptors (ORs) and/or OR-dependent processes are under clock control. This study measured single-unit responses in different antennal sensillae from wild-type, clock mutant, odorant-receptor mutant, and G protein-coupled receptor kinase 2 (Gprk2) mutant flies to examine the cellular and molecular mechanisms that drive rhythms in olfaction. Spontaneous spike amplitude, but not spontaneous or odor-induced firing frequency, is under clock control in ab1 and ab3 basiconic sensillae and T2 trichoid sensillae. Mutants lacking odorant receptors in dendrites display constant low spike amplitudes, and the reduction or increase of levels of GPRK2 in OSNs results in constant low or constant high spontaneous spike amplitudes, respectively. It is concluded that spike amplitude is controlled by circadian clocks in basiconic and trichoid sensillae and requires GPRK2 expression and the presence of functional ORs in dendrites. These results argue that rhythms in GPRK2 levels control OR localization and OR-dependent ion channel activity and/or composition to mediate rhythms in spontaneous spike amplitude (Krishnan, 2008).

One hypothesis to explain rhythms in the amplitude of spontaneous spikes and electroanntenogram rhythms (EAGs) is that ion channel activity and/or composition is under circadian control. Drosophila ORs have been found to form heteromeric odor-gated and cyclic-nucleotide-activated cation channels. It has been demonstrated that ORs accumulate in OSN dendrites in a circadian fashion, where OR abundance peaks near the middle of the night and is low during the day (Tanoue, 2008). These rhythms are dependent on the levels of GPRK2 and coincide with rhythms in the amplitude of both EAGs and spontaneous spikes (Tanoue, 2008). Taken together, these results suggest a model whereby GPRK2 controls the abundance and/or activity of OR-dependent odor-gated cation channels in OSN dendrites, which in turn alter membrane conductance to generate rhythms in the amplitude of spontaneous spikes and EAG responses. The possibility cannot be excluded that the clock modulates other molecular or cellular targets to generate rhythms in EAG and spike amplitude such as other ion channels expressed in OSNs, the composition of sensillar lymph, and/or the size and shape of OSNs (Krishnan, 2008).

Though a clear ecological explanation for rhythms in spike amplitude is not understood, one could hypothesize that the circadian clock could tune the olfactory system to a higher gain level (higher signal-to-noise ratio) by modulating spike amplitude irrespective of the stimulus preferentially in the subjective night. For instance, rhythms in OSN spike amplitude might produce increased activity in downstream neurons during the night and decreased activity during the day in response to the same stimulus. Recent evidence demonstrates that locomotor activity of paired male and female Drosophila is increased during the subjective night and is dependant on an intact olfactory system (Fujii, 2007). In addition, behavioral responses to odors in Drosophila are lower during the day than at night and are controlled by the circadian clock (Zhou, 2005). These phenomena may represent behavioral consequences of this electrophysiological rhythm that could provide an advantage in courtship, food acquisition, or predator avoidance. These data are consistent with circadian-clock-dependent rhythms in mating activity (Sakai, 2001). The phase of the peak in spontaneous spikes in trichoids could translate to heightened behavioral activity associated with mating during the subjective night. Thus, the results suggest that spike amplitude, in addition to firing frequency, can also encode meaningful information in the peripheral OSNs, which is transmitted to higher processing centers of CNS that mediate behavioral responses to odors (Krishnan, 2008).

G protein-coupled receptor kinase 2 promotes high-level Hedgehog signaling by regulating the active state of Smo through kinase-dependent and kinase-independent mechanisms in Drosophila.

G protein-coupled receptor kinase 2 (Gprk2/GRK2) plays a conserved role in modulating Hedgehog (Hh) pathway activity, but its mechanism of action remains unknown. This study provides evidence that Gprk2 promotes high-level Hh signaling by regulating Smoothened (Smo) conformation through both kinase-dependent and kinase-independent mechanisms. Gprk2 promotes Smo activation by phosphorylating Smo C-terminal tail (C-tail) at Ser741/Thr742, which is facilitated by PKA and CK1 phosphorylation at adjacent Ser residues. In addition, Gprk2 forms a dimer/oligomer and binds Smo C-tail in a kinase activity-independent manner to stabilize the active Smo conformation, and promotes dimerization/oligomerization of Smo C-tail. Gprk2 expression is induced by Hh signaling, and Gprk2/Smo interaction is facilitated by PKA/CK1-mediated phosphorylation of Smo C-tail. Thus, Gprk2 forms a positive feedback loop and acts downstream from PKA and CK1 to facilitate high-level Hh signaling by promoting the active state of Smo through direct phosphorylation and molecular scaffolding (Chen, 2010).

A genetic modifier screen for novel Hh signaling components identified Gprk2 as a positive regulator of Smo. Gprk2 was shown to be required for high but not low levels of Hh signaling activity. Evidence was provided that Gprk2 is a Smo kinase and that Gprk2 promotes maximal Smo activity by phosphorylating S₇₄₁/T₇₄₂ in Smo C-tail. Furthermore, a kinase-independent function of Gprk2 in Hh signaling was uncovered. Gprk2 forms a dimer/oligomer and binds Smo C-tail to promote the active state of Smo. Thus, this study reveals a novel mechanism for regulating a GPCR-like protein by GRK (Chen, 2010).

Previous studies suggest that Drosophila Gprk2 and mammalian GRK2 are involved in Smo phosphorylation because their knockdown in cultured cells either increased Smo mobility on SDS-PAGE or decreased metabolic labeling of Smo by γ-³²p-ATP. However, these studies did not distinguish whether Gprk2/GRK2 phosphorylates Smo directly or indirectly through regulating other kinases. Neither did they reveal any biological relevance of Gprk2/GRK2-mediated phosphorylation in Hh signaling, since the relevant phosphorylation sites on Smo were not identified. In an in vitro kinase assay using purified substrates and a recombinant GRK, this study found that Smo is phosphorylated by GRK at S₇₄₁/T₇₄₂ and S₁₀₁₃/S₁₀₁₅. A mutagenesis study demonstrated that phosphorylation at S₇₄₁/T₇₄₂ is required for optimal Smo activation. Indeed, a previous study showed that Smo is phosphorylated at S₇₄₁/T₇₄₂ in cultured cells in the presence of Hh. In further agreement with the functional significance of S₇₄₁/T₇₄₂ phosphorylation, conserved S/T residues are found at the corresponding location in other insect Smo proteins (FlyBase) (Chen, 2010).

Interestingly, the in vitro kinase assay revealed that phosphorylation of S₇₄₁/T₇₄₂ by Gprk2 is regulated by PKA/CK1 phosphorylation at adjacent Ser residues, including S₇₄₀, S₇₄₃, and S₇₄₆. Previous studies in mammalian systems suggest that GRKs tend to phosphorylate S/T residues embedded in an acidic environment. Phosphorylation at S₇₄₀, S₇₄₃, and S₇₄₆ improves the acidic environment for S₇₄₁/T₇₄₂, which may account for the observed stimulation of S₇₄₁/T₇₄₂ phosphorylation by PKA/CK1. Indeed, mutating S₇₄₀, S₇₄₃, and S₇₄₆ to Ala abolished PKA/CK1-mediated stimulation of S₇₄₁/T₇₄₂ phosphorylation, whereas converting these residues to acidic residues mimicked PKA/CK1-mediated stimulation. As Hh induces Smo phosphorylation by PKA and CK1, phosphorylation at S₇₄₁/T₇₄₂ by Gprk2 is likely to be stimulated by Hh in vivo (Chen, 2010).

Although phosphomimetic mutation at S₇₄₁/T₇₄₂ promotes Smo activity, it does not bypass the requirement for Gprk2 for optimal Smo activation because SmoSDGPSD failed to induce ectopic en expression in Gprk2 mutant discs. This implies that Gprk2 promotes Hh signaling through a mechanism in parallel to S₇₄₁/T₇₄₂ phosphorylation. It is possible that Gprk2 might act at an additional step downstream from Smo activation by phosphorylating intracellular Hh signaling components, or at the level of Smo activation by phosphorylating Smo at additional sites that have been missed by the in vitro kinase assay. However, the finding that the constitutively active form of Smo lacking the autoinhibitory domain (SAID: SmoΔ661-818) is insensitive to Gprk2 inactivation suggests that Gprk2 acts mainly at the level of Smo, although the possibility cannot be ruled out that Gprk2 may also play a minor role downstream from Smo. Interestingly, it was found that the kinase-dead form of Gprk2 (Gprk2^KM) can rescue the activity defect of SmoSDGPSD in Gprk2 mutants, demonstrating that Gprk2 also regulates Smo in a phosphorylation-independent manner. The observation that Gprk2^KM does not rescue the activity defect of SmoSD123 in Gprk2 mutants suggests that the phosphorylation-dependent and phosphorylation-independent mechanisms act in parallel rather than redundantly to promote Smo activation. Furthermore, evidence was obtained that Gprk2 interacts with the SAID independently of its kinase activity. Therefore, it is proposed that Gprk2 promotes Smo activation by counteracting Smo autoinhibition through binding to and phosphorylating the SAID (Chen, 2010).

At least two paralleled mechanisms have been attributed to Smo activation by Hh: (1) Smo cell surface accumulation, and (2) conformation change in Smo C-tail. Intriguingly, it was found that loss of Gprk2 resulted in increased rather than decreased Smo levels in cells that are not exposed to Hh or are exposed to low levels of Hh. However, unlike Hh stimulation, which preferentially stabilizes Smo on the cell surface, Gprk2 inactivation appears to stabilize Smo both inside the cell and on the cell surface. Furthermore, in the presence of high levels of Hh where Smo is accumulated at high levels on the cell surface, Gprk2 inactivation does not cause any discernible changes in either the level or subcellular distribution of Smo. Thus, the reduced Smo activity in Gprk2 mutant cells exposed to high levels of Hh is unlikely to be due to a change in Smo level or subcellular localization (Chen, 2010).

It is not clear what role Gprk2-mediated down-regulation of Smo levels might play in Hh signaling, although this may reflect an ancient mechanism by which GRK family kinases 'desensitize' GPCRs. In this regard, Gprk2-mediated down-regulation could serve as a mechanism to restrict the basal level of Hh signaling activity or to terminate or tune down Hh signaling activity once the Hh signal is withdrawn. However, this negative role of Gprk2 could be masked by its positive role. The mechanism by which Gprk2 down-regulates Smo levels remains unclear, although the kinase activity of Gprk2 appears to be required. Gprk2 could phosphorylate Smo and/or other proteins to promote Smo internalization and degradation. High levels of Hh could counteract Gprk2-mediated down-regulation of Smo by preventing Gprk2-meidated Smo internalization or by promoting Smo recycling (Chen, 2010).

FRET analysis provided strong evidence that Gprk2 is required for Smo to adopt and/or maintain its active conformation in response to Hh stimulation. A previous study suggested that Hh induces a conformational switch in Smo C-tail that is mediated by PKA and CK1 phosphorylation. In the absence of Hh, the Smo C-tail adopts a closed conformation in which the tail folds back, resulting in a close proximity between the C terminus and the third intracellular loop. The closed conformation is maintained, at least in part, through intramolecular electrostatic interactions between the multiple Arg clusters in the SAID and multiple acidic clusters near the C terminus. Hh-induced phosphorylation at PKA and CK1 sties disrupts the intramolecular electrostatic interactions, resulting in unfolding of the C-tail, which is reflected by a decreased intramolecular FRET (FRET^L3C). In addition, phosphorylation promotes dimerization of two C-tails within a Smo homodimer, leading to increased proximity of the two C termini, as reflected by an increased C-terminal FRET (FRET^C). Multiple intermediate conformational states may exist, depending on the levels of Smo phosphorylation, as increasing the number of phosphomimetic mutations progressively decreased FRET^L3C and gradually increased FRET^C. It was found that both an Hh-induced decrease in FRET^L3C and an Hh-induced increase in FRET^C were compromised by loss of Gprk2, suggesting that Gprk2 is critical for Smo to adopt and/or maintain the fully open conformation (Chen, 2010).

How does Gprk2 regulate Smo conformation? Genetic and FRET analyses demonstrated that Gprk2 promotes high levels of Hh signaling activity and regulates Smo conformation through both phosphorylation-dependent and phosphorylation-independent mechanisms. Furthermore, this study found that Gprk2 self-associates, binds the SAID, and promotes self-association of Smo C-tail. Interestingly, both Gprk2/SAID interaction and S₇₄₁/T₇₄₂ phosphorylation by Gprk2 are enhanced by PKA/CK1 phosphorylation. Taken together, the following model is proposed to account for the regulation of Smo conformation by Gprk2. In response to Hh, PKA/CK1-mediated phosphorylation of Smo C-tail promotes its unfolding and dimerization; however, in the absence of Gprk2, the open conformational state of Smo is unstable and may exist in equilibrium with the closed and/or partially open conformational states. Phosphorylation of Smo by PKA/CK1 promotes the binding of Gprk2 to the SAID and phosphorylation at S₇₄₁/T₇₄₂, both of which may stabilize Smo in the fully open and active conformation by preventing refolding of Smo C-tail and by 'cross-linking' the two C-tails within a Smo dimer via dimerization of Gprk2. In essence, Gprk2 may function as a 'molecular clamp' to promote the clustering of Smo C-tails. It is also possible that Gprk2 could cross-link two or more Smo dimers to form high-order oligomers, which might be essential for high levels of Hh signaling activity. This study thus reveals an unanticipated complexity in the regulation of Smo conformational states, and provides the first evidence that Smo conformation states are regulated by not only phosphorylation and intramolecular interactions, but also intermolecular interactions. It is possible that the closed conformation state of Smo is also regulated by intermolecular interactions in addition to intramolecular interactions. For example, it has been shown that Fu can directly bind the Smo C terminus in the absence of Hh, and this interaction may help stabilize the closed conformation of Smo C-tail. Indeed, disrupting Smo/Fu interaction led to increased basal activity of Smo (Chen, 2010).

Recent studies have emphasized the differences between vertebrate and Drosophila Hh signaling mechanisms. The sequence divergence between Drosophila and vertebrate Smo proteins and the lack of conserved PKA/CK1 phosphorylation sites in vertebrate Smo proteins have led to the proposal that vertebrate Smo proteins are activated through fundamentally distinct mechanisms. Nevertheless, a previous study revealed that Shh induces a conformational change in mSmo similar to that of dSmo, and forced clustering of mSmo also leads to pathway activation). GRK2 has been implicated as a positive regulator of the Shh pathway, and mSmo phosphorylation is affected by GRK2 silencing, although direct phosphorylation of mSmo by GRK2 has not been demonstrated. It is possible that GRK2 may substitute the role of PKA and CK1 and act as a major Smo kinase in vertebrates to promote the active Smo conformation. Alternatively, GRK2 may act in conjunction with other GRKs and/or yet-to-be-identified kinases to regulate Smo conformation, subcellular localization, and activity in vertebrates. The relatively weak phenotypes exhibited by GRK2 mutants are consistent with the latter possibility. This study also raised an interesting possibility that GRK2 may regulate mSmo not only by phosphorylation, but also by a kinase-independent mechanism such as a protein-protein interaction. Further investigation of the mechanism by which GRK2 and other kinases regulate mSmo will shed an important light on how vertebrate Smo activation is achieved (Chen, 2010).

Drosophila G-protein-coupled receptor kinase 2 regulates cAMP-dependent Hedgehog signaling

G-protein-coupled receptor kinases (GRKs) play a conserved role in Hedgehog (Hh) signaling. In several systems, GRKs are required for efficient Hh target gene expression. Their principal target appears to be Smoothened (Smo), the intracellular signal-generating component of the pathway and a member of the G-protein-coupled receptor (GPCR) protein family. In Drosophila, a GRK called Gprk2 is needed for internalization and downregulation of activated Smo, consistent with the typical role of these kinases in negatively regulating GPCRs. However, Hh target gene activation is strongly impaired in gprk2 mutant flies, indicating that Gprk2 must also positively regulate Hh signaling at some level. To investigate its function in signaling, several different readouts of Hh pathway activity were analyzed in animals or cells lacking Gprk2. Surprisingly, although target gene expression was impaired, Smo-dependent activation of downstream components of the signaling pathway was increased in the absence of Gprk2. This suggests that Gprk2 does indeed play a role in terminating Smo signaling. However, loss of Gprk2 resulted in a decrease in cellular cAMP concentrations to a level that was limiting for Hh target gene activation. Normal expression of target genes was restored in gprk2 mutants by stimulating cAMP production or activating the cAMP-dependent Protein kinase A (Pka). These results suggest that direct regulation of Smo by Gprk2 is not absolutely required for Hh target gene expression. Gprk2 is important for normal cAMP regulation, and thus has an indirect effect on the activity of Pka-regulated components of the Hh pathway, including Smo itself (Cheng, 2012).

Based on existing evidence, the role of GRKs in Hh signaling is complex. Elimination of Gprk2 in flies leads to increased accumulation of Smo at the cell surface, generally a sign of high-level signaling and consistent with the typical function of GRKs in negatively regulating GPCRs. However, activation of Hh target genes is lost, indicating that Gprk2 plays a positive role in the pathway. Based on analysis of how signaling downstream of Smo is affected in the absence of Gprk2, this study draws three main conclusions that provide insight into this apparent contradiction. First, Gprk2 does indeed act as a negative regulator of the Hh pathway by limiting accumulation of active Smo. Second, Gprk2 activity is required for normal regulation of cellular cAMP levels, and thus Pka activity. Third, much of the positive effect of Gprk2 on Hh pathway activation is indirect, through promotion of Pka-dependent Smo phosphorylation and activation (Cheng, 2012).

GRKs regulate homologous desensitization of GPCRs, thereby limiting their signaling activity. Consequently, GRK loss often leads to increased surface receptor levels and exaggerated signaling responses. Consistent with this, previous studies have shown that Gprk2 downregulates Smo subsequent to its activation. This study finds that Smo-dependent activation of the canonical Hh pathway is increased at a cellular level in the absence of Gprk2, in a manner roughly proportional to the increase in Smo levels. This is the first demonstration that Gprk2 does in fact restrict Smo activity. Taken together the evidence suggests that Smo undergoes Gprk2-dependent homologous desensitization; that in the absence of Gprk2, Smo activity is not dramatically impaired; and that the accumulation of active Smo leads to more total pathway activation. As a result, Ci¹⁵⁵ is stabilized and enters the nucleus in gprk2-mutant Hh-responding cells, as in wild-type cells. However, high-threshold target genes are not expressed (Cheng, 2012).

Some of the same features of Hh pathway misregulation are found in other pathway mutants. Loss of Fu leads to similar effects on Ci stabilization and target gene expression. In contrast to fu mutants, SuFu is strongly phosphorylated and Ci¹⁵⁵ accumulates in the nucleus in gprk2 mutants. Manipulation of SuFu and Gα activity clearly points to distinct underlying causes of the signaling defects in these mutants. In dally and lipophorin (lpp; Rfabg -- FlyBase) mutants, Ci¹⁵⁵ stabilization and nuclear import are uncoupled from target gene expression, as in the gprk2 mutants. Knockdown of lpp lipoprotein particle production in particular has some similar, if more severe, consequences as loss of gprk2 - ectopic Smo stabilization and nuclear accumulation of Ci¹⁵⁵ (although in cells not exposed to Hh), without activating target genes. Although the phenotypic similarities suggest that Gprk2 could work through the same mechanism, there are important differences that suggest this is unlikely. For example, in contrast to gprk2 mutants, high-threshold target genes are expressed in the absence of Dally and Lpp, although over a narrower range. Loss of lpp appears to impair the ability of Ptc to silence Smo in the absence of Hh, whereas Ptc regulates Smo normally in the absence of Gprk2. Furthermore, the genetic and biochemical evidence for Gprk2 acting directly on Smo is compelling. Instead of a direct mechanistic link, what these mutants may have in common is accumulation of a not-fully active form of Smo. In lpp and dally mutants, this is due to inappropriate Smo activation in the absence of Hh, whereas in gprk2 mutants it reflects the failure to downregulate Smo in Hh-responding cells (Cheng, 2012).

The reduction of cAMP levels caused by loss of Gprk2 is a general effect, occurring not only in adult animals but during development and in cultured cells as well. Importantly, the results clearly implicate this cAMP misregulation in the impairment of Hh target gene expression. The sensitivity of gprk2 mutants to alterations in cAMP levels or Pka activity indicates that these are limiting for target gene expression in the mutants. This is not the case in fu mutants, highlighting the specificity of the effect (Cheng, 2012).

An important outstanding issue is why cAMP levels decrease in gprk2 mutants. Given that Smo can signal through Gα_i, excessive Smo-Gα_i coupling (as observed for other GPCRs in GRK knockout mice) would be the most obvious explanation. However, the fact that elimination of Smo in Gprk2-depleted S2 cells does not restore cAMP levels to normal means that there must be other factors. This is consistent with the magnitude of the drop in cAMP levels observed in gprk2 mutant larvae, which would be difficult to explain solely in terms of Smo activity (Cheng, 2012).

An alternative explanation is that misregulation of G-protein-dependent signaling by GPCRs other than Smo in the gprk2 mutants affects cAMP levels and output of the Hh pathway. Individual GRKs can regulate many GPCRs and loss of a single GRK can have a major impact on cAMP production, as previously observed in GRK3 knockout mice. Furthermore, cross-talk between the Sonic Hh pathway and Gα-coupled GPCRs has been demonstrated in mammals. A more global misregulation of Gprk2-regulated GPCRs could thus explain the changes in cAMP levels and impairment of Hh signaling seen in the absence of this kinase (Cheng, 2012).

The gprk2 mutants reveal an important positive role for Pka in Hh signaling. Both genetic and biochemical analyses suggest that reduced Pka-dependent phosphorylation and activation of Smo contributes to the Hh signaling defect in gprk2 mutants. How can the seemingly contradictory observations that elimination of Gprk2 increases Smo-dependent canonical pathway activity while also reducing Smo activation? In the absence of Gprk2, activated Smo accumulates to ~3-fold higher levels than normal be reconciled. The increase in total canonical pathway activation can most easily be explained by the increase in Smo levels or duration of its activity. However, because of reduced Pka phosphorylation, individual Smo molecules may be unable to achieve the most active conformation required for full Ci activation (Cheng, 2012).

If the positive effect of Pka were mediated entirely through the three phosphorylation sites in Smo, one would expect Smo^SD123 to be at least as effective as Gα_s or Pka in rescuing target gene expression in the gprk2 mutants. That this seems not to be the case implies that a Pka target other than the three sites in Smo is required for maximal target gene activation. This is consistent with observations in other systems. Despite evidence for a positive role for Pka in vertebrate Hh signaling, the three sites in the Smo C-terminus are not conserved and there is no clear evidence that Pka phosphorylates vertebrate Smo proteins. Analyses in Drosophila embryos also point to the existence of a positively acting Pka target other than the three sites in Smo. This target is unlikely to be Ci, as Pka strongly inhibits Ci¹⁵⁵ both by promoting its cleavage and reducing its specific activity. In fact, the stimulatory effects of Pka on Hh target gene transcription in embryos appear to be mediated by promoter sequences distinct from known Ci binding sites (Cheng, 2012).

The conclusion that Gprk2 affects Hh signaling indirectly through Pka appears to contradict a recent study that found that direct binding and phosphorylation of Smo by Gprk2 are required for high-level signaling. In fact, both mechanisms may normally contribute to Smo activation. Mutation of the Gprk2 sites at serine-740 and -741 (GPS1) to alanine reduced the ability of overexpressed Smo^SD123 to activate ectopic en expression, pointing to the importance of phosphorylation at these sites for maximal Smo activity. It may be that other kinases can substitute for Gprk2 to phosphorylate these sites in some circumstances. It has been previously shown that the second Drosophila GRK, Gprk1, does contribute to Hh pathway regulation at permissive temperature in the absence of Gprk2, making it a potential candidate. However, unlike Gprk2, Gprk1 does not associate with Smo, nor has it been able to demonstrate direct regulation of Smo by this kinase, suggesting that its effects may also be indirect. It is also possible that both Gprk2 and Pka can phosphorylate the GPS1 sites, or that Pka acting through a distinct positively acting target renders Hh target genes easier to activate, bypassing the requirement for GPS1 phosphorylation. In any case, it is clear from the results that the Gprk2-dependent changes in Smo phosphorylation extend well beyond the four mapped Gprk2 sites. The full effects, both direct and indirect, of Gprk2 on Smo and their respective contributions to Smo activation will need to be carefully defined (Cheng, 2012).

EVOLUTIONARY HOMOLOGS

Identification and characterization of GRKs

Guanine nucleotide binding protein (G-protein)-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G-protein-coupled receptors such as the beta 2-adrenergic receptor and rhodopsin. The best characterized members of this family include the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase. To identify additional members of the GRK family, the polymerase chain reaction was used to amplify human heart cDNA using degenerate oligonucleotide primers from highly conserved regions unique to the GRK family. The isolation has been reported of a cDNA that encodes a 590-amino acid protein kinase, termed GRK5, which has 34.8% and 47.2% amino acid identities with beta ARK and rhodopsin kinase, respectively. Interestingly, GRK5 has an even higher homology with Drosophila GPRK-2 (71.0% identity) and the recently identified human IT11 (69.1% identity). Northern blot analysis of GRK5 with selected human tissues reveals a message of approximately 3 kilobases with highest levels in heart, placenta, lung > skeletal muscle > brain, liver, pancreas > kidney. GRK5, overexpressed in Sf9 insect cells using the baculovirus system, is able to phosphorylate rhodopsin in a light-dependent manner. In addition, GRK5 neither contains a consensus sequence for isoprenylation like rhodopsin kinase nor is activated by G-protein beta gamma subunits like beta ARK1. Thus, GRK5 represents a member of the GRK family that likely has a unique physiological role (Kunapuli, 1993).

G protein-coupled receptor kinases (GRK), such as the beta-adrenergic receptor kinase (beta ARK) and rhodopsin kinase, specifically phosphorylate the activated form of G protein-coupled receptors. To identify additional members of the GRK family, a human heart cDNA library was screened by low stringency hybridization using the catalytic domains of two beta ARK isoforms. A cDNA has been isolated that encodes a 576-amino-acid protein kinase, termed GRK6, that has significant homology with GRK5 (70.1% amino acid identity), IT11 (68.5%), rhodopsin kinase (47.1%), and beta ARK (37.4%). RNA blot analysis of GRK6 with selected human tissues reveals two distinct mRNAs of 3 and 2.4 kilobases with a distribution very similar to that of beta ARK (i.e. brain, skeletal muscle > pancreas > heart, lung, kidney, placenta > liver). GRK6, overexpressed in Sf9 insect cells using the baculovirus system, is able to phosphorylate both the beta 2-adrenergic receptor and rhodopsin in a stimulus-dependent fashion, although it is significantly less active then beta ARK on these substrates. These data extend the family of GRKs and suggest that GRK6 may have a substrate specificity quite distinct from beta ARK and rhodopsin kinase (Benovic, 1993).

A novel member of the family of G protein-coupled receptor kinases (GRKs), named GRK5, has been cloned from bovine taste epithelium. The cDNA sequence predicts a 590-amino acid protein with high overall similarity to rhodopsin kinase. GRK5 mRNA is found most abundantly in lung, heart, retina, and lingual epithelium, but is expressed very little in brain, liver, kidney, or testis. GRK5 expressed in Sf9 cells was purified to apparent homogeneity. GRK5 major autophosphorylation sites were mapped to Ser484 and Thr485. Purified GRK5 phosphorylates rhodopsin in a light-dependent manner and beta 2-adrenergic receptor in an agonist-dependent manner; GRK5 phosphorylates the C-terminal tail regions of both receptor proteins. GRK5 possesses neither a CAAX motif specifying protein prenylation like rhodopsin kinase nor similarity to the G protein beta gamma-subunit binding domain of beta-adrenergic receptor kinases. GRK5 phosphorylation of rhodopsin or beta 2-adrenergic receptor is not stimulated by G protein beta gamma-subunits. The GRK5 protein does not undergo agonist-dependent translocation from cytosol to membranes as do beta-adrenergic receptor kinase and rhodopsin kinase, but rather appears to associate with membranes constitutively. GRK5 thus appears functionally similar to other characterized GRKs, but has distinct regulatory properties which may be important for its cellular function (Premont, 1994).

GRK6 is the most recently identified member of the GRK family and displays higher homology with GRK5 (70.1% amino acid identity) and IT11 (68.5%) compared to beta ARK (37.4%) and rhodopsin kinase (47.1%). To further characterize GRK6, it has been overexpressed in Sf9 cells and purified to homogeneity by sequential chromatography on SP-Sepharose and heparin-Sepharose columns. GRK6 shares a number of in vitro characteristics with GRK5, including potent inhibition by heparin and dextran sulfate, hyperstimulation by polycations, and preference for phosphorylation of non-acidic peptides. Rhodopsin and the beta 2-adrenergic and m2 muscarinic cholinergic receptors serve as stimulus-dependent substrates for GRK6, but with stoichiometries significantly lower than achieved by GRK5 and beta ARK. Additionally, GRK6 does not undergo significant autophosphorylation even though it contains residues identical to those that are autophosphorylated in GRK5 and rhodopsin kinase. These data extend knowledge of a growing family of receptor-specific kinases and suggest that GRK6 has a substrate specificity distinct from beta ARK, rhodopsin kinase, and GRK5 (Loudon, 1994).

Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 has suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist in any of four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase (Premont, 1996).

G protein-coupled receptor kinases (GRKs) desensitize G protein-coupled receptors by phosphorylating activated receptors. The six known GRKs have been classified into three subfamilies based on sequence and functional similarities. Examination of the mouse GRK4 subfamily (GRKs 4, 5, and 6) suggests that mouse GRK4 is not alternatively spliced in a manner analogous to human or rat GRK4, whereas GRK6 undergoes extensive alternative splicing to generate three variants with distinct carboxyl termini. Characterization of the mouse GRK 5 and 6 genes reveals that all members of the GRK4 subfamily share an identical gene structure, in which 15 introns interrupt the coding sequence at equivalent positions in all three genes. Surprisingly, none of the three GRK subgroups (GRK1, GRK2/3, and GRK4/5/6) shares even a single intron in common, indicating that these three subfamilies are distinct gene lineages that have been maintained since their divergence over 1 billion years ago. Comparison of the amino acid sequences of GRKs from various mammalian species indicates that GRK2, GRK5, and GRK6 exhibit a remarkably high degree of sequence conservation, whereas GRK1 and particularly GRK4 have accumulated amino acid changes at extremely rapid rates over the past 100 million years. The divergence of individual GRKs at vastly different rates reveals that strikingly different evolutionary pressures apply to the function of the individual GRKs (Premont, 1999).

Mutation of GRKs

Tentative identification of the G protein-coupled receptor kinase 2 and 5 (GRK2 and GRK5) sites of phosphorylation of the beta2-adrenergic receptor (betaAR) has been reported based on in vitro phosphorylation of recombinant receptor. Phosphorylated residues identified for GRK2 are threonine 384 and serines 396, 401, and 407. GRK5 phosphorylates these four residues as well as threonine 393 and serine 411. To determine if mutation of these sites alters desensitization, betaARs were constructed in which the threonines and serines of the putative GRK2 and GRK5 sites were substituted with alanines. These constructs were further modified to eliminate the cAMP-dependent protein kinase (PKA) consensus sites. Mutants betaARs were transfected into HEK 293 cells, and standard kinetic parameters were measured following 10 microM epinephrine treatment of cells. The mutant and wild type (WT) receptors are all desensitized 89%-94% after 5 min of epinephrine stimulation and 96%-98% after a 30-min pretreatment. There were no significant changes observed for any of the mutant betaARs relative to the WT in the extent of epinephrine-induced internalization (77%-82% after 30 min). Epinephrine treatment for 1 min induces a rapid increase in the phosphorylation of the GRK5 and PKA- mutant betaARs as well as the WT. It is concluded that sites other than the GRK2 and GRK5 sites identified by in vitro phosphorylation are involved in mediating the major effects of the in vivo GRK-dependent desensitization of the betaAR (Seibold, 1998).

G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibit mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation are enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine is also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resist oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity (Gainetdinov, 1999).

Domain structure of GRKs

Inhibition of G protein-coupled receptor kinases (GRKs) by Ca2+-binding proteins is a general mechanism of GRK regulation. While GRK1 (rhodopsin kinase) is inhibited by the photoreceptor-specific Ca2+-binding protein recoverin, other GRKs can be inhibited by Ca2+-calmodulin. To dissect the mechanism of this inhibition at the molecular level, the GRK domains involved in Ca2+-binding protein interaction have been localized using a series of GST-GRK fusion proteins. GRK1, GRK2, and GRK5, which represent the three known GRK subclasses, were each found to possess two distinct calmodulin-binding sites. These sites have been localized to the N- and C-terminal regulatory regions within domains rich in positively charged and hydrophobic residues. In contrast, the unique N-terminally localized GRK1 site for recoverin has no clearly defined structural characteristics. Interestingly, while the recoverin and calmodulin-binding sites in GRK1 do not overlap, recoverin-GRK1 interaction is inhibited by calmodulin, most likely via an allosteric mechanism. Further analysis of the individual calmodulin sites in GRK5 suggests that the C-terminal site plays the major role in GRK5-calmodulin interaction. While specific mutation within the N-terminal site has no effect on calmodulin-mediated inhibition of GRK5 activity, deletion of the C-terminal site attenuates the effect of calmodulin on GRK5, and the simultaneous mutation of both sites renders the enzyme calmodulin-insensitive. These studies provide new insight into the mechanism of Ca2+-dependent regulation of GRKs (Levay, 1998).

Stimulation and inhibition of GRKs

GRK5, a recently identified member of the GRK family, undergoes a rapid phospholipid-stimulated autophosphorylation to a stoichiometry of approximately 2 mol of phosphate/mol of GRK5. The ability of phospholipids to stimulate autophosphorylation is largely blocked by a glutathione S-transferase fusion protein containing the last 102 amino acids of GRK5 (amino acids 489-590), suggesting that this is a primary region involved in GRK5/phospholipid interaction. Phosphoamino acid determination and mutagenesis studies demonstrate that autophosphorylation of GRK5 occurs primarily at residues Ser-484 and Thr-485. Expression and characterization of a mutant GRK5 that does not autophosphorylate (S484A and T485A) reveals that the mutant has a approximately 15-20-fold reduced ability to phosphorylate the beta 2-adrenergic receptor and rhodopsin compared to wild type GRK5. These results suggest that phospholipid-stimulated autophosphorylation may represent a novel mechanism for membrane association and regulation of GRK5 activity (Kunapuli, 1994).

Regulation of GRKs by Ca2+-binding proteins such as calmodulin (CaM) was examined. Gbetagamma-activated GRK2 and GRK3 are inhibited by CaM to similar extents, while a 50-fold more potent inhibitory effect is observed on GRK5. Inhibition by CaM is strictly dependent on Ca2+ and is prevented by the CaM inhibitor CaMBd. Since Gbetagamma, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gbetagamma-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduces GRK5 binding to the membrane. The mechanism of GRK5 inhibition appears to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases (Chuang, 1996).

The G protein-coupled receptor kinases (GRKs) phosphorylate agonist occupied G protein-coupled receptors and play an important role in mediating receptor desensitization. The localization of these enzymes to their membrane incorporated substrates is required for their efficient function and appears to be a highly regulated process. Phosphatidylinositol 4, 5-bisphosphate (PIP2) enhances GRK5-mediated beta-adrenergic receptor (betaAR) phosphorylation by directly interacting with this enzyme and facilitating its membrane association. GRK5-mediated phosphorylation of a soluble peptide substrate is unaffected by PIP2, suggesting that the PIP2-enhanced receptor kinase activity arises as a consequence of this membrane localization. The lipid binding site of GRK5 exhibits a high degree of specificity and appears to reside in the amino terminus of this enzyme. Mutation of six basic residues at positions 22, 23, 24, 26, 28, and 29 of GRK5 ablates the ability of this kinase to bind PIP2. This region of the GRK5, which has a similar distribution of basic amino acids to the PIP2 binding site of gelsolin, is highly conserved between members of the GRK4 subfamily (GRK4, GRK5, and GRK6). Indeed, all the members of the GRK4 subfamily exhibit PIP2-dependent receptor kinase activity. The membrane association of betaARK (beta-adrenergic receptor kinase) (GRK2) is mediated, in vitro, by the simultaneous binding of PIP2 and the betagamma subunits of heterotrimeric G proteins to the carboxyl-terminal pleckstrin homology domain of this enzyme. Thus, five members of the GRK family bind PIP2, betaARK (GRK2), betaARK2 (GRK3), GRK4, GRK5, and GRK6. However, the structure, location, and regulation of the PIP2 binding site distinguishes the betaARK (GRK2 and GRK3) and GRK4 (GRK4, GRK5, and GRK6) subfamilies (Pitcher, 1996).

To assess a potential general role for PKC in regulating GRK function, the ability of PKC to phosphorylate GRK5 was characterized. GRK5 can be rapidly and stoichiometrically phosphorylated by PKC in vitro. Intact cell studies reveal that GRK5 is also phosphorylated when transiently expressed in COS-1 cells following treatment with the PKC activator, phorbol 12-myristate 13-acetate. In vitro analysis reveals two major sites of PKC phosphorylation within the C-terminal 26 amino acids of GRK5. GRK5 phosphorylation by PKC dramatically reduces its ability to phosphorylate both receptor (light-activated rhodopsin) and non-receptor (casein and phosvitin) substrates. Kinetic analysis reveals an approximately 5-fold increased Km and approximately 3-fold decreased Vmax for rhodopsin, with no change in the Km for ATP. The reduced affinity of PKC-phosphorylated GRK5 for rhodopsin is also evident in a decreased ability to bind to rhodopsin-containing membranes, while direct binding of GRK5 to phospholipids appears unaltered. These results suggest that PKC might play an important role in modulating the ability of GRK5 to regulate receptor signaling and that GRK phosphorylation by PKC may serve as a disparate mechanism for regulating GRK activity (Pronin, 1997a).

GRK2 and GRK5 can be phosphorylated and either activated or inhibited, respectively, by protein kinase C. Calmodulin, another mediator of calcium signaling, is a potent inhibitor of GRK activity with a selectivity for GRK5. Calmodulin inhibition of GRK5 is mediated via a reduced ability of the kinase to bind to both receptor and phospholipid. Interestingly, calmodulin also activates autophosphorylation of GRK5 at sites distinct from the two major autophosphorylation sites on GRK5. Moreover, calmodulin-stimulated autophosphorylation directly inhibits GRK5 interaction with receptor even in the absence of calmodulin. Using glutathione S-transferase-GRK5 fusion proteins either to inhibit calmodulin-stimulated autophosphorylation or to bind directly to calmodulin, it was determined that an amino-terminal domain of GRK5 (amino acids 20-39) is sufficient for calmodulin binding. This domain is abundant in basic and hydrophobic residues, characteristics typical of calmodulin binding sites, and is highly conserved in GRK4, GRK5, and GRK6. These studies suggest that calmodulin may serve a general role in mediating calcium-dependent regulation of GRK activity (Pronin, 1997b).

GRK2-mediated receptor phosphorylation is preceded by the agonist-dependent membrane association of this enzyme. Previous in vitro studies with purified proteins have suggested that this translocation may be mediated by the recruitment of GRK2 to the plasma membrane by its interaction with the free betagamma subunits of heterotrimeric G proteins (G betagamma). This mechanism operates in intact cells and specificity is imparted by the selective interaction of discrete pools of G betagamma with receptors and GRKs. Treatment of Cos-7 cells transiently overexpressing GRK2 with a beta-receptor agonist promotes a 3-fold increase in plasma membrane-associated GRK2. This translocation of GRK2 is inhibited by the carboxyl terminus of GRK2, a known G betagamma sequestrant. Furthermore, in cells overexpressing both GRK2 and G beta1 gamma2, activation of lysophosphatidic acid receptors leads to the rapid and transient formation of a GRK/G betagamma complex. That G betagamma specificity exists at the level of the GPCR and the GRK is indicated by the observation that a GRK2/G betagamma complex is formed after agonist occupancy of the lysophosphatidic acid and beta-adrenergic but not thrombin receptors. In contrast to GRK2, GRK3 forms a G betagamma complex after stimulation of all three GPCRs. This G betagamma binding specificity of the GRKs is also reflected at the level of the purified proteins. Thus the GRK2 carboxyl terminus binds G beta1 and G beta2 but not G beta3, while the GRK3 fusion protein binds all three G beta isoforms. This study provides a direct demonstration of a role for G betagamma in mediating the agonist-stimulated translocation of GRK2 and GRK3 in an intact cellular system and demonstrates isoform specificity in the interaction of these components (Daaka, 1997).

GRKs are subject to post-translational regulation. For example, GRK5 activity is strongly inhibited by protein kinase C phosphorylation and by Ca2+-calmodulin binding. Ca2+-calmodulin binding also promotes GRK5 autophosphorylation, which further contributes to kinase inhibition. Two important structural domains have been identified in GRK5, a phospholipid binding domain (residues 552-562) and an autoinhibitory domain (residues 563-590), that significantly contribute to GRK5 localization and function. The C-terminal region of GRK5 (residues 563-590) contains residues autophosphorylated in the presence of calmodulin as well as the residues phosphorylated by protein kinase C. Deletion of this domain increases the apparent affinity of GRK5 for receptor substrates 3-4-fold but has no effect on nonreceptor substrates. These findings define residues 563-590 of GRK5 as an autoinhibitory domain with efficacy that is regulated by phosphorylation. Another C-terminal domain in GRK5 that appears to be functionally important is found between residues 552 and 562. Deletion of this region significantly inhibits kinase phosphorylation of membrane-bound receptor substrates but has no effect on soluble substrates. Additional studies reveal that this domain is critical for GRK5 interaction with phospholipids and for the intracellular localization of the kinase. Interestingly, similar regions in GRK4 and GRK6 appear to be palmitoylated (and involved in membrane interaction), suggesting evolutionary conservation of the function of this domain (Pronin, 1998).

G protein-coupled receptor kinases (GRKs) phosphorylate G protein-coupled receptors, thereby terminating receptor signaling. Alpha-actinin potently inhibits all GRK family members. In addition, calcium-bound calmodulin and phosphatidylinositol 4,5-bisphosphate (PIP2), two regulators of GRK activity, coordinate with alpha-actinin to modulate substrate specificity of the GRKs. In the presence of calmodulin and alpha-actinin, GRK5 phosphorylates soluble, but not membrane-incorporated substrates. In contrast, in the presence of PIP2 and alpha-actinin, GRK5 phosphorylates membrane-incorporated, but not soluble substrates. Thus, modulation of alpha-actinin-mediated inhibition of GRKs by PIP2 and calmodulin has profound effects on both GRK activity and substrate specificity (Freeman, 2000).

Degradation of GRKs

G-protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of G-protein-coupled receptors (GPCRs). GRK2 expression is altered in several pathological conditions, but the molecular mechanisms that modulate GRK2 cellular levels are largely unknown. GRK2 is degraded rapidly by the proteasome pathway. This process is enhanced by GPCR stimulation and is severely impaired in a GRK2 mutant that lacks kinase activity (GRK2-K220R). ß-arrestin function and Src-mediated phosphorylation of GRK2 are critically involved in GRK2 proteolysis. Overexpression of ß-arrestin triggers GRK2-K220R degradation based on its ability to recruit c-Src, since this effect is not observed with ß-arrestin mutants that display an impaired c-Src interaction. The presence of an inactive c-Src mutant or of tyrosine kinase inhibitors strongly inhibits co-transfected or endogenous GRK2 turnover, respectively, and a GRK2 mutant with impaired phosphorylation by c-Src shows a markedly retarded degradation. This pathway for the modulation of GRK2 protein stability puts forward a new feedback mechanism for regulating GRK2 levels and GPCR signaling (Penela, 2001).

These results are consistent with the notion that GRK2-dependent binding of ß-arrestin to GPCRs allows the recruitment of c-Src to the receptor signaling complex at the plasma membrane, leading to phosphorylation of GRK2 on tyrosine residues and its targeting for degradation. This model is in agreement with the rapid ß-arrestin and c-Src recruitment following ß2AR stimulation, and with the agonist-stimulated phosphorylation of GRK2 by c-Src. Under basal conditions, ß-arrestin recruitment to the plasma membrane would be promoted by the activated state of different endogenous GPCRs and/or by the reported basal activity of overexpressed ß2AR. In the presence of GPCR agonists, an acceleration of the GRK2 degradation rate is detected, consistent with a more efficient ß-arrestin and c-Src translocation to the receptor complex. Although detailed knowledge of the sequential assembly of these proteins in a multimolecular complex is lacking, and other molecular interactions may participate in c-Src binding to the receptor complex and GRK2 tyrosine phosphorylation, the proposed model is consistent with the co-immunoprecipitation of ß-arrestin and c-Src, of GRK2 and ß-arrestin and of GRK2 and c-Src. Disruption of the ß-arrestin-c-Src interaction with specific mutants or inhibition of the phosphorylation step by dominant-negative Src or a GRK2 mutant lacking critical phosphorylation sites results, as predicted by this model, in a marked reduction in GRK2 degradation (Penela, 2001).

GRKs target adrenergic and Dopaminergic receptors

Persistent stimulation of the beta 1-adrenergic receptor (beta 1AR) engenders, within minutes, diminished responsiveness of the beta 1 AR/adenylyl cyclase signal transduction system. This desensitization remains incompletely defined mechanistically, however. The hypothesis that agonist-induced desensitization of the beta 1AR (like that of the related beta 2AR) involves phosphorylation of the receptor itself, by cAMP-dependent protein kinase (PKA) and the beta-adrenergic receptor kinase (beta ARK1) or other G protein-coupled receptor kinases (GRKs), was tested. Both Chinese hamster fibroblast and 293 cells demonstrate receptor-specific desensitization of the beta 1 AR within 3-5 min. Both cell types also express beta ARK1 and the associated inhibitory proteins beta-arrestin-1 and beta-arrestin-2, as assessed by immunoblotting. Agonist-induced beta 1AR desensitization in 293 cells correlates with a 2 +/- 0.3-fold increase in phosphorylation of the beta 1AR, determined by immunoprecipitation of the beta 1AR from cells metabolically labeled with 32P(i). This agonist-induced beta 1AR phosphorylation derives approximately equally from PKA and GRK activity, as judged by intact cell studies with kinase inhibitors or dominant negative beta ARK1 (K220R) mutant overexpression. Desensitization, likewise, is reduced by only approximately 50% when PKA is inhibited in the intact cells. Overexpression of rhodopsin kinase, beta ARK1, beta ARK2, or GRK5 significantly increases agonist-induced beta 1AR phosphorylation and concomitantly decreases agonist-stimulated cellular cAMP production. Furthermore, purified beta ARK1, beta ARK2, and GRK5 all demonstrate agonist-dependent phosphorylation of the beta 1AR. Consistent with a GRK mechanism, receptor-specific desensitization of the beta 1AR is enhanced by overexpression of beta-arrestin-1 and -2 in transfected 293 cells. It is concluded that rapid agonist-induced desensitization of the beta 1AR involves phosphorylation of the receptor by both PKA and at least beta ARK1 in intact cells. Like the beta 2AR, the beta 1AR appears to bind either beta-arrestin-1 or beta-arrestin-2 and to react with rhodopsin kinase, beta ARK1, beta ARK2, and GRK5 (Freedman, 1995).

Although previous studies have implicated the cytoplasmic tail of the beta2-adrenergic receptor (beta2AR) as the site of GRK-mediated phosphorylation, the identities of the phosphorylated residues were unknown. The sites of GRK2- and GRK5-mediated beta2AR phosphorylation have been identified in this study. The phosphorylation sites of both serine/threonine kinases reside exclusively in a 40-amino acid peptide located at the extreme carboxyl terminus of the beta2AR. Of the seven phosphorylatable residues within this peptide, six are phosphorylated by GRK5 (Thr-384, Thr-393, Ser-396, Ser-401, Ser-407, and Ser-411) and four are phosphorylated by GRK2 (Thr-384, Ser-396, Ser-401, and Ser-407) at equivalent phosphorylation stoichiometries (approximately 1.0 mol Pi/mol receptor). In addition to the GRK5-specific phosphorylation of Thr-393 and Ser-411, differences in the distribution of phosphate between sites are observed for GRK2 and GRK5. Increasing the stoichiometry of GRK2-mediated beta2AR phosphorylation from approximately 1.0 to 5.0 mol Pi/mol receptor increases the stoichiometry of phosphorylation of Thr-384, Ser-396, Ser-401, and Ser-407 rather than increasing the number of phosphoacceptor sites. The location of multiple GRK2 and GRK5 phosphoacceptor sites at the extreme carboxyl terminus of the beta2AR is highly reminiscent of GRK1-mediated phosphorylation of rhodopsin (Fredericks, 1996).

The alpha2-adrenergic receptor (alpha2AR) subtype alpha2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate alpha2C10 in an agonist-dependent manner is the betaAR kinase (betaARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the alpha2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by betaARK. However, the substrate specificities of the GRKs for phosphorylating alpha2AR subtypes are not known. Differential capacities of various GRKs to phosphorylate alpha2C10 and alpha2C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors. COS-7 cells were co-transfected with alpha2C10 or alpha2C4 without or with the following GRKs: betaARK, betaARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the alpha2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. BetaARK and betaARK2 were both found to phosphorylate alpha2C10 to equal extents. GRK5 and GRK6 did not phosphorylate alpha2C10. In contrast to the findings with alpha2C10, alpha2C4 is not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing alpha2C10 or alpha2C4 and selected GRKs were consistent with these phosphorylation results. With the marked expression of these receptors, no agonist-promoted desensitization is observed in the absence of GRK co-expression. However, desensitization is imparted to alpha2C10 by co-expression of betaARK but not GRK6, while alpha2C4 failed to desensitize with co-expression of betaARK. These results indicate that short term agonist-promoted desensitization of alpha2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform (Jewell-Motz, 1996).

The alpha1B-adrenergic receptor (alpha1BAR), its truncated mutant T368, different G protein-coupled receptor kinases (GRK) and arrestin proteins were transiently expressed in COS-7 or HEK293 cells alone and/or in various combinations. Coexpression of beta-adrenergic receptor kinase (betaARK) 1 (GRK2) or 2 (GRK3) can increase epinephrine-induced phosphorylation of the wild type alpha1BAR above basal as compared to that of the receptor expressed alone. Overexpression of the dominant negative betaARK (K220R) mutant impairs agonist-induced phosphorylation of the receptor. Overexpression of GRK6 can also increase epinephrine-induced phosphorylation of the receptor, whereas GRK5 enhances basal but not agonist-induced phosphorylation of the alpha1BAR. Increasing coexpression of betaARK1 or betaARK2 results in the progressive attenuation of the alpha1BAR-mediated response on polyphosphoinositide (PI) hydrolysis. However, coexpression of betaARK1 or 2 at low levels does not significantly impair the PI response mediated by the truncated alpha1BAR mutant T368, lacking the C terminus, which is involved in agonist-induced desensitization and phosphorylation of the receptor. Similar attenuation of the receptor-mediated PI response is also observed for the wild type alpha1BAR, but not for its truncated mutant, when the receptor is coexpressed with beta-arrestin 1 or beta-arrestin 2. Despite their pronounced effect on phosphorylation of the alpha1BAR, overexpression of GRK5 or GRK6 do not affect the receptor-mediated response. In conclusion, these results provide the first evidence that betaARK1 and 2 as well as arrestin proteins might be involved in agonist-induced regulation of the alpha1BAR. They also identify the alpha1BAR as a potential phosphorylation substrate of GRK5 and GRK6. However, the physiological implications of GRK5- and GRK6-mediated phosphorylation of the alpha1BAR remain to be elucidated (Diviani, 1996).

To explore the potential role played by the GRKs in the regulation of the rat dopamine D1A receptor, whole cell phosphorylation experiments and cAMP assays were performed in 293 cells cotransfected with the receptor alone or with various GRKs (GRK2, GRK3, and GRK5). The agonist-dependent phosphorylation of the rat D1A receptor is substantially increased in cells overexpressing GRK2, GRK3, or GRK5. Moreover, cAMP formation upon receptor activation is differentially regulated in cells overexpressing either GRK2, GRK3, and GRK5 under conditions that elicited similar levels of GRK-mediated receptor phosphorylation. Cells expressing the rat D1A receptor with GRK2 and GRK3 display a rightward shift of the dopamine dose-response curve with little effect on the maximal activation when compared with cells expressing the receptor alone. In contrast, cells expressing GRK5 display a rightward shift in the EC50 value with an additional 40% reduction in the maximal activation when compared with cells expressing the receptor alone. Thus, the dopamine D1A receptor can serve as a substrate for various GRKs and GRK-phosphorylated D1A receptors display a differential reduction of functional coupling to adenylyl cyclase. These results suggest that the cellular complement of G protein-coupled receptor kinases may determine the properties and extent of agonist-mediated responsiveness and desensitization (Tiberi, 1996).

GRKs target other G protein-coupled receptors

To identify GRK(s) that play a role in homologous desensitization of the thyrotropin (TSH) receptor, thyroid cDNA was amplified by polymerase chain reaction using degenerate oligonucleotide primers from highly conserved regions in GRK family. GRK5 is found in the predominant isoform expressed in the thyroid. Rat GRK5 cDNA was then isolated, which encodes a 590-amino acid protein with 95% homology to human and bovine homologs. Northern blot identified GRK5 mRNA of approximately 3, 8, and 10 kilobases with highest expression levels in lung > heart, kidney, colon > thyroid. In functional studies using a normal rat thyroid FRTL5 cells, overexpression of GRK5 suppresses basal cAMP levels and augments the extent of TSH receptor desensitization, whereas suppression of endogenous GRK5 expression by transfecting the antisense GRK5 construct increases basal cAMP levels and attenuates the extent of receptor desensitization. Although exogenously overexpressed GRK6 also enhances TSH receptor desensitization, it is concluded that GRK5, the predominant GRK isoform in the thyroid, appears to be mainly involved in homologous desensitization of the TSH receptor (Nagayama, 1996).

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. The present study investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. These cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 are rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells demonstrate the following: (1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase; (2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold; (3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and (4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. These results show that the desensitization of the FSH-induced response involves the GRK/arrestin system (Troispoux, 1999).

The Xenopus oocyte expression system to examine the regulation of rat kappa opioid receptor (rKOR) function by G protein receptor kinases (GRKs). Kappa agonists increase the conductance of G protein-activated inwardly rectifying potassium channels in oocytes co-expressing KOR with Kir3.1 and Kir3.4. In the absence of added GRK and beta-arrestin 2, desensitization of the kappa agonist-induced potassium current is modest. Co-expression of either GRK3 or GRK5 along with beta-arrestin 2 significantly increases the rate of desensitization, whereas addition of either beta-arrestin 2, GRK3, or GRK5 alone has no effect on the KOR desensitization rate. The desensitization is homologous since co-expressed delta opioid receptor-evoked responses are not affected by KOR desensitization. The rate of GRK3/beta-arrestin 2-dependent desensitization is reduced by truncation of the C-terminal 26 amino acids, KOR(Q355Delta). In contrast, substitution of Ala for Ser within the third intracellular loop [KOR(S255A,S260A, S262A)] does not reduce the desensitization rate. Within the C-terminal region, KOR(S369A) substitution significantly attenuates desensitization, whereas the KOR(T363A) and KOR(S356A,T357A) point mutations do not. These results suggest that co-expression of GRK3 or GRK5 and beta-arrestin 2 produce homologous, agonist-induced desensitization of the kappa opioid receptor by a mechanism requiring the phosphorylation of the serine 369 of rKOR (Appleyard, 1999).

Examination of the structure of [Arg(8)]-vasopressin receptors (AVPRs) and oxytocin receptors (OTRs) suggests that G protein-coupled receptor kinases (GRKs) and protein kinase C (PKC) are involved in their signal transduction. To explore the physical association of AVPRs and OTRs with GRKs and PKC, wild types and mutated forms of these receptor subtypes were stably expressed as green fluorescent protein fusion proteins and analyzed by fluorescence, immunoprecipitation, and immunoblotting. Addition of a C-terminal GFP tag does not interfere with ligand binding, internalization, and signal transduction. After agonist stimulation, PKC dissociates from the V(1)R, does not associate with the V(2)R, but associates with the V(3)R and the OTR. After AVP stimulation, only GRK5 briefly associates with AVPRs following a time course that varies with the receptor subtype. No GRK associates with the OTR. Exchanging the V(1)R and V(2)R C termini alters the time course of PKC and GRK5 association. Deletion of the V(1)R C terminus results in no PKC association and a ligand-independent sustained association of GRK5 with the receptor. Deletion of the GRK motif prevents association and reduces receptor phosphorylation. Thus, agonist stimulation of AVP/OT receptors leads to receptor subtype-specific interactions with GRK and PKC through specific motifs present in the C termini of the receptors (Berrada, 2000).

Investigating the parathyroid hormone (PTH) receptor --> inositol phosphate pathway, it has been found that GRKs can inhibit receptor signaling already under nonphosphorylating conditions. GRKs phosphorylated the PTH receptor in membranes and in intact cells; the order of efficacy was GRK2>GRK3>GRK5. Transient transfection of GRKs with the PTH receptor into COS-1 cells inhibits PTH-stimulated inositol phosphate generation. Such an inhibition also is seen with the kinase-negative mutant GRK2-K220R and also for a C-terminal truncation mutant of the PTH receptor that could not be phosphorylated. Several lines of evidence indicate that this phosphorylation-independent inhibition is exerted by an interaction between GRKs and receptors: (1) this inhibition is not mimicked by proteins binding to G proteins, phosducin, and GRK2 C terminus; (2) GRKs cause an agonist-dependent inhibition (= desensitization) of receptor-stimulated G protein GTPase-activity (this effect also is seen with the kinase-inactive GRK2-mutant and the phosphorylation-deficient receptor mutant), and (3) GRKs bind directly to the PTH receptor. These data suggest that signaling by the PTH receptor already is inhibited by the first step of homologous desensitization, the binding of GRKs to the receptors (Dicker, 1999).

Although endothelin-1 can elicit prolonged physiologic responses, accumulating evidence suggests that rapid desensitization affects the primary G protein-coupled receptors mediating these responses, the endothelin A and B receptors (ETA-R and ETB-R). The mechanisms by which this desensitization proceeds remain obscure, however. Because some intracellular domain sequences of the ETA-R and ETB-R differ substantially, the possibility that these receptor subtypes might be differentially regulated by G protein-coupled receptor kinases was examined. Homologous, or receptor-specific, desensitization occurs within 4 min both in the ETA-R-expressing A10 cells and in 293 cells transfected with either the human ETA-R or ETB-R. In 293 cells, this desensitization corresponds temporally with agonist-induced phosphorylation of each receptor. Agonist-induced receptor phosphorylation is not substantially affected by PKC inhibition but is reduced 40% by GRK inhibition, effected by a dominant negative GRK2 mutant. Inhibition of agonist-induced phosphorylation abrogates agonist-induced ETA-R desensitization. Overexpression of GRK2, -5, or -6 in 293 cells augments agonist-induced ET-R phosphorylation approximately 2-fold, but each kinase reduces receptor-promoted phosphoinositide hydrolysis differently. While GRK5 inhibits ET-R signaling by only approximately 25%, GRK2 inhibits ET-R signaling by 80%. Congruent with its superior efficacy in suppressing ET-R signaling, GRK2, but not GRK5, co-immunoprecipitates with the ET-Rs in an agonist-dependent manner. It is concluded that both the ETA-R and ETB-R can be regulated indistinguishably by GRK-initiated desensitization. It is proposed that because of its affinity for ET-Rs, GRK2 is the most likely of the GRKs to initiate ET-R desensitization (Freedman, 1997).

GRKs function in the sequestration/internalization of receptors

A study was performed of the agonist-dependent sequestration/internalization of dopamine D2 receptor (the long form D2L and short form D2S), which were transiently expressed in COS-7 and HEK 293 cells with or without G-protein-coupled receptor kinases (GRK2 or GRK5). In COS-7 cells expressing D2 receptors alone, virtually no sequestration is observed with or without dopamine (< 4%). When GRK2 is coexpressed, 50% of D2S receptors and 36% of D2L receptors are sequestered by treatment with 10(-4) M dopamine for 2 h, whereas no sequestration is observed in cells expressing the dominant negative form of GRK2 (DN-GRK2). When GRK5 is coexpressed, 36% of D2S receptors are sequestered following the same treatment. The agonist-dependent and GRK2-dependent sequestration of D2S receptors is reduced markedly in the presence of hypertonic medium containing 0.45 M sucrose, suggesting that the sequestration follows the clathrin pathway. Internalization of D2S receptors was also assessed by immunofluorescence confocal microscopy. Translocation of D2 receptors from the cell membrane to intracellular vesicles is observed following the treatment with dopamine from HEK 293 cells only when GRK2 is coexpressed. D2S receptors expressed in HEK 293 cells are phosphorylated by GRK2 in an agonist-dependent manner. These results indicate that the sequestration of D2 receptors occurs only through a GRK-mediated pathway (Ito, 1999).

GRKs regulate cytoskeletal function

GRK2 is a microtubule-associated protein and tubulin is identified as a novel GRK2 substrate. GRK2 is associated with microtubules purified from bovine brain, forms a complex with tubulin in cell extracts, and colocalizes with tubulin in living cells. Furthermore, an endogenous tubulin kinase activity that copurifies with microtubules has properties similar to GRK2 and is inhibited by anti-GRK2 monoclonal antibodies. Indeed, GRK2 phosphorylates tubulin in vitro with kinetic parameters very similar to those for phosphorylation of the agonist-occupied beta2-adrenergic receptor, suggesting a functionally relevant role for this phosphorylation event. In a cellular environment, agonist occupancy of GPCRs, which leads to recruitment of GRK2 to the plasma membrane and its subsequent activation, promotes GRK2-tubulin complex formation and tubulin phosphorylation. These findings suggest a novel role for GRK2 as a GPCR signal transducer mediating the effects of GPCR activation on the cytoskeleton (Pitcher, 1998).

G protein-coupled receptor kinases (GRKs) initiate pathways leading to the desensitization of agonist-occupied G-protein-coupled receptors (GPCRs). The cytoskeletal protein actin binds and inhibits GRK5. Actin inhibits the kinase activity directly, reducing GRK5-mediated phosphorylation of both membrane-bound GPCRs and soluble substrates. GRK5 binds actin monomers with a Kd of 0.6 microM and actin filaments with a Kd of 0. 2 microM. Mutation of 6 amino acids near the amino terminus of GRK5 eliminates actin-mediated inhibition of GRK5. Calmodulin binds to the amino terminus of GRK5 and displaces GRK5 from actin. Calmodulin inhibits GRK5-mediated phosphorylation of GPCRs, but not soluble substrates such as casein. Thus in the presence of actin, calmodulin determines the substrate specificity of GRK5 by preferentially allowing phosphorylation of soluble substrates over membrane-bound substrates (Freeman, 1998).

Although the beta-adrenergic receptor kinase (betaARK) mediates agonist-dependent phosphorylation and desensitization of G protein-coupled receptors, recent studies suggest additional cellular functions. During attempts to identify novel betaARK interacting proteins, it was found that the cytoskeletal protein tubulin can specifically bind to a betaARK-coupled affinity column. In vitro analysis demonstrates that betaARK and G protein-coupled receptor kinase-5 (GRK5) are able to stoichiometrically phosphorylate purified tubulin dimers with a preference for beta-tubulin and, under certain conditions, the betaIII-isotype. Examination of the GRK/tubulin binding characteristics revealed that tubulin dimers and assembled microtubules bind GRKs, whereas the catalytic domain of betaARK contains the primary tubulin binding determinants. In vivo interaction of GRK and tubulin is suggested by the following: (1) co-purification of betaARK with tubulin from brain tissue; (2) co-immunoprecipitation of betaARK and tubulin from COS-1 cells; and (3) co-localization of betaARK and GRK5 with microtubule structures in COS-1 cells. In addition, GRK-phosphorylated tubulin is found preferentially associated with the microtubule fraction during in vitro assembly assays suggesting potential functional significance. These results suggest a novel link between the cytoskeleton and GRKs that may be important for regulating GRK and/or tubulin function (Carman, 1998).

G protein-coupled receptor kinases (GRKs) have been principally characterized by their ability to phosphorylate and desensitize G protein-coupled receptors. However, recent studies suggest that GRKs may have more diverse protein/protein interactions in cells. Based on the identification of a consensus caveolin binding motif within the pleckstrin homology domain of GRK2, the direct binding of purified full-length GRK2 to various glutathione S-transferase-caveolin-1 fusion proteins was studied, and a specific interaction of GRK2 with the caveolin scaffolding domain was discovered. Interestingly, analysis of GRK1 and GRK5, which lack a pleckstrin homology domain, revealed in vitro binding properties similar to those of GRK2. Maltose-binding protein caveolin and glutathione S-transferase-GRK fusion proteins were used to map overlapping regions in the N termini of both GRK2 and GRK5 that appear to mediate conserved GRK/caveolin interactions. In vivo association of GRK2 and caveolin was suggested by co-fractionation of GRK2 with caveolin in A431 and NIH-3T3 cells and was further supported by co-immunoprecipitation of GRK2 and caveolin in COS-1 cells. Functional significance for the GRK/caveolin interaction was demonstrated by the potent inhibition of GRK-mediated phosphorylation of both receptor and peptide substrates by caveolin-1 and -3 scaffolding domain peptides. These data reveal a novel mode for the regulation of GRKs that is likely to play an important role in their cellular function (Carman, 1999).

Smoothened signal transduction is promoted by G protein-coupled receptor kinase 2

Deregulation of the Sonic hedgehog pathway has been implicated in an increasing number of human cancers. In this pathway, the seven-transmembrane (7TM) signaling protein Smoothened regulates cellular proliferation and differentiation through activation of the transcription factor Gli. The activity of mammalian Smoothened is controlled by three different hedgehog proteins, Indian, Desert, and Sonic hedgehog, through their interaction with the Smoothened inhibitor Patched. However, the mechanisms of signal transduction from Smoothened are poorly understood. This study shows that a kinase which regulates signaling by many 'conventional' 7TM G-protein-coupled receptors, G protein-coupled receptor kinase 2 (GRK2), participates in Smoothened signaling. Expression of GRK2, but not catalytically inactive GRK2, synergizes with active Smoothened to mediate Gli-dependent transcription. Moreover, knockdown of endogenous GRK2 by short hairpin RNA (shRNA) significantly reduces signaling in response to the Smoothened agonist SAG and also inhibits signaling induced by an oncogenic Smoothened mutant, Smo M2. GRK2 promotes the association between active Smoothened and beta-arrestin 2. Indeed, Gli-dependent signaling, mediated by coexpression of Smoothened and GRK2, is diminished by beta-arrestin 2 knockdown with shRNA. Together, these data suggest that GRK2 plays a positive role in Smoothened signaling, at least in part, through the promotion of an association between beta-arrestin 2 and Smoothened (Meloni, 2006).

Physiological functions of GRKs

Transgenic mice were generated with cardiac-specific overexpression of the G protein-coupled receptor kinase-5 (GRK5), a serine/threonine kinase most abundantly expressed in the heart compared with other tissues. Animals overexpressing GRK5 show marked beta-adrenergic receptor desensitization in both the anesthetized and conscious state compared with nontransgenic control mice, while the contractile response to angiotensin II receptor stimulation is unchanged. In contrast, the angiotensin II-induced rise in contractility is significantly attenuated in transgenic mice overexpressing the beta-adrenergic receptor kinase-1, another member of the GRK family. These data suggest that myocardial overexpression of GRK5 results in selective uncoupling of G protein-coupled receptors and demonstrate that receptor specificity of the GRKs may be important in determining the physiological phenotype (Rockman, 1996).

Pressure overload cardiac hypertrophy in the mouse is achieved following 7 days of transverse aortic constriction. This is associated with marked beta-adrenergic receptor (beta-AR) desensitization in vivo, as determined by a blunted inotropic response to dobutamine. Extracts from hypertrophied hearts have approximately 3-fold increase in cytosolic and membrane G protein-coupled receptor kinase (GRK) activity. Incubation with specific monoclonal antibodies to inhibit different GRK subtypes showed that the increase in activity can be attributed predominately to the beta-adrenergic receptor kinase (betaARK). Although overexpression of a betaARK inhibitor in hearts of transgenic mice does not alter the development of cardiac hypertrophy, the beta-AR desensitization associated with pressure overload hypertrophy is prevented. To determine whether the induction of betaARK occurs because of a generalized response to cellular hypertrophy, betaARK activity was measured in transgenic mice homozygous for oncogenic ras overexpression in the heart. Despite marked cardiac hypertrophy, no difference in betaARK activity was found in these mice overexpressing oncogenic ras compared with controls. Taken together, these data suggest that betaARK is a central molecule involved in alterations of beta-AR signaling in pressure overload hypertrophy. The mechanism for the increase in betaARK activity appears not to be related to the induction of cellular hypertrophy but to possibly be related to neurohumoral activation (Choi, 1997).

The function of G protein-coupled receptor kinases (GRKs) in the regulation of thrombin-activated signaling in endothelial cells was studied. GRK2, GRK5, and GRK6 isoforms are expressed predominantly in endothelial cells. The function of these isoforms was studied by expressing wild-type and dominant negative (dn) mutants in endothelial cells. The responses to thrombin, which activates intracellular signaling in endothelial cells by cleaving the NH(2) terminus of the G protein-coupled proteinase-activated receptor-1 (PAR-1), was examined. Changes in phosphoinositide hydrolysis and intracellular Ca²⁺ concentration in response to thrombin were studied as well as the state of endothelial activation. In the latter studies, the transendothelial monolayer electrical resistance, a measure of the loss of endothelial barrier function, was measured in real time. Of the three isoforms, GRK5 overexpression is selective in markedly reducing the thrombin-activated phosphoinositide hydrolysis and increased intracellular Ca²⁺. GRK5 overexpression also inhibited the thrombin-induced decrease in endothelial monolayer resistance by 75%. These effects of GRK5 overexpression occur in association with the specific increase in the thrombin-induced phosphorylation of PAR-1. In contrast to the effects of GRK5 overexpression, the expression of the dn-GRK5 mutant produces a long-lived increase in intracellular Ca²⁺ in response to thrombin, whereas dn-GRK2 has no effect. These results indicate the crucial role of the GRK5 isoform in the mechanism of thrombin-induced desensitization of PAR-1 in endothelial cells (Tiruppathi, 2000).

Metabotropic glutamate receptors (mGluRs) constitute a unique subclass of G protein-coupled receptors (GPCRs) that bear little sequence homology to other members of the GPCR superfamily. The mGluR subtypes that are coupled to the hydrolysis of phosphoinositide contribute to both synaptic plasticity and glutamate-mediated excitotoxicity in neurons. The expression of mGluR1a in HEK 293 cells leads to agonist-independent cell death. Since G protein-coupled receptor kinases (GRKs) desensitize a diverse variety of GPCRs, whether GRKs contributes to the regulation of both constitutive and agonist-stimulated mGluR1a activity and thereby prevents mGluR1a-mediated excitotoxicity associated with mGluR1a overactivation was examined. The co-expression of mGluR1a with GRK2 and GRK5, but not GRK4 and GRK6, reduces both constitutive and agonist-stimulated mGluR1a activity. Agonist-stimulated mGluR1a phosphorylation is enhanced by the co-expression of GRK2 and is blocked by two different GRK2 dominant-negative mutants. Furthermore, GRK2-dependent mGluR1a desensitization protects against mGluR1a-mediated cell death, at least in part by blocking mGluR1a-stimulated apoptosis. These data indicate that as with other members of the GPCR superfamily, a member of the structurally distinct mGluR family (mGluR1a) serves as a substrate for GRK-mediated phosphorylation and that GRK-dependent 'feedback' modulation of mGluR1a responsiveness protects against pathophysiological mGluR1a signaling (Dale, 2000).

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response is abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. Beta-arrestin-1 transfection also induces a significantly decrease in GIP-stimulated cAMP production, and this effect is greater with cotransfection of both GRK2 and beta-arrestin-1 than with either alone. In betaTC3 cells, expression of GRK2 or beta-arrestin-1 attenuates GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion is not affected. GRK2 and beta-arrestin-1 messenger RNAs are expressed endogenously in betaTC3 and L293 cells. Overexpression of GRK2 enhances agonist-induced GIPR phosphorylation, but receptor endocytosis is not affected by cotransfection with GRKs or beta-arrestin-1. These results suggest a potential role for GRK2/beta-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR (Tseng, 2000).

The myometrial beta-adrenergic receptor (beta-AR)-adenylyl cyclase pathway is markedly desensitized at the end of pregnancy in the rat. Whether changes in the amount and/or the activity of G protein-coupled receptor kinase (GRK) occurs at the same period of pregnancy was tested. Using Northern and Western blotting, GRK2, GRK5, GRK6, and a small amount of GRK3 were identified in late pregnant rat myometrium. GRK activity, as measured by in vitro phosphorylation of rhodopsin, is detected in both cytosolic and plasma membrane fractions. Interestingly, in the 6-10 h preceding parturition, there is a substantial increase (+190%) of myometrial membrane-associated GRK activity. This is associated with an increase in membrane GRK2 immunoreactivity. Such alterations occur concomitantly with uncoupling of beta-AR, as assessed by quantification of high-affinity binding receptors. These data suggest that GRK activity increase may be one of the mechanisms underlying alterations in the coupling between beta-AR and adenylyl cyclase and may thus contribute to the initiation of myometrial contractions at term (Simon, 2001).

Effects of Mutation or Deletion

Adult viable mutations of decapentaplegic, or its putative receptor saxophone, cause homozygous females to produce short rounded eggs with abnormal anterior structures. A new female sterile mutation that effects G protein-coupled receptor kinase 2 function, fs(3)06936, has been isolated in a P element mutagenesis screen that causes similar effects on egg shape. Mature oocytes produced by homozygous fs(3)06936 females are slightly shorter and more rounded than wild type. Although positioned normally, dorsal appendages in fs(3)06936 eggs are generally shorter and broader than wild type, and the two appendages on a single egg chamber frequently differ in length. The operculum is oriented more vertically than in wild type, giving the eggs a 'square-ended' appearance, but the chorion within the operculum retains its distinctive appearance and the micropyle forms. Nurse cells often fail to completely transfer their contents into the oocyte, leaving residual material that may interfere with anterior end formation. Thus fs(3)06936 appears to affect specific aspects of egg formation without grossly altering the major pattern axes of the egg (Schneider, 1997).

Two additional ovarian defects have suggested that fs(3)06936 also functions at earlier stages of oogenesis. (1) Homozygous fs(3)06936 egg chambers degenerate during vitellogenic stages (stages 8-10A) much more frequently than expected. 26.8% of ovarioles from 4-day-old fs(3)06936 females contained a degenerating vitellogenic chamber compared to only 0.7% of wild-type ovarioles. (2) Egg chamber formation slows or ceases entirely within a significant number of fs(3)06936 ovarioles. 5.2% of the mutant ovarioles contained only 0-2 egg chambers instead of the 6-7 that are present in wild type. Germaria in such ovarioles are often smaller and thinner than in wild type, like the germaria of agametic ovarioles. Although cyst production normally declines in old females, 4-day-old wild-type females contained no similar ovarioles (Schneider, 1997).

The fs(3)06936 mutant exhibits additional defects that indicate roles for this gene outside of the ovaries. Homozygous fs(3)06936 females lay a small number of eggs, but those that are laid display a maternal effect that is partially rescued by zygotic fs(3)06936⁺ expression. 23.7% of embryos produced by homozygous females hatch when crossed to wild-type males, compared to only 10.3% following crosses to homozygous males. The unhatched eggs displayed a wide variety of defects including twisted gastrulation, fused adjacent segments, and perforated dorsal and ventral cuticle. These defects are more severe when the embryos lack both maternal and zygotic fs(3)06936⁺ function (Schneider, 1997).

G protein-coupled receptor kinase 2 is required for rhythmic olfactory responses in Drosophila

The Drosophila circadian clock controls rhythms in the amplitude of odor-induced electrophysiological responses that peak during the middle of night. These rhythms are dependent on clocks in olfactory sensory neurons (OSNs), suggesting that odorant receptors (ORs) or OR-dependent processes are under clock control. Because responses to odors are initiated by heteromeric OR complexes that form odor-gated and cyclic-nucleotide-activated cation channels, whether regulators of ORs were under circadian-clock control was tested. The levels of G protein-coupled receptor kinase 2 (Gprk2) messenger RNA and protein cycle in a circadian-clock-dependent manner with a peak around the middle of the night in antennae. Gprk2 overexpression in OSNs from wild-type or cyc⁰¹ flies elicits constant high-amplitude electroantennogram (EAG) responses to ethyl acetate, whereas Gprk2 mutants produce constant low-amplitude EAG responses. ORs accumulate to high levels in the dendrites of OSNs around the middle of the night, and this dendritic localization of ORs is enhanced by GPRK2 overexpression at times when ORs are primarily localized in the cell body. These results support a model in which circadian-clock-dependent rhythms in GPRK2 abundance control the rhythmic accumulation of ORs in OSN dendrites, which in turn control rhythms in olfactory responses. The enhancement of OR function by GPRK2 contrasts with the traditional role of GPRKs in desensitizing activated receptors and suggests that GPRK2 functions through a fundamentally different mechanism to modulate OR activity (Tanoue, 2008).

Many sensory systems are regulated by the circadian clock. Various insects including flies, moths, and cockroaches show circadian rhythms in odor-dependent electrophysiological and behavioral responses. In mammals, the firing rate of isolated mouse olfactory bulb neurons is regulated by the circadian clock, as are odor-evoked brain activity waves (e.g., event-related potentials [ERPs]) in humans. Daily rhythms in neuronal activity or sensitivity have been reported for other sensory systems, such as the visual and auditory systems (Tanoue, 2008).

The circadian clock modulates olfactory responses in Drosophila: Robust electroantennogram (EAG) responses are seen during the middle of the night, and weak EAG responses are seen during the middle of the day (Krishnan, 1999). These rhythms in EAG responses are controlled by the olfactory sensory neurons (OSNs) in Drosophila, which act as independent peripheral circadian oscillators (Tanoue, 2004). Colocalization of the circadian oscillator and a rhythmic output to the OSNs indicates that the abundance and/or activity of odorant receptors (ORs) and/or OR-dependent processes are under clock control. Drosophila ORs are seven-transmembrane-domain proteins that share some structural similarities with G protein-coupled receptors (GPCRs). However, recent studies demonstrate that Drosophila ORs have an inverted membrane topology compared to canonical GPCRs and function as odor-gated and cyclic-nucleotide-activated cation channels. To understand how the clock modulates odor-dependent responses, it was determined whether factors that modulate ORs were regulated by the circadian clock (Tanoue, 2008).

G protein-coupled receptor kinases (GPRKs) and arrestins act to terminate GPCR signaling in mammals, thereby protecting cells from receptor overstimulation. GPRK-phosophorylated GPCRs are internalized by arrestin and subsequently degraded or recycled. Two Gprk genes, Gprk1 and Gprk2, have been reported in Drosophila. Gprk1 messenger RNA (mRNA) is enriched in photoreceptor cells, and expression of a Gprk1 dominant-negative mutant in photoreceptors increases the amplitude of electroretinogram (ERG) responses (Lee, 2004). Gprk2 is required for egg and wing morphogenesis, as well as embryogenesis in Drosophila (Molnar, 2007; Schneider, 1997). In mammals, seven Gprk genes are divided into three subfamilies on the basis of sequence homology: the rhodopsin kinase or visual Gprk subfamily (Gprk1 and Gprk7), the β-adrenergic receptor kinase subfamily (Gprk2 and Gprk3), and the Gprk4 subfamily (Gprk4, Gprk5, and Gprk6). Gprk3 knockout mice are unable to mediate odor-induced desensitization of odorant receptors (Peppel, 1997). In contrast, loss of Gprk2 function in C. elegans olfactory sensory neurons results in reduced chemosensory behavior, suggesting that Gprk2 is necessary for GPCR signaling (Fukuto, 2004). These results suggest that GPRKs play different roles in vertebrate and invertebrate olfaction (Tanoue, 2008).

This study reports that Gprk2 expression is regulated by circadian clocks in antennae and that GPRK2 drives circadian rhythms in olfactory responses by enhancing OR accumulation in the dendrites of basiconic sensilla. Gprk2 mRNA and protein expression levels were high around the middle of the night, coincident with the peak of olfactory responses. Flies that overexpress Gprk2 in OSNs show constant high EAG responses to ethyl acetate during 12 hr light:12 hr dark (LD) cycles and accumulate high levels of ORs in OSN dendrites, whereas hypomorphic Gprk2 mutants show constant low EAG responses to ethyl acetate during LD. On the basis of these results, it is proposed that GPRK2 mediates cycles of OR accumulation in OSN dendrites to generate rhythms in EAG responses (Tanoue, 2008).

Gprk2 is a circadian output gene whose mRNA and protein peak during the middle of the night in antennae. This phase of mRNA expression is similar to that of per, tim, and other genes driven directly by CLK-CYC binding to E-box regulatory sequences. However, CLK-CYC-dependent genes are expressed at constitutively high levels in per⁰¹ and tim⁰¹ mutants and constitutively low levels in Clk^Jrk and cyc⁰¹ mutants, whereas Gprk2 is expressed at low levels in per⁰¹, tim⁰¹, and cyc⁰¹ mutants. Several rhythmically expressed transcripts identified by microarray analysis of heads have low levels of expression in per⁰¹ and Clk^Jrk mutants, but the mechanism governing their rhythmic expression has not been explored (Tanoue, 2008).

Analysis of arrhythmic clock mutants indicates that cycling levels of Gprk2 mRNA give rise to rhythms in GPRK2 protein. The levels of GPRK2 protein cycle in phase with Gprk2 mRNA and correspond to rhythms in EAG responses. Other kinases such as Double-time (Dbt), Shaggy (Sgg)/GSK3, and Casein kinase 2 (CK2) in Drosophila are constitutively expressed proteins, whereas GPRK2 is the first example of a rhythmically expressed kinase. However, other kinases such as Erk-MAP kinase and Calcium/calmodulin-dependent protein kinase II in the chicken retina are rhythmically activated due to phosphorylation and control cGMP-gated ion channels in cone photoreceptors (Tanoue, 2008).

Cycling levels of GPRK2 are coincident with rhythms in EAG responses; GPRK2 levels and EAG responses peak around the middle of the night and are at their lowest levels during the middle of the day. When GPRK2 levels are constitutively low, as in per⁰¹, tim⁰¹, and cyc⁰¹ mutants, EAG responses are also low. In addition, levels of GPRK2 are at or below the normal wild-type trough in Gprk2⁶⁹³⁶ and Gprk2^EY09213 mutants and generate EAG responses that are at or below those at the wild-type trough. GPRK2 levels are barely detectable in Gprk2^pj1 antennae. These results suggest that Gprk2^pj1 is not a null allele and raise the possibility that a Gprk2 null mutant will lack EAG responses altogether. Such a result would demonstrate that Gprk2 is required for olfactory responses per se. In contrast, constitutive overexpression of Gprk2 produces constant high EAG responses in both wild-type and cyc⁰¹ flies, demonstrating that high levels of GPRK2 can effect high amplitude EAG responses independent of other clock-dependent factors. Taken together, these results argue that Gprk2 levels control the amplitude of EAG responses. If so, this would imply that low levels of GPRK2 present in the Gprk2⁶⁹³⁶ mutant do not cycle in abundance (Tanoue, 2008).

Given that GPRK2 levels regulate the amplitude of EAG responses, what is the mechanism through which GPRK2 controls EAG response amplitude? The traditional targets of GPRKs are GPCRs. In the mammalian olfactory system, GPRK3 desensitizes ORs by triggering their internalization. These results suggest that Drosophila Gprk2 is necessary for EAG responses, and, taken together with C. elegans Gprk2 function, they indicate that GPRKs play a different role in invertebrate olfaction than in vertebrate olfaction. The subcellular localization of ORs is high in dendrites of basiconic sensilla at ZT17 and low at ZT5, but the abundance of ORs in these dendrites at ZT5 can be driven to high levels by increasing GPRK2 expression. These results support a model in which the circadian clock generates a rhythm of Gprk2 expression, which in turn generates rhythms in the amplitude of EAG responses by promoting OR accumulation (and consequently odor-gated cation-channel formation) in OSN dendrites from basiconic sensillae. GPRK2-dependent rhythms in the amplitude of spontaneous spikes are also seen in OSNs, thus demonstrating that the clock controls basic (i.e., odor-independent) properties of the OSN membrane. It is possible that the rhythmic localization of odor-gated cation channels to OSN dendrites accounts for rhythms in the amplitude of spontaneous spikes. The results can't exclude the possibility that cyclic expression of other genes also contribute to rhythms in EAG responses. mRNA cycling was tested for several genes that could potentially modulate EAG responses, including arrestin 2, Gprk1, and kurtz arrestin; arrestin 2 mRNA levels cycle, but neither Gprk1 or kurtz arrestin mRNA levels cycle. Given that microarray analysis was done on fly heads depleted of antennae, microarray analysis of antennae may reveal other rhythmically expressed genes that contribute to EAG rhythms (Tanoue, 2008).

Myc-tagged ORs did not accumulate to high levels in the dendrites of trichoid sensilla at ZT17. Trichoid sensilla have different functions than basiconic sensilla; T1 trichoid sensilla detect the pheromone 11-cis-vaccenyl acetate (cVA), whereas the basiconic sensilla recognize food and plant odors. It could be that the circadian clock regulates OSN activity differently in basiconic sensilla and trichoid sensilla, although the possibility cannot be excluded that detection of Myc-tagged ORs in dendrites failed because of low expression levels in trichoid sensillae, poor permeability of Myc antibody into trichoid sensilla, or the long, thin geometry of trichoid sensillae (Tanoue, 2008).

In summary, Drosophila Gprk2 mRNA and protein expression is under clock control in antennae. The levels of GPRK2 protein determine the amplitude of EAG responses to ethyl acetate in basiconic sensillae; high levels generate high-amplitude EAGs, and low levels produce low-amplitude EAGs. This result suggests that GPRK2 directly or indirectly enhances OR activity, in contrast to the inhibition of olfactory signaling by Gprk3 in mammals. Given that the most severe Drosophila Gprk2 mutant still produces low-amplitude EAG responses, a complete loss of Gprk2 function may lack EAG responses altogether and be required for olfaction. High levels of GPRK2 enhance OR localization to dendrites of basiconic sensillae and support a model in which rhythms in GPRK2 levels drive rhythms in OR localization to dendrites that ultimately mediates rhythms in EAG responses (Tanoue, 2008).

Gprk2 adjusts Fog signaling to organize cell movements in Drosophila gastrulation

Gastrulation of Drosophila melanogaster proceeds through sequential cell movements: ventral mesodermal (VM) cells are induced by secreted Fog protein to constrict their apical surfaces to form the ventral furrow, and subsequently lateral mesodermal (LM) cells involute toward the furrow. How these cell movements are organized remains elusive. This study observed that LM cells extend apical protrusions and then undergo accelerated involution movement, confirming that VM and LM cells display distinct cell morphologies and movements. In a mutant for the GPCR kinase Gprk2, apical constriction expands to all mesodermal cells and the involution movement is abolished. In addition, the mesodermal cells halt apical constriction prematurely in accordance with the aberrant accumulation of Myosin II. Epistasis analyses revealed that the Gprk2 mutant phenotypes are dependent on the fog gene. Overexpression of Gprk2 suppresses the effects of excess Cta, a downstream component of Fog signaling. Based on these findings, it is proposed that Gprk2 attenuates and tunes Fog-Cta signaling to prevent apical constriction in LM cells and to support appropriate apical constriction in VM cells. Thus, the two distinct cell movements in mesoderm invagination are not predetermined, but rather are organized by the adjustment of cell signaling (Fuse, 2013).

In the Gprk2 mutant embryos, cell movements triggered by Fog signaling were compromised. fog is genetically epistatic to Gprk2, indicating that Gprk2 functions by acting on Fog signaling. LM cells undergo apical constriction in the Gprk2 mutant, suggesting that Gprk2 normally inhibits Fog signaling in LM cells. Premature termination of apical constriction and abnormal accumulation of Myosin were also observed in the Gprk2 mutant, suggesting that Gprk2 adjusts Fog signaling to an appropriate level in VM cells. Thus, Gprk2 regulates Fog signalingin a cell group-dependent manner. But what are the underlying molecular mechanisms (Fuse, 2013)?

It is known that GPCR kinase phosphorylates the C-terminal region of GPCR, and regulates GPCR signaling by multiple mechanisms. The phosphorylated GPCR dissociates from the G protein and is internalized from the plasma membrane. This produces a negative-feedback loop for GPCR signaling. Theoretically, the negative-feedback loop stabilizes the signaling and generates biphasic output from fluctuating inputs: OFF for low inputs and ON for high inputs. It is speculated that Gprk2 might phosphorylate a GPCR and might generate biphasic output for Fog signaling in a spatial manner: OFF in LM cells and ON in VM cells. The Fog receptor is expected to be a GPCR, since a G protein (Cta) functions downstream of Fog. Identification of the Fog receptor would help to clarify the molecular functions of Gprk2 (Fuse, 2013).

The kinase activity of Gprk2 is essential for gastrulation. Although it is not yet known what substrates are phosphorylated by Gprk2 in this process, one might be Gprk2 itself because it was observed that Gprk2 protein was phosphorylated in S2 cultured cells and that the phosphorylation was abolished in the K338R mutant of Gprk2. Autophosphorylation of other GPCR kinases has been demonstrated previously and is thought to stimulate their binding to GPCR. Autophosphorylation of Gprk2 might play a similar role (Fuse, 2013).

In addition to its kinase activity, GPCR kinase has an RGS domain, which exhibits GAP (GTPase activating protein) activity and functions in recycling of the Gα protein. Therefore, whether Gprk2 exhibits GAP activity for Cta is an intriguing issue. Indeed, this possibility was supported by genetic data showing that Gprk2 suppresses the effect of Cta overexpression, but not that of Cta Q303L, the GTP-bound form of Cta protein. Cta Q303L might not be subject to the inhibitory effect (GAP activity) of Gprk2, although the alternative explanation has not been ruled out that the inhibition of Cta Q303L might require more Gprk2 protein than does the inhibition of wild-type Cta. Considering that GPCR kinase regulates GPCR signaling by multiple mechanisms, it is suggested that the repression of Cta activity might be one of several means by which Gprk2 regulates Fog signaling (Fuse, 2013).

Fog signaling stimulates the apical localization of Myosin, which generates a force to constrict the apical cell surface. In the wild-type embryo, Myosin protein appears and disappears at the apical surface in a dynamic pattern that they described as 'pulsed coalescence'. In the Gprk2 mutant, Myosin continued to accumulate on the entire apical surface of mesodermal cells. Similar phenomena were also observed in Cta-overexpressing ectodermal cells, and this phenotype was suppressed by simultaneous expression of Gprk2. It is suggested that Gprk2 normally attenuates Fog-Cta signaling to an appropriate level, and such refinement might contribute to controlling the dynamics of Myosin protein (Fuse, 2013).

Previous studies showed that Gprk2 acts in Hedgehog (Hh) signaling for imaginal disc patterning. In this process, Gprk2 phosphorylates a GPCR, Smoothened, and potentiates Hh signaling. Thus, Gprk2 plays roles in multiple signaling pathways in various contexts during development (Fuse, 2013).

The movements of LM cells were characterized, and were found to extended apical protrusions. Some examples have been documented of the extension of protrusions by epithelial cells, such as dorsal ectodermal cells of embryos and wing disc cells of larvae in Drosophila. However, the mechanisms that induce the protrusion and the roles of protrusion in directional cell movement are not understood. Since it was observed that apical protrusions in LM cells always pointed toward the ventral furrow and that cells close to the furrow extended longer protrusions than cells distant from it, it is speculated that the apical protrusion might be induced by the apically constricting neighbors. Indeed, in cta mutant embryos the apical protrusions did not always point toward mid-ventral, but rather frequently pointed toward the slight depressions that were formed at random positions by uncoordinated apical constriction. One possibility is that mechanical or chemical signals that emanate from apically constricting cells might induce apical protrusions in surrounding cells (Fuse, 2013).

Apical protrusions became apparent when LM cells started to accelerate toward the ventral furrow. From this observation, it is supposed that the directional protrusion might contribute to the movement of LM cells. Given that the apical protrusion of LM cells is analogous to the pseudopod of cultured cells, the apical protrusion might act as a scaffold for pulling the cell body into the furrow. The fact that the apical protrusion was also observed in some ectodermal cells, which never undergo involution movement, suggests that the apical protrusion is not sufficient to induce involution movement and that other mechanisms might regulate the cell movement in parallel (Fuse, 2013).

Drosophila mesoderm invagination is driven by sequential movements of different cells. The apical constriction of VM cells is one of the essential movements in this process. It is expected that the involution movement of LM cells might be another of the cell movements driving mesoderm invagination. The movements of different cells would probably influence each other in a complex manner. Observations of LM movements might be explained by such a coordination of cell movements. For example, the apical constriction of VM cells might stretch LM cells and thereby prevent LM cell apical constriction, as previously suggested. VM cells might then continue to move inward and pull LM cells toward the ventral furrow. In addition, ectodermal cells might generate a force to push mesodermal cells inward. These possibilities are not mutually exclusive. Further analyses are required to clarify the role of each cell movement and the effect of coordinated movements in mesoderm invagination (Fuse, 2013).

In the Gprk2 mutant, LM cells underwent apical constriction instead of involution movement. Given the inhibition of Fog signaling in the wild-type LM cells, involution movement might be a default state of mesodermal cells without Fog signaling. As noted above, in the fog and cta mutant, apical constriction occurs in some VM cells, and involution-like movement operates in an uncoordinated manner. These uncoordinated cell movements finally result in disorganized, but nearly complete, mesoderm invagination. Thus, apical constriction and involution movements seem to be alternative choices for mesodermal cells, and robust mesoderm invagination might progress via either type of cell movement. In normal Drosophila embryos, cell movements are spatially and temporally organized, and such organization might ensure the correct shape of gastrulae (Fuse, 2013).

Cell movements in gastrulation show diversity among insects. For example, mosquito embryos undergo only apical constriction and no apparent involution process. Locust embryos undergo neither apical constriction nor involution, but instead utilize the delamination of individual mesodermal cells. Compared with gastrulation in these insects, Drosophila gastrulation is a more complex process and is completed within a shorter time (15 minutes compared with hours). The highly organized cell movements in Drosophila might enable this rapid completion of gastrulation. The molecular mechanisms underlying the evolution of insect gastrulation are an intriguing issue for future studies (Fuse, 2013).

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Search PubMed for articles about Drosophila G protein-coupled receptor kinase 2

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date revised: 20 December 2013

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