Sleep is vital for most animals, yet its mechanism and funwction remain unclear. This study found that permeability of the BBB (blood-brain barrier)-the organ required for the maintenance of homeostatic levels of nutrients, ions, and other molecules in the brain-is modulated by sleep deprivation (SD) and can cell-autonomously effect sleep changes. Increased BBB permeability was observed in known sleep mutants as well as in acutely sleep-deprived animals. In addition to molecular tracers, SD-induced BBB changes also increased the penetration of drugs used in the treatment of brain pathologies. After chronic/genetic or acute SD, rebound sleep or administration of the sleeping aid gaboxadol normalized BBB permeability, showing that SD effects on the BBB are reversible. Along with BBB permeability, RNA levels of the BBB master regulator moody are modulated by sleep. Conversely, altering BBB permeability alone through glia-specific modulation of moody, g&slpha;o, loco, lachesin, or i>neuroglian -each a well-studied regulator of BBB function-was sufficient to induce robust sleep phenotypes. These studies demonstrate a tight link between BBB permeability and sleep and indicate a unique role for the BBB in the regulation of sleep (Axelrod, 2023).

This study establish the BBB as a sleep regulatory center in Drosophila. It was observed that the BBB dynamically and rapidly—both functionally and molecularly—responds to acute as well as chronic-genetic SD and that altering BBB function alone, through mutations of the moody signaling pathway, can affect sleep, suggesting that the BBB senses sleep need and responds to it by altering its permeability. The correct BBB permeability state seems to be required for normal sleep, as disrupting BBB function causes sleep loss (Axelrod, 2023).

In normal physiology, the BBB controls influx and efflux of molecules in and out of the brain, including ions, amino acids, neurotransmitters, vitamins, proteins, carbohydrates, lipids, and hormones. Transport is facilitated via various mechanisms which include active transporters, transcytosis, and passive diffusion. The function of the BBB is not static but is affected by circadian rhythms, neuronal activity, and the metabolism. the data add sleep as an additional parameter modulating BBB function, as has been suggested by studies in mammals, and this study found that the relationship between the BBB and sleep is reciprocal. The results show that the BBB rapidly and dynamically responds to SD by increasing its permeability and that permeability increases conversely cause sleep loss. It is conceivable that BBB permeability changes allow the passage of one or multiple molecules that affect sleep into or out of the brain. Defective xenobiotic exclusion of steroid hormones from the brain via the BBB has been shown to affect sleep in Drosophila, demonstrating that molecules outside the CNS can affect sleep. Future studies may determine whether the BBB mediates a rapid global switch from a sleeping to an awake brain, for example, by controlling ionic diffusion and potassium concentrations, which could broadly raise neuronal activation thresholds and lead to the observed extracellular ionic changes in the sleeping mammalian brain (Axelrod, 2023).

The BBB creates a highly controlled microenvironment in the brain, perturbations of which affect neuronal function, as evidenced by neuropathologies at least in part caused by BBB disruption including stroke, Alzheimer's, Multiple Sclerosis, epilepsy and brain tumor metastasis. BBB disruption also occurs during normal aging, under chronic stress and during systemic inflammation and infection; however, whether the relationship between BBB disruption and these pathologies is causal or merely correlative is less clear. The magnitude of BBB changes in disease states can be an order of magnitude larger than the observed changes after sleep loss described in this study, illustrating the physiological relevance of these experiments. All of the pathologies listed above are also marked by sleep disruption, which in some instances increases the risk for developing the disease in the first place and the severity of which correlates with disease severity. The fly ortholog of a human autism gene has been reported to act in the BBB and elicit sleep fragmentation via developmental hyperserotonemia. However, a direct involvement of the BBB in sleep regulation had not been shown until this present study (Axelrod, 2023).

BBB and sleep disruptions have been, so far independently, associated with causing illness, and this study provides additional evidence for the notion that the two processes are linked. If BBB permeability and sleep regulation are fundamentally connected as part of the same system, it will be interesting to study how they relate to disease risk, development, and prognosis, and whether interventions that affect one also augment the other (Axelrod, 2023).

Adequate drug delivery to the brain is a problem plaguing the treatment of many neuropathologies. This puts strong importance on finding possible regulatory sites for controlling the BBB as a means of finding better ways to deliver drugs to the brain. The BBB has distinct mechanisms to exclude molecules from the brain including xenobiotic transporters in the cell membrane that efflux most small hydrophobic molecules taken up transcellularly through the membrane, and the paracellular barrier formed by junctions between the BBB forming cells. The latter creates a diffusion barrier for hydrophilic molecules in a size-dependent manner with a maximum passage size of about 0.5 kDa. Due to these mechanisms, the BBB poses a major obstacle to treatment of brain pathologies, because 100% of large molecules (>0.5 kDa), like the brain cancer drug vinblastine (molecular weight: 1 kDa), and 98% of small molecule drugs (<0.5 kDa), like the non-steroidal anti-inflammatory drug paracetamol, cannot penetrate it in therapeutic amounts. Current solutions to this problem include pharmacologically increasing the diffusion barrier permeability, implantation of ultrasonic devices to temporarily permeabilize the diffusion barrier, fusing molecules to receptor ligands to engage the transcellular route, or intracranial administration via injection. Provided the observed effects are conserved in humans, a patient's sleep and nighttime administration of drugs could have a significant effect on drug penetration into the CNS and possibly treatment outcomes (Axelrod, 2023).

In Drosophila, the BBB's subperineurial glia form a layer of polarized epithelial cells, with septate junctions between them creating a seal for water-soluble molecules over 500 Da. The observation that SD rapidly induces increases in BBB permeability raises the question: What are the molecular and cellular mechanisms that underlie the remodeling of septate junctions on the scale of hours? The Moody GPCR pathway has been shown to play a key role in the development of the epithelial barrier, as well as its maintenance in adulthood. Moody activates a signaling cascade which, via PKA activation, controls the developmental assembly of the septate junction belt in a highly coordinated spatiotemporal manner that involves rearrangements of the actin cytoskeleton and vesicular trafficking. This study has shown that RNAi-based suppression of moody pathway genes causes increased BBB permeability. Strong downregulation of moody was observed in every genetic sleep mutant tested. These findings suggest that altered moody pathway function may be primarily responsible for the leaky barriers of these mutants (Axelrod, 2023).

During SD and recovery, it is conceivable that similar processes occur. Indeed, it has been shown that perturbation of endocytosis at the Drosophila BBB alters sleep, possibly interfering with Moody-activated remodeling during sleep/wake cycles. Scute SD has been shown to rapidly depress moody expression in wild-type Drosophila, which is accompanied by increased BBB permeability. Moody is expressed on the neuronal-facing apical cell surface of the subperineurial glia. While its ligand is not known, this topology could permit neuronal states related to sleep to be communicated to glia via this GPCR pathway. As many known sleep genes in Drosophila have been shown to be expressed in neurons and this study observed BBB changes upon sleep-suppressing neuronal activation, neuron-glia signaling seems likely to be part of the process, and it will be interesting to elucidate the involved signaling molecules including Moody's ligand (Axelrod, 2023).

The two-step model of sleep regulation posits that circadian rhythm and sleep homeostasis control the timing of sleep. Sleep homeostasis is a process that controls sleep duration to meet an organism's daily sleep need. Rebound sleep after SD is interpreted as proof of the existence of sleep homeostasis. The physiological and cellular nature of the sleep homeostat is-like the nature of sleep itself-the subject of current research. Sleep homeostasis has been shown to be affected by oxidative stress, neuronal, glial, and microglial activity, neuropeptide signaling, infection, the circadian clock, temperature, and starvation. Synaptic downscaling, glymphatic clearance of toxins, peripheral glucose homeostasis, reactive oxygen species clearance, and inhibition of motor function have been proposed as processes occurring during sleep homeostasis, constituting functions of sleep. As at least some of these processes must involve yet to be identified body-brain signals, future studies will reveal how the BBB changes shown here affect known or novel functions of sleep (Axelrod, 2023).

Together the data indicate a system where an increase of sleep need is measured by the BBB, resulting in an adjustment of Moody levels, an opening of the BBB, and the initiation of rebound sleep. This process is likely to involve neuronal to BBB signaling (dopaminergic SD also alters the BBB, and many of the sleep mutants are expressed in neurons) as well as yet-to-be-characterized molecular exchange across the BBB (Axelrod, 2023).

REGULATION

Transcriptional Regulation

To test whether CNS glia expression of loco depends on the function of pointed, beta-Galactosidase expression directed by the rC56 enhancer was assayed in a pointed mutant background. Initially expression directed by the rC56 element at early stage 12 in the progeny of the longitudinal glioblast appears to be slightly weaker in the mutant. During later stages of CNS development, a reduction in the expression level as well as in the number of cells expressing the rC56 reporter gene can be detected. In stage 16 mutant embryos, the activity of the rC56 enhancer is most prominently reduced or absent in the longitudinal glial cells, whereas A and B glial cells and the VUM glial cells appear relatively unaffected. The influence of pointed on the rC56 enhancer is less pronounced in the posteriormost 2-3 neuromeres. Furthermore, the rC56 reporter can be ectopically activated by ectopic expression of pointed P1. This suggests that the lacZ gene located in the rC56 P-element is under at least partial control of a pointed-dependent genomic enhancer element (Granderath, 1999).

In Drosophila, lateral glial cell development is initiated by the transcription factor encoded by glial cells missing. gcm activates downstream transcription factors such as repo and pointed, which subsequently control terminal glial differentiation. The gene loco has been identified as a potential target gene of pointed and is involved in terminal glial differentiation. It encodes an RGS domain protein expressed specifically by the lateral glial cells in the developing embryonic CNS. The loco promoter and the control of the glial-specific transcription pattern has been analyzed. Using promoter-reporter gene fusions, a 1.9 kb promoter element capable of directing the almost complete loco gene expression pattern has been identified. Sequence analysis suggests the presence of Gcm and Pointed DNA binding sites. Following in vitro mutagenesis of these sites their relevance in vivo has been demonstrated. The expression of loco is initially dependent on gcm. During subsequent stages of embryonic development Gcm and Pointed appear to activate loco transcription synergistically. In addition, at least two other factors appear to repress loco expression in the ectoderm and in the CNS midline cells (Granderath, 2000).

Two alternative modes are presented as to how loco transcription might be regulated. In the simple model, a linear array of transcriptional regulators results in the correct expression of loco. gcm acts on top of this cascade and activates pointed, which in turn leads to glial-specific loco expression. Alternatively, loco gene activation might be biphasic. Initially gcm concomitantly activates both loco and pointed. In a second phase, gcm and pointed act synergistically on the loco promoter to mediate high levels of glial-specific loco expression. The data favour the latter model (Granderath, 2000).

The 1.9 kb Rrk promoter element is capable of directing expression of a lacZ reporter in the complete loco expression domain. The Rrk fragment itself appears to contain more than one crucial regulatory element. The US1 construct, which overlaps the Rrk fragment, which harbors two gcm binding sites located in the 5' part of the Rrk fragment, directs glial expression resembling the expression of loco in a pointed mutant background. The 3' sequences of the Rrk fragment are found in the Nrk fragment. This promoter fragment, which harbours one gcm and one pointed binding site, is not able to confer any glial expression. Only the complete Rrk fragment is able to direct the entire loco transcriptional profile, pointing to synergistic effects of proteins binding to the 3' and 5' portions of the Rrk element. This notion is supported by the observation that pointed cannot activate the Rrk element when both Gcm binding sites GBS1 and GBS3 are deleted. Ectopic expression of either gcm or pointed alone within the neuroectoderm leads to sporadic activation of the Rrk enhancer, suggesting the presence of both gcm and pointed responsive elements. Coexpression of gcm and pointed in the rhomboid expression pattern shows two interesting results: (1) it is evident that cells within neuroectoderm activate the Rrk reporter fragment very strongly, showing that the two transcription factors act synergistically; (2) it is important to note that although comparably high levels of gcm and pointed are found in the CNS midline and the mesodermal cells, they never activate the Rrk reporter (Granderath, 2000).

Coexpression of gcm and pointed can also direct expression of the Nrk reporter. Within the Nrk fragment only one Gcm and one Pointed binding site are found, 370 bp apart. gcm is the master regulatory gene controlling lateral glial cell development. The gene pointed is not expressed in mutant gcm embryos suggesting that pointed expression depends on gcm. However, only coexpression of pointed and gcm leads to an efficient activation of the Rrk enhancer, indicating that gcm can not efficiently activate pointed transcription in the neuroectoderm. Despite the fact that pointedP1 is thought to act as a transcriptional activator it appears that cofactors such as gcm are required to allow full activation. This observation parallels results obtained in vertebrate systems, where it has been suggested that the binding of cofactors is a mechanism to relieve auto-inhibition of ETS proteins. pointed is expressed in many tissues during development and activates very different sets of genes (e.g., depending on the cells in which pointedP1 is expressed it activates tracheal, epidermal, neuronal or glial development). Thus, interaction with different tissue-specific coactivators might be an important step in selecting the appropriate downstream target genes (Granderath, 2000). Direct coactivation of glial target genes by both gcm and pointedP1 is possibly not confined to loco; the analysis of a second pointed-dependent enhancer element has revealed the presence of putative binding sites for both gcm and pointed (Granderath, S. and Klambt, C., unpublished data cited in Granderath, 2000).

The synergistic activation of loco by Gcm and Pointed could suggest that Pointed might be able to recruit or stabilize Gcm at the regulatory regions of terminal differentiation genes. This would lead to an increased expression of the respective genes but concomitantly could also disrupt the positive auto-regulatory feedback loop found for the gcm gene. This would provide a possible mechanism as to how the positive auto-regulation of gcm is terminated. How loco expression is maintained in vivo remains to be addressed (Granderath, 2000).

Terminal differentiation of glial cells is controlled by pointed. Two different isoforms are generated from the pointed locus, PointedP1 and PointedP2. They share the DNA binding domain and during embryonic CNS development they are expressed in the lateral glia (PointedP1) or the midline glia (PointedP2). Despite the common DNA binding activity, the two factors activate non-overlapping sets of target genes in the different glial cell types. The mechanism by which the selection of glial PointedP1 and PointedP2 target genes occurs appears to be complex. A simple model would be to postulate that specific, as yet unidentified cofactors are expressed either in the neuroectoderm or the CNS midline cells. However, in the midline, PointedP2 function can be substituted by PointedP1. This might be explained by postulating that PointedP1 is able to interact with a pointedP2 coactivator. Besides Gcm, additional factors appear to be required to specify PointedP1 target genes, because the coexpression of PointedP1 and Gcm in the CNS midline is not sufficient to evoke any Rrk reporter gene expression. Alternatively, the discrimination of PointedP1 and PointedP2 target genes might be mediated by transcriptional repressors. Two such proteins are known to be expressed in the CNS midline: Single minded and Abrupt. No potential Single minded binding sites were found in the Rrk construct. One potential Abrupt binding site (CTTAATTAA at position 1537-1547 of the Rrk fragment) was predicted by DNA sequence analysis. However, disruption of this site does not alter the reporter gene expression directed by the Rrk fragment in vivo. Thus, if Abrupt directly acts on the lococ1 promoter, it must bind to a different site in the Rrk fragment. Abrupt apparently represses Rrk-mediated expression (and possibly expression of other gcm-dependent genes) only in the apodemata, which might explain the muscle attachment defects observed in abrupt mutant embryos. In the CNS midline, however, the function of abrupt is not required for the repression of loco. Thus, additional experiments are required to determine which mechanisms are used in vivo to discriminate between lateral and midline glial gene expression (Granderath, 2000).

The Drosophila gene dead ringer (dri) [also known as retained (retn)] encodes a nuclear protein with a conserved DNA-binding domain termed the ARID domain (AT-rich interaction domain). dri is expressed in a subset of longitudinal glia in the Drosophila embryonic central nervous system and dri forms part of the transcriptional regulatory cascade required for normal development of these cells. Analysis of mutant embryos reveals a role for dri in formation of the normal embryonic CNS. Longitudinal glia arise normally in dri mutant embryos, but they fail to migrate to their final destinations. Disruption of the spatial organization of the dri-expressing longitudinal glia accounts for the mild defects in axon fasciculation observed in the mutant embryos. The axon phenotype includes incorrectly bundled and routed connectives, and axons that sometimes join the wrong bundle or cross from one tract to another. Consistent with the late phenotypes observed, expression of the glial cells missing (gcm) and reversed polarity (repo) genes was found to be normal in dri mutant embryos. However, from stage 15 of embryogenesis, expression of locomotion defects (loco) and prospero (pros) was found to be missing in a subset of LG. This suggests that loco and pros are targets of Dri transcriptional activation in some LG. It is concluded that dri is an important regulator of the late development of longitudinal glia (Shandala, 2003).

What is the molecular basis of the mutant phenotype found in dri mutants? Dri is a transcription factor, so the link between loss of dri function and the failure to differentiate properly is likely to be indirect, mediated through misregulation of dri targets required for normal longitudinal glial development. The most informative data came from an analysis of the position of dri in the glial transcriptional regulatory cascade. In general terms, dri activity was found to be downstream of gcm and repo, and independent of pnt and cut. It was also found to be upstream of two genes, loco and pros, which are essential for normal development of some glial cells. In this developmental context dri acts as an activator of downstream targets (Shandala, 2003).

The requirement for Dri in the activation of loco is unexpected. loco has been found to be a transcriptional target of Pnt but not of Repo, while dri expression depends on Repo and not on Pnt. It is possible that expression of loco is co-dependent on Pnt and Dri in some cells and that the reduced level of dri expression observed in repo mutants is enough to permit loco expression (Shandala, 2003).

The genetic analysis presented here strengthens the hypothesis that there are different genetic controls for different subsets of dorsal glia. For example, dri expression in all glial cells requires GCM activation, but only some of them requires Repo. The Repo-independent dri-positive cells, two per hemineuromere, appear to correspond to the A and B subperineural glia (A/B SPG). These derive from neuroglioblast NB1.1, suggesting that Repo is required for the expression of dri only in cells derived from the lateral glioblasts. Unlike dri, pnt and its downstream target loco are not expressed in the medialmost cell body glia, which do not have a lateral glioblast origin. This suggests that there are different pathways for pnt and dri induction downstream of gcm (Shandala, 2003).

At least some of these hierarchical transcriptional interactions may explain the phenotypes observed. The axon and mild positional defects of glia in dri mutants resemble phenotypes of other known late gliogenesis factors, such as those observed in pnt, repo, loco or pros embryos. It is known that early distribution of the glycoprotein Neuroglian is perturbed in pros mutant embryos. loco encodes a regulator of G-protein signalling (RGS) that has been shown to bind to a Gαi-subunit and could regulate a G-protein signalling pathway involved in LG migratory behavior. In addition, expression of the Drosophila FGF receptor Heartless in LG, and similarities between the loco and heartless mutant phenotypes, leaves open the possibility that FGF could trigger final migration of glia along the longitudinal connectives. This hypothesis is strengthened by the recent finding that subcellular redistribution of Neuroglian from the plasma membrane to cytoplasm, which normally happens during final glial migration to enwrap axon bundles, is disrupted in heartless mutants. Alternatively, it remains possible that additional targets of dri mediate the role of this gene in longitudinal glial differentiation (Shandala, 2003).

These studies add dri to the list of genes, including pnt, repo, loco and pros, that exhibit phenotypes that are much milder than those of the gcm, glide2 and Drop/Ltt genes at the head of the dorsal glia hierarchy. It appears that diversification of these downstream regulators produces different types of glial cells. Nonetheless, each plays an essential role in driving the required behavior of glial cells during CNS development. In the case of the Dri transcription factor, this role includes fine tuning the cell shape and migration characteristics of longitudinal glia that enable them to establish a normal axon scaffold (Shandala, 2003).

Protein Interactions

RGS domains directly interact with G-protein alpha subunits, displaying a remarkable degree of specificity (De Vries, 1995; Berman, 1996; Druey, 1996; Hunt, 1996; Watson, 1996). If Loco indeed functions as a regulator of G-protein signalling, the presence of a G-protein would be anticipated in the lateral glial cells. The expression of Galphas, Galphai and Galphao RNAs was examined in the embryonic nerve cord: the Galphai subunit appears to be specifically expressed in the glial cells (Wolfgang, 1991). Further evidence for the interaction of Loco and Galphai was found in a yeast two-hybrid screen. A cDNA clone of Drosophila Galphai was used as ‘bait’ for interacting proteins. The Gai gene was fused in frame at its N terminus to a gene encoding a LexA DNA-binding domain. Yeast that express this fusion were transformed with a library carrying Drosophila cDNAs fused to a gene for a transcriptional activation domain. Clones that encoded putative Galphai-interacting proteins were identified by the ability of the transformed yeast colonies to express a LEU2 gene that contained LexA-dependent regulatory elements and the interaction was confirmed by reintroducing the putative positive clones into yeast that carried the LexA-Gai fusion. Six non-overlapping sets of interacting clones have been identified. Four non-identical loco clones have been recovered, with C-terminal fragments of various lengths fused to the lexA gene. The longest fragment begins at residue 443 of the predicted Loco c2 protein and includes the RGS domain; the shortest encodes only 199 amino acid residues, those that extend C-terminal from residue 977 of the predicted Loco c2 protein and includes the final 43 amino acids of the conserved region D closest to the C terminus. Thus LOCO appears to be an RGS domain protein specific for Galphai (Granderath, 1999).

Locomotion defects, together with Pins, regulates heterotrimeric G-protein signaling during Drosophila neuroblast asymmetric divisions

Heterotrimeric G proteins mediate asymmetric division of Drosophila neuroblasts. Free Gßgamma appears to be crucial for the generation of an asymmetric mitotic spindle and consequently daughter cells of distinct size. However, how Gßgamma is released from the inactive heterotrimer remains unclear. This study shows that Locomotion defects (Loco) interacts and colocalizes with Galphai and, through its GoLoco motif, acts as a guanine nucleotide dissociation inhibitor (GDI) for Galphai. Simultaneous removal of the two GoLoco motif proteins, Loco and Pins, results in defects that are essentially indistinguishable from those observed in Gß13F or Ggamma1 mutants, suggesting that Loco and Pins act synergistically to release free Gßgamma in neuroblasts. Furthermore, the RGS domain of Loco can also accelerate the GTPase activity of Galphai to regulate the equilibrium between the GDP- and the GTP-bound forms of Galphai. Thus, Loco can potentially regulate heterotrimeric G-protein signaling via two distinct modes of action during Drosophila neuroblast asymmetric divisions (Yu, 2005).

Heterotrimeric G proteins have been shown to be involved in controlling distinct microtubule-dependent processes in one-cell embryos of C. elegans. Gßgamma is important for correct centrosome migration around the nucleus and spindle orientation, while Galpha subunits, GOA-1 and GPA-16, are required for asymmetric spindle positioning. Recent studies have shown that the GoLoco-motif-containing proteins, GPR1/2, act as GDIs for GOA-1 and GPA-16 to translate polarity cues, mediated by the asymmetrically localized Par proteins, into asymmetric spindle positioning in the C. elegans zygote (Colombo, 2003; Gotta, 2003; Srinivasan, 2003). In Drosophila NBs, heterotrimeric G proteins Gß13F and Ggamma1 are required for the asymmetric localization/stability of the apical components and, hence, the formation of an asymmetric spindle (Yu, 2003b). This is likely to be achieved through the generation of free Gßgamma since depletion of Gßgamma function by overexpression of wild-type Galphai/Galphao or loss of Gß13F or Ggamma1 function can lead to the generation of a symmetric and centrally placed mitotic spindle, and NBs frequently divide to produce daughter cells of similar size (henceforth referred to as 'similarsized divisions,'). Thus, generation of free Gßgamma is crucial for NB asymmetric divisions. However, it is not clear whether Gßgamma mediates spindle geometry independently of the Galpha subunit(s) or alternatively by controlling the localization of Galpha subunit(s) and/or the GoLoco proteins. Pins has previously been shown to act as a GDI to facilitate the dissociation of Gßgamma from heterotrimers by binding to and stabilizing the GDP-bound form of Galphai (GDP-Galphai). However, paradoxically, loss of pins function does not produce the severe spindle defects seen in the Gß13F or Ggamma1 mutant NBs, suggesting that the absence of the Pins GDI activity does not prevent the generation of free Gßgamma. Similarly, loss of Galphai, while causing defects in spindle orientation and the localization of the basal proteins up to metaphase, like pins loss of function, also does not cause the severe spindle asymmetry defects seen in Gß13F or Ggamma1 mutant NBs; however, it remains possible that additional Galpha subunits may be involved in this process (Yu, 2005 and references therein).

This study shows that locomotion defects (loco), a gene previously shown to be required for glial cell differentiation and dorsal-ventral patterning, encodes a novel component of the NB apical complex that exhibits both guanine nucleotide dissociation inhibitor (GDI) and GTPase-activating protein (GAP) activities for Galphai. Loco interacts with GDP-Galphai through its GoLoco motif and forms a complex with Galphai in vivo. Loco colocalizes with Galphai and Pins at the apical cortex of NBs throughout mitosis and is required for the asymmetric localization/stabilization of Pins/Galphai. Analyses of various double-mutant NBs suggest that Loco, like Pins and Galphai, functions redundantly with the Baz/DaPKC pathway in regulating spindle geometry. Interestingly, loss of both loco and pins functions leads to similar-sized divisions in the majority of NBs, similar to that seen in either Gß13F or Ggamma1 mutants, suggesting that activation of Gßgamma is mediated in a redundant manner by both Loco and Pins. These data therefore provide functional support for the idea that the activation of heterotrimeric G-protein signaling through the generation of free Gßgamma, crucial for NB asymmetric divisions, can occur via a receptor-independent mechanism by using multiple GDIs that functionally overlap. Moreover, Loco can, through its RGS domain, also function as a GAP to regulate the balance between GDP-Galphai and GTP-Galphai. Hence, both the GDI and GAP functions of Loco are important for NBs to regulate the activities of Galphai and Gßgamma (Yu, 2005).

Previous studies have shown that heterotrimeric G-protein components play important roles in NB asymmetric divisions. This study considers the issues of how heterotrimeric G-protein activation might be mediated during NB asymmetric divisions and the roles that Gßgamma, GTP-Galphai, and GDP-Galphai play in this process. Loco is shown to be a novel asymmetrically localized component of the NB asymmetric division machinery that possesses both GDI and GAP activities for Galphai. Evidence is provided that indicates that the redundant GDI activities of Pins and Loco lead to the generation of free Gßgamma, which plays a crucial role for the formation of an asymmetric mitotic spindle and daughter cells of distinct size. Based on loss-of-function phenotype, Galphai appears to play a less important role than Gßgamma in this process; however, the proper balance between the levels of GTP- and GDP-bound forms of Galphai, which may be mediated, at least in part, by the GAP activity of Loco, is crucial for the asymmetric localization of Pins and Insc. It is important to note that there may exist additional Galpha subunit(s) that might functionally overlap with Galphai in the generation of an asymmetric spindle. Therefore the possibility that Gßgamma might mediate asymmetric spindle geometry by regulating the localization Galpha subunit(s) (and GoLoco proteins) cannot be excluded at this point (Yu, 2005).

Heterotrimeric G proteins are classically known to transmit extracellular signals to targets within the cell through seven transmembrane, G-protein coupled receptors (GPCRs). Upon ligand binding, GPCR acts as a GEF to stimulate release of GDP from the Galpha subunit, which, in turn, is converted to the GTP-bound form. GTP-Galpha and Gßgamma dissociate and activate their respective effectors to initiate downstream signaling. G-protein signaling is attenuated through the hydrolysis of GTP to GDP by the GTPase activity of Galpha, which is accelerated by GAPs, which often contain an RGS domain. GDP-Galpha can reassociate with and inactivate Gßgamma (Yu, 2005).

Analyses of loss of function of Gß13F and Ggamma1 as well as gain of function of Galphai in NBs have provided compelling support for the view that free Gßgamma is required for the asymmetric localization/stability of both apical pathway components as well as the generation of asymmetric spindle and daughter cell size. Galphai is required primarily for the asymmetric localization of Pins and makes only a minor contribution in regulating spindle geometry and asymmetric daughter cell size. The mechanism by which heterotrimeric G-protein activation (generation of free Gßgamma) is mediated in NBs has been unclear. The fact that no G-protein-coupled receptors (GPCRs) have been implicated in NB asymmetric divisions, the apparent intrinsic polarity exhibited by cultured NBs, as well as the observed GDI activity associated with Pins have raised the possibility that heterotrimeric G-protein activation may occur via a receptor-independent mechanism since GoLoco-containing molecules like Pins should be able to generate free Gßgamma from the heterotrimeric complex by competing for binding to GDP-Galphai. However, loss of pins does not cause the majority of NBs to produce daughters of similar size and is therefore inconsistent with a failure to activate G-protein signaling (Yu, 2005).

This apparent contradiction is resolved by observations that indicate that receptor-independent activation of heterotrimeric G-protein signaling may be mediated through the GDI activities of both Pins and Loco. Like Pins, Loco can interact with GDP-Galphai through its GoLoco motif and form an in vivo complex with Galphai. In NBs, Loco colocalizes with Galphai and Pins at the apical cortex throughout mitosis. Removal of maternal and zygotic loco leads to delocalization of Pins/Galphai. Analysis of double mutants indicates that Loco functions redundantly with the Baz/DaPKC pathway with respect to the generation of differential daughter size. Simultaneous loss of both loco and pins results in phenotypic defects essentially indistinguishable from those seen in Gß13F or Ggamma1 loss-of-function NBs. These observations indicate that receptor-independent activation of heterotrimeric G proteins during Drosophila NB asymmetric division may be achieved through the actions of the two functionally redundant GDI activities of Pins and Loco (Yu, 2005).

In addition to its GDI activity, Loco also possesses an RGS domain that exhibits GAP activity for Galphai in vitro, suggesting that Loco can regulate Galphai via two distinct modes of action, both as a GDI and as a GAP. These studies suggest that Gßgamma, activated by the GDI activity of Pins and Loco, is crucial for NBs to produce daughters of unequal size, while the equilibrium between GDP-Galphai and GTP-Galphai, regulated, at least in part, by the GAP activity of Loco, is required for the localization of Insc/Pins/Loco at the apical cortex in NBs. When the equilibrium is shifted toward GTP-Galphai, that is, when GalphaiQ205L (the constitutively GTP-bound form) is expressed in the absence of endogenous wild-type Galphai, Pins becomes delocalized/destabilized because it requires binding to GDP-Galphai to localize to the cell cortex; however, the ability to generate an asymmetric spindle and unequal-size daughters is not compromised since Gßgamma function should not be compromised. Conversely, when the equilibrium is shifted toward GDP-Galphai, through the ectopic expression of GalphaiG204A (the constitutively GDP-bound form) in the absence of endogenous wild-type Galphai, free Gßgamma fails to be generated and defects similar to those seen in Gß13F or Ggamma1 loss of function result (Yu, 2005).

While the Loco-associated GAP activity can facilitate the conversion of GTP-Galphai to GDP-Galphai in NBs, how might the reverse reaction be catalyzed without invoking the involvement of a GPCR-associated GEF activity? A possible nonreceptor GEF that can fulfill this role may be the Drosophila homolog of the mammalian Ric-8A (Synembrin; see Drosophila Ric-8). Mammalian Ric-8A has been shown to act as a nonreceptor GEF for Galphao, Gq, and Galphai₁ subunits. Ric-8A is evolutionarily conserved from worm to mammals. More recent reports on C. elegans RIC-8 suggest that it functions as a GEF to regulate asymmetric divisions in the zygote for the Galpha subunits (GOA-1 and GPA-16). The fly homolog, DmRic-8, is indeed able to associate with Galphai and is involved in NB asymmetric divisions (Yu, 2005).

While receptor-independent activation of heterotrimeric G-protein signaling appears to be a mechanism conserved between fly and nematode, there are clear differences between the two systems. In the nematode zygote, previous studies have suggested that the Galpha subunits, GOA-1 and GPA-16, are required for generation of a net pulling force from the posterior cortex that leads to the displacement of the mitotic spindle toward the posterior cortex. Either (possibly both) of the GoLoco/GPR motif proteins, GPR1/2, which are enriched at the posterior pole of the zygote (Colombo, 2003; Gotta, 2003), can act as GDIs to asymmetrically activate heterotrimeric G-protein signaling. The Galpha subunits and GPR1/2 both appear to act downstream of the PAR proteins and their inactivation using RNAi results in identical spindle phenotypes that resemble those seen in par-2 mutants for which a reduction in cortical spindle forces have been directly demonstrated (Colombo, 2003; Gotta, 200). More recently, it has been reported that loss of ric-8 function also disrupts the movement of the posterior centrosome, suggesting that RIC-8 acts in the same pathway as GPR-1/2 to establish Galpha-dependent force generation, whereas loss of function of rgs-7, encoding a GAP protein for GOA-1, leads to overly vigorous posterior spindle rocking and more exaggerated size difference between two daughter cells, indicating that Galpha passes through the GTP-bound state during its activity cycle to regulate the force in one-cell-stage nematode embryos. In contrast, Gßgamma does not appear to regulate spindle displacement in the worm zygote (Yu, 2005).

For Drosophila NBs, spindle geometry and displacement appear to be regulated to a large extent through Gßgamma activation by the GoLoco proteins Loco and Pins. The spindle defects associated with loco/pins double loss-of-function NBs resemble those seen in the Gß13F and Ggamma1 mutants. However, it is clear that in Gß13F and Ggamma1 mutants there is a small degree of residual asymmetry in the size of the NB daughters; this residual size difference can be removed by the additional loss of baz function. There is no evidence implicating a major role for Galphai in spindle asymmetry since loss of Gi has relatively mild effects. However, the possibility that multiple Galpha subunits redundantly regulate NB spindle geometry cannot be ruled out (Yu, 2005).

Furthermore, in contrast to the C. elegans zygote where heterotrimeric G-protein signaling acts downstream of the PAR polarity cues, the precise hierarchical relationship between the heterotrimeric G proteins and the PAR proteins in Drosophila NBs is more complex. Some observations can be interpreted, at least formally, to suggest that free Gßgamma acts upstream of the apical components, since mutations in Gß13F and Ggamma1 cause delocalization of Pins/Loco/Galphai and affect the stability (intensity) of the Baz and DaPKC apical crescents. However, reduced levels of Baz and DaPKC can nevertheless asymmetrically localize and maintain residual levels of asymmetry despite the loss of free Gßgamma, suggesting that some aspects of NB asymmetry and PAR polarity cues act in parallel or upstream of heterotrimeric G proteins. This study provides evidence that in Drosophila NBs, both Loco and Pins contribute toward the generation of free Gßgamma and the asymmetric localization of Pins/Loco/Galphai depends not only on Gßgamma but also the right balance of GDP-Galphai and GTP-Galphai. It remains to be seen whether in NBs Gßgamma mediates the formation of an asymmetric spindle by regulating Galpha subunits (Yu, 2005).

Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein

Glia mediate a vast array of cellular processes and are critical for nervous system development and function. Despite their importance in neurobiology, glia remain understudied and the molecular mechanisms that direct their differentiation are poorly understood. Rap/Fzr is the Drosophila homolog of the mammalian Cdh1, a regulatory subunit of the anaphase-promoting complex/cyclosome (APC/C). APC/C is an E3 ubiquitin ligase complex well characterized for its role in cell cycle progression. This study uncovered a novel cellular role for Rap/Fzr. Loss of rap/fzr function leads to a marked increase in the number of glia in the nervous system of third instar larvae. Conversely, ectopic expression of UAS-rap/fzr, driven by repo-GAL4, results in the drastic reduction of glia. Data from clonal analyses using the MARCM technique show that Rap/Fzr regulates the differentiation of surface glia in the developing larval nervous system. Genetic and biochemical data further indicate that Rap/Fzr regulates glial differentiation through its interaction with Loco, a regulator of G-protein signaling (RGS) protein and a known effector of glia specification. It is proposed that Rap/Fzr targets Loco for ubiquitination, thereby regulating glial differentiation in the developing nervous system (Kaplow, 2008).

The APC/C is a multi-subunit ubiquitination complex that has been well characterized for its role in regulating mitotic exit. Rap/Fzr/Cdh1 is an activator of APC/C and plays a key role in the regulation of mitosis by targeting cell cycle regulators, such as cyclins and cyclin-dependent kinases, for ubiquitination. This study uncover a novel role for Rap/Fzr in the regulation of glia differentiation. Loss-of-function rap/fzr mutants display an increase in glia number and a corresponding decrease in neuronal number. Conversely, targeted overexpression of Rap/Fzr in glia leads to a severe reduction in glia number with a corresponding increase in neuronal number. This change in glia and neuron number occurs without significantly altering the mitotic index. Similarly, Pereanu (2005) reported a change in glial cell number in the larval brain without a significant change in mitotic index and suggested that the additional glial cells arise from the differentiation of secondary neuro-glioblasts located in the surface of the brain. Clonal analysis data derived from MARCM experiments suggest that Rap/Fzr specifically regulates differentiation of a subset of glia, the surface glia. Several lines of evidence presented here support the model that Rap/Fzr regulates gliogenesis by targeting the RGS protein, Loco, for ubiquitination. First, genetic interaction studies show that a single copy of the loco mutation is a dominant suppressor of both the rap/fzr rough-eye phenotype and the glial phenotype in the larval brain. Second, biochemical data show an interaction between Rap/Fzr and Loco in larval brain tissue and that Loco is ubiquitinated in larval extracts. Third, results from immunolocalization experiments show that Rap/Fzr and Loco colocalize within surface glia in the postembryonic larval brain. It is concluded that Loco is targeted for ubiquitination by Rap/Fzr through its D-box and/or KEN box, two signature ubiquitination-targeting motifs recognized by the APC/C (Kaplow, 2008).

Loco has been previously shown to be a positive effector of glia development during Drosophila embryogenesis. Recently, Loco has also been reported to have a role during the asymmetric cell division of embryonic neuroblasts. The current results suggest a new role for Loco in postembryonic development of Drosophila CNS and, specifically, in glial differentiation. It is proposed that the cellular level of Loco in the postembryonic GMC is a key positive effector in the binary switch model of glia-neuron differentiation. In this model, Rap/Fzr negatively regulates glia number by targeting Loco for ubiquitination and eventual proteosomal degradation. The model further predicts that alteration in the rap/fzr gene dosage would change cellular levels of Loco, with resulting effects on glia number. In larval neuroblasts, compartmentalization of Loco within GMCs may be critical in promoting a glial cell lineage. The results showed that, in the larval brain, Loco is colocalized with Miranda and Rap/Fzr in the basal axis, whereas during asymmetric division of embryonic neuroblasts, Loco is expressed in the apical axis. Although Miranda is a known mediator of asymmetric division of embryonic neuroblasts and a specific marker for larval neuroblasts, its function in postembryonic development has not been completely elucidated. Colocalization of Loco with Miranda and Rap/Fzr suggests a possible functional role for these molecules during postembryonic neuroblast division (Kaplow, 2008).

Collectively, these data suggest that Rap/Fzr regulates glia differentiation during two phases of development: (1) initially, Rap/Fzr controls the proliferation and self-renewal of dividing neuroblasts, and (2) subsequently, Rap/Fzr regulates the differentiation of GMCs. This model is consistent with evidence from other studies showing that proliferation of larval neuroblasts is controlled by other components of the APC/C, such as ida (a subunit of the APC/C), and Aurora-A kinase, a known target of APC/C-mediated ubiquitination during mitotic progression. Since work by Slack (2006) has shown a possible role for ida and, in turn, for the APC/C during neuroblast division, it would be interesting to determine if additional components of the APC/C have roles during later phases of development. Preliminary analysis of glia number in morula/APC2 (a catalytic subunit of APC/C) mutants showed a significant increase in glia number similar to rap/fzr loss-of-function mutants. However, the precise roles of additional components of the APC/C, a complex of 11 subunits, during glial differentiation have yet to be elucidated. While the results suggest that Rap/Fzr regulates neuroblast number by targeting Loco for degradation, Rap/Fzr may also regulate neuroblast self-renewal through its interactions with other proteins such as Aurora-A kinase. In Drosophila larval neuroblasts, Aurora-A kinase is an important regulator of neuroblast self-renewal and is known to be a substrate for APC/C in vertebrates (Kaplow, 2008).

The data presented in this article support a model in which components of the ubiquitin ligase complex, APC/C, mediate a post-translational regulatory mechanism critical to the glial differentiation program. During the past 2 years, other studies have also reported novel roles for the APC/C and its components during nervous system development, independent of its function during cell cycle regulation. Studies have demonstrated a role for Cdh1, the mammalian homolog of Rap/Fzr, in axon growth through its interaction with the transcriptional corepressor SnoN. Furthermore, in vitro cell culture studies using neuroblastoma cell lines and silencing of Cdh1 in postmitotic cerebellar granule neurons demonstrate that the DNA-binding protein inhibitor of differentiation 2 (Id2) is a target for Cdh1-mediated ubiquitination. The current results show that Rap/Fzr is involved in glia differentiation and are consistent with other data that demonstrate that Cdh1 targets transcriptional regulators involved in the differentiation program of the developing nervous system. Thus, in addition to its role in the regulation of cell cycle progression, Rap/Fzr/Cdh1 promotes neuron formation and inhibits gliogenesis. These studies here lend further support to the idea that ubiquitination functions as a key regulatory mechanism during nervous system development (Kaplow, 2008).

DEVELOPMENTAL BIOLOGY

Embryonic

Two enhancer trap lines, 3-109 and rC56, were selected based on their specific beta-Galactosidase expression in the lateral CNS glia. Both lines show identical beta-Galactosidase expression patterns and carry a P-element insertion at the cytological position 94B/C. In embryos carrying the rC56 enhancer trap insertion, first beta-Galactosidase expression can be detected in early stage 12 in cells which, based on their position, appear to be the progeny of the lateral glioblast. Interestingly, at this early stage these cells appear to be already different from surrounding cells. The anterior pair of progeny expresses elevated levels of beta-Galactosidase. As CNS development continues, these cells migrate medially and divide. By the end of embryogenesis, most glial cells except the midline glia express beta-Galactosidase. In addition, beta-Galactosidase expression can be detected in the dorsal leading edge cells in the lateral ectoderm (Granderath, 1999).

The mapping of exons by restriction analysis and genomic sequencing reveals two different loco variants differing in their 5' ends (transcripts c1 and c2). In situ hybridisation experiments with transcript-specific digoxigenin-labelled cDNA probes show that both LOCO RNA classes are expressed in the embryo. loco-c1 transcription is very weak and is detected only after prolonged incubation (6-12 hours) in the staining solution. Using a 200 bp loco-c1- specific probe, expression can be first detected in late stage 12 embryos. In stage 16 embryos, loco-c1 RNA is found in the dorsal leading edge cells in the lateral ectoderm, in the tracheal cells and in the lateral glial cells within the CNS. Except for loco expression in tracheal cells, this corresponds well with the beta-Galactosidase expression pattern observed for the two P-element insertions in the loco gene. loco-c2 transcripts are found only in scattered cells in the lateral ectoderm. Based on their position, these cells might correspond to PNS progenitor cells. No expression can be detected in the CNS (Granderath, 1999).

EFFECTS OF MUTATION

Two non-complementing lethal mutations, loco^delta13 and loco^delta293, were recovered using P-element excision. In loco^delta13, the proximal deletion breakpoint lies within the P-element leaving the lacZ gene intact. However, the relative level of beta-Galactosidase expression in different glial cells appears to be altered and, in loco^delta13 embryos, the longitudinal glial cells express only low levels of beta-Galactosidase. This indicates that glia-specific enhancer elements reside upstream of the rC56 enhancer trap insertion whereas a transcriptional activator acting specifically in the longitudinal glial cells must reside 3' of the rC56 insertion. The breakpoint in loco^delta13 was cloned and is at least 7 kb downstream of the loco gene. A small inversion as well as a deletion of about 2 kb of genomic sequence is associated with the loco^delta293 allele. Here, putative promotor sequences as well as the first exon are deleted. Both mutations are homozygous embryonic lethal. Additional loco alleles were obtained following EMS mutagenesis. These additional mutations are lethal in trans to both loco excision mutations at 29°C. Based on the complementation analyses, they were placed into an allelic series. At 25°C, loco M1 (but none of the other EMS-induced alleles) produces adult escapers when in trans to loco^delta133 or loco^delta293 . About 10% of the expected numbers of transheterozygous adults appear. They often fail to eclose from the opened puparium. Eclosed flies show a paralytic phenotype and drop into the food and die. If such flies are rescued from the food, they show a severe impairment of spontaneous locomotor activity and display a ‘shaking’ phenotype. Response to mechanical stimulation (e.g. after stimulation of thoracic bristles) is weak in loco^M1/loco^delta29 and undetectable in loco^M1/loco^delta13, indicating that D13 is a stronger allele than D293. Similar phenotypes, albeit with lower expressivity, are seen in flies heterozygous for loco^M1 and other EMS-induced alleles at lower temperatures. All adult escapers die after a maximum of 2 days (Granderath, 1999).

At least two transcripts of loco are expressed in oogenesis: (1) loco-c2 is observed in the anterior-dorsal follicle cells and is downstream of the epidermal growth factor receptor signaling pathway, initiated in the oocyte; (2) loco-c3 is a new transcript of loco that is expressed in the nurse cells from stage 6 onwards. Disrupting loco in follicle cells results in ventralized eggs, while disrupting loco in nurse cells results in short eggs, due to defective dumping of the nurse cell cytoplasm into the oocyte (Pathirana, 2002).

The observed egg phenotypes laid by loco³⁷¹ homozygous females suggest a role for loco in DV axis formation of the egg. This data was corroborated by heatshock induced anti- sense-loco experiments. However, the mutant analysis was not straightforward. Although molecular studies clearly showed that mutants had been generated in the loco gene and there were no other P-elements in the stocks, the complementation analysis did not show a more severe phenotype when newly generated mutants were crossed to the existing deficiencies in the region. loco is a large gene with several differently spliced forms, which would suggest a complex mechanism of gene regulation. Preliminary analysis with antibodies has also shown there are different isoforms of the protein present at distinct developmental stages suggesting different roles for different protein isoforms. As a negative regulator of G-protein levels, the types of protein expressed would be critical in maintaining equilibrium in signaling systems. All these factors could contribute to the observed behavior of loco³⁷¹ which has a small insertion and retains rearranged parts of the P-element in the loco gene. This would be likely to interfere with expression of some transcripts but not others. This misregulation would lead to unusual complementation analysis, since having two copies of a gene that is expressed in the wrong cells or at the wrong time, or as the wrong isoform is likely to be more developmentally disruptive than a single copy. This would explain why homozygotes are mostly lethal and hemizygotes are not (Pathirana, 2002).

Heatshock antisense results were crucial for interpreting how the different mutant phenotypes were generated. The different heatshock lines generate transcripts in different cell types. This allowed loco to be disrupted separately in the germ-line and follicle cells. loco-c3 is expressed in the nurse cells and, when disrupted, results in a dumpless phenotype resulting in smaller than normal eggs being laid. When loco-c2 is disrupted in the anterior-dorsal follicle cells, this results in a range of dorsal defects in the eggs. The anterior-dorsal egg defects vary from dorsal appendages, which are fused at the base, to appendages fused along the whole length, to completely absent appendages. These results suggest a role for G-protein signaling in DV pattern formation in oogenesis (Pathirana, 2002).

The role of RGS proteins is to negatively regulate G-protein signaling. It seems likely that G-protein signaling has a role in DV axis formation. In the absence of loco there is a loss of DV polarity in the egg and embryo. This phenotype is similar to the one induced by Gurken mutants and suggests that in the absence of loco repression EGFR signaling occurs. Since RGS genes negatively regulate G-protein signaling, one can assume that in wild type ovaries G-protein signaling inhibits EGFR signalling but the presence of loco prevents this inhibition in anterior-dorsal follicle cells. This adds to the complexity of regulation of the EGFR pathway and suggests that the tight regulation or modulation of EGFR is critical for the correct sequence of morphological events to occur in the specification of follicle cell fates along the DV axis with time (Pathirana, 2002).

How does loco fit into the existing EGF pathway? loco is downstream of Egfr/torpedo in the follicle cells and appears to be activated both at high and moderate levels of torpedo activation. loco is downstream of pointed, a target of Egf signaling. loco has been identified in a screen for genes downstream of pointed in specific subsets of cells in the central nervous system (CNS). pointed P1 and P2 are expressed in the anterior-dorsal follicle cells in oogenesis. The expression of pointed in oogenesis is dynamic, expression first being observed in the germarium, then later at stage 8, downstream of torpedo, in the posterior follicle cells and again at stage 10, also downstream of torpedo, in anterior-dorsal follicle cells. Since pointed is observed in anterior-dorsal follicle cells in a similar pattern to loco at stage 10, it is possible that loco is downstream of pointed at this particular stage of oogenesis. The relationship between loco and pointed was analyzed in these cells. Using a pointed 1/UAS sense fly line, expression of pointed was driven in all the follicle cells using a T155 GAL4 driver. In situ hybridization to RNA in the GAL4/UAS-pointed ovaries, using a pointed probe, clearly shows high levels of pointed expression in all the follicle cells that cover the oocyte at stage 10. The expression of loco-c2 was examined in egg chambers where pointed was being ectopically expressed. A normal spatial distribution was seen in anterior-dorsal follicle cells at stage 10, though levels of expression were somewhat reduced. This reduction in expression is not uniform over the anterior-dorsal region, with the anterior-most follicle cells maintaining their normal level of expression. At later stages the expression pattern is slightly different from wild type, since there is a patch of cells expressing loco in the dorsal position that has not migrated as far anteriorly as would be expected at this stage. This is due to overexpression of pointed in oogenesis resulting in failure to make dorsal appendages. This suggests that cells normally expressing loco do not migrate as far as in wild type egg chambers. This experiment shows that there is not a simple relationship between loco and pointed. Ectopic pointed expression does disrupt the normal loco-c2 expression pattern at stage 10, with its level of expression dropping in the anterior-dorsal follicle cells, except in the anterior-most follicle cells. This indicates that loco-c2 is downstream of pointed, although not directly (Pathirana, 2002).

The DV defects in twist expression observed in the embryos of eggs laid by fly line loco³⁷¹ help clarify the role of loco in oogenesis, showing that disruption of maternal loco in oogenesis results in the disruption of native twist expression, a marker for ventral cell fate, and clear DV defects are observed in the embryos that fail to hatch, as many fail to form normal guts. The range of patterning defects observed are related to the disruption of native twist expression. This suggests that maternally expressed loco is required for normal embryonic development. It cannot be distinguished if the failure of embryos to develop properly and the observed defects in twist expression in the early embryo result from the mutant follicle cells affecting the developing egg chamber and oocyte or if the maternal contribution of loco from the nurse cells deposited in the egg is being needed for zygotic development of the D/V axis. However, the twist expression observed only in the activation of the embryo does suggest that disruption of loco in anterior-dorsal follicle cells may affect embryonic patterning. Dorsal follicle cell identity is determined as the follicle cells migrate posteriorly over the egg chamber, coming into contact with the localized gurken signal over the oocyte nucleus. If EGFR signaling is disrupted in follicle cells during the course of this migration, an embryo that has abnormal D/V patterning along only part of its AP axis would result (Pathirana, 2002).

The small egg phenotype observed in both the mutant lines and heatshock antisense lines indicates that loco is required for cytoplasmic dumping from the nurse cells to the oocyte. Several mutants have been identified that are involved in this process, and the phenotypes can be attributed to two defective processes. The first is the failure of the ring canals to grow, restricting the flow of cytoplasm from the nurse cells to the oocyte. The second is a change in the actin cytoskeleton, with the nurse cells failing to centralize the nurse cell nuclei. When this happens the nuclei block the ring canals, preventing dumping. Which process requires loco during nurse cell dumping remains to be determined (Pathirana, 2002).

Thus, two roles for the loco gene in oogenesis have been identified; cytoplasmic dumping and DV axis formation. Goalpha mRNA is present in oocyte and nurse cells and Gsalpha protein in follicle cells; Gialpha mRNA is present in nurse cells and the oocyte, with Gialpha protein present in stage 10 anterior-dorsal follicle cells and the oocyte. The expression patterns of the different Galpha subunits correlate with the expression patterns of the loco isoforms, further suggesting a role for G-protein signaling in nurse cell dumping and a link between G-protein signaling and the EGFR pathway in follicle cells (Pathirana, 2002).

GPCR signaling is required for blood-brain barrier formation in Drosophila

The blood-brain barrier of Drosophila is established by surface glia, which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions. The mechanisms underlying the formation of this barrier remain obscure. The G protein-coupled receptor (GPCR) Moody, the G protein subunits Galphai and Galphao, and the regulator of G protein signaling Loco are required in the surface glia to achieve effective insulation. The data suggest that the four proteins act in a complex common pathway. At the cellular level, the components function by regulating the cortical actin and thereby stabilizing the extended morphology of the surface glia, which in turn is necessary for the formation of septate junctions of sufficient length to achieve proper sealing of the nerve cord. This study demonstrates the importance of morphogenetic regulation in blood-brain barrier development and places GPCR signaling at its core (Schwabe, 2005).

The Drosophila nerve cord is ensheathed by a thin single-layer epithelium, which in turn is surrounded by an acellular layer of extracellular matrix material. Ultrastructural analysis has revealed that septate junctions (SJs) between the epithelial cells are responsible for the insulation of the nerve cord. Fate-mapping studies have shown that the nerve cord is enveloped by glia expressing the glial-specific marker Repo, but to date there has been no direct proof that it is these surface glia that form intercellular SJs and thus the insulating sheath. Moreover, the time course for the formation of the sheath and of the SJ-mediated seal has not been established (Schwabe, 2005).

Several assays were developed to follow the morphogenesis of the surface glial sheath. Due to the onset of cuticle formation, immunohistochemistry becomes unreliable after 16 hr of development. Live imaging of GFP-tagged marker proteins was therefore used to visualize cell shapes, in particular the actin cytoskeleton marker GFP/RFP-Moesin and the SJ marker Neuroglian (Nrg)-GFP. Nrg-GFP expressed under its own promoter and RFP-Moesin driven by repo-Gal4 are colocalized in the same cells, establishing that the SJ-forming cells are repo positive and thus conclusively demonstrating the insulating function of the surface glia. To probe the permeability of the transcellular barrier, fluorescent dye was injected into the body cavity and dye penetration into the nerve cord was quantified by determining mean pixel intensity in sample sections (Schwabe, 2005).

The surface glia are born in the ventrolateral neuroectoderm and migrate to the surface of the developing nerve cord, where they spread until they touch their neighbors (17 hr of development). The glia then join to form a contiguous sheet of square or trapezoidal cells, tiled to form three-cell corners. SJ material is visible as a thin contiguous belt by 18 hr but continues to accumulate until the end of embryogenesis. Similar to other secondary epithelia, the surface glia do not form a contiguous adherens-junction belt (zonula adherens), but only spotty, inconsistent adherens junctions were seen, as visualized by Armadillo-GFP (driven by own promoter). At 16 hr, the fluorescent dye freely penetrates into the nerve cord, but by 20 hr the nerve cord is completely sealed. The completion of the seal thus coincides with the onset of visible movements in the late embryo (Schwabe, 2005).

To further gauge the dye-penetration assay, embryos mutant for known septate-junction components were examined: Neurexin IV, which is required for blood-nerve barrier formation in the PNS; Neuroglian, and the sodium-pump component Nervana 2, for which only a role in the earlier formation of the ectodermal seal has been demonstrated. In all three mutants, severe penetration of dye was found, well after the nerve cord is sealed in wild-type (22 hr). These findings provide further evidence that the sealing of the nerve cord is achieved by SJs and suggest that the components of the ectodermal SJs are required for the function of surface glial SJs as well (Schwabe, 2005).

In a genome-wide screen for glial genes, using FAC sorting of GFP-labeled embryonic glia and Affymetrix microarray expression analysis, two novel GPCRs, Moody (CG4322) and Tre1 (CG3171) were identified. Both are orphan receptors belonging to the same novel subclass of Rhodopsin-family GPCRs. Their expression was examined by RNA in situ hybridization; different subtypes of glia in the embryonic nerve cord can be distinguished based on their position and morphology. In the CNS, moody is expressed in surface glia from embryonic stage 13 onward (10 hr); in addition to cells surrounding the nerve cord (subperineurial glia), this includes cells lining the dorsoventral channels (channel glia). moody is also expressed in the ensheathing glia of the PNS (exit and peripheral glia). Both CNS and PNS expression of moody are lost in mutants for the master regulator of glial fate, glial cells missing (gcm^N17), confirming that they are indeed glial. tre1 is expressed in all longitudinal glia and a subset of surface glia, as well as in cells along the midline. As expected, the (lateral) glial expression is lost in gcm mutants, while midline expression is not. Both moody and tre1 are also expressed outside the nervous system in a largely mutually exclusive manner, specifically in the germ cells, the gut, and the heart (Schwabe, 2005).

Several additional G protein signaling components are found in the surface glia. The six extant Gα genes show broad and overlapping expression in embryogenesis, with three of them (Go, Gq, and Gs) expressed throughout the nervous system and Gi expressed more specifically in surface glia. Gβ13F and Gγ1 are ubiquitously expressed during embryogenesis. Finally, the RGS loco is uniformly expressed in early embryos due to a maternal contribution but is then transcriptionally upregulated in surface and longitudinal glia, as well as in other tissues outside the nervous system. The nervous-system expression of loco is lost in gcm mutants. The presence of both Moody and Loco protein in the surface glia is confirmed using immunohistochemistry, but at 17 hr of development, when staining is feasible, the protein levels are still quite low (Schwabe, 2005).

In sum, the GPCR Moody, the RGS Loco, and Gi are differentially expressed in surface glia. This expression precedes and accompanies the morphogenesis and sealing of the surface glial sheath (Schwabe, 2005).

Protein expression and distribution of the GPCR signaling components were examined in greater detail in third-instar larval nerve cords. By this stage, the surface glia have doubled in size and show robust protein expression of GPCR signaling and SJ components (Schwabe, 2005).

Moody immunostaining is found at the plasma membrane, where it shows strong colocalization with the SJ marker Nrg-GFP. Loco immunostaining is punctate and more dispersed throughout the cytoplasm, with some accumulation at the plasma membrane, where it colocalizes with Moody. To avoid fixation and staining artifacts, fluorescent-protein fusions (Moody-mRFP; Loco-GFP) were generated and expressed using moody-Gal4, which drives weak surface glial expression. In the live nerve-cord preparations, Loco-GFP is much less dispersed and shows strong colocalization with Moody-mRFP at the plasma membrane (Schwabe, 2005).

In the absence of a known ligand, the coupling of G proteins to receptors is difficult to establish, but their binding to RGS proteins is readily determined. Loco physically binds to and negatively regulates Gi, and vertebrate Loco homologs (RGS12/14) have been shown to negatively regulate Gi/Go. In S2 tissue-culture assays, it was found that Loco binds to Gi and Go, but not to Gs and Gq. Double-label immunohistochemistry confirms that both Gi and Go are expressed in the surface glia (Schwabe, 2005).

Thus, Loco physically interacts with Gi and Go and shows subcellular colocalization with Moody, suggesting that the four signaling components are part of a common molecular pathway (Schwabe, 2005).

Using dye penetration as the principal assay, whether the GPCR signaling components that are expressed in surface glia play a role in insulation was examined. moody genomic (Δ17; Bainton, 2005) and RNAi mutants show similar, moderate insulation defects. The embryos are able to hatch but show mildly uncoordinated motor behavior and die during larval or pupal stages. The dye-penetration defect of moody^Δ17 is completely rescued by genomic rescue constructs containing only the moody ORF. Both moody splice forms (α and β; Bainton, 2005) are able to rescue the defect independently, as well as in combination. tre1 genomic (Kunwar, 2003) and RNAi mutants show no significant dye-penetration defect and no synergistic effects when combined with moody using RNAi. Thus, despite the close sequence similarity of the two GPCRs and their partially overlapping expression in surface glia, only moody plays a significant role in insulation. Overexpression of moody causes intracellular aggregation of the protein (Schwabe, 2005).

loco is expressed both maternally and zygotically. loco zygotic nulls are paralytic and, on the basis of an ultrastructural analysis, a disruption of the glial seal, has been suggested. In a dye-penetration assay, loco zygotic null mutants show a strong insulation defect, which can be rescued by panglial expression of Loco in its wt or GFP-tagged form. The extant null allele of loco (Δ13) did not yield germline clones; therefore loco RNAi was used to degrade the maternal in addition to the zygotic transcript. In loco RNAi embryos, dye penetration is indeed considerably more severe. Overall, insulation as well as locomotor behavior is affected much more severely in loco than in moody and is close in strength to the SJ mutants. Overexpression of loco is phenotypically normal (Schwabe, 2005).

Thus, positive (moody) and negative (loco) regulators of G protein signaling show qualitatively similar defects in loss of function, suggesting that both loss and gain of signal are disruptive to insulation. Such a phenomenon is not uncommon and is generally observed for pathways that generate a localized or graded signal within the cell (Schwabe, 2005).

Both Gi and Go have a maternal as well as a zygotic component. Gi zygotic null flies survive into adulthood but show strong locomotor defects. In Gi maternal and zygotic null embryos show a mild dye-penetration defect, which is markedly weaker than that of moody, suggesting redundancy among Gα subunits. To further probe Gi function, the wt protein (Gi-wt) as well as a constitutively active version (Gi-GTP) were overexpressed in glia using repo-Gal4; such overexpression presumably leads to a masking of any local differential in endogenous protein distribution. Expression of Gi-wt results in very severe dye penetration, while overexpression of Gi-GTP is phenotypically normal. Only Gi-wt but not Gi-GTP can complex with Gβγ; overexpression of Gi-wt thus forces Gβγ into the inactive trimeric state. This result therefore suggests that the phenotypically crucial signal is not primarily transduced by activated Gi but rather by free Gβγ. Similar results have been obtained in the analysis of Gi function in asymmetric cell division (Schwabe, 2005).

Go null germline clones do not form eggs and do not survive in imaginal discs, indicating an essential function for cell viability (Katanaev, 2005). Therefore animals with glial overexpression of constitutively active (Go-GTP), constitutively inactive (Go-GDP), and wt (Go-wt) Go (Katanaev, 2005) were examined. Overexpression of Go-GDP, which cannot signal but binds free Gβγ, leads to severe dye penetration, again pointing to a requirement for Gβγ in insulation. However, Go-GTP and Go-wt show a moderate effect, suggesting that signaling by active Go does contribute significantly to insulation, in contrast to active Gi (Schwabe, 2005).

Overall, it was found that all four GPCR signaling components expressed in surface glia are required for insulation, further supporting the notion that the four components are part of a common pathway. The phenotypic data suggest that this pathway is complex: two Gα proteins, Gi and Go, are involved, but with distinct roles: activated Go and Gβγ appear to mediate most of the signaling to downstream effectors, while activated Gi seems to function primarily as a positive regulator of Gβγ. The loss of moody appears much less detrimental than the loss of free Gβγ (through overexpression of Gi-wt or Go-GDP); this is inconsistent with a simple linear pathway and points to additional input upstream or divergent output downstream of the G proteins. Finally, it was consistently observed that both loss (moody, Gi null, and Go-GDP) and gain (loco and Go-GTP) of signal are disruptive to insulation, suggesting that the G protein signal or signals have to be localized within the cell (Schwabe, 2005).

These complexities of G protein signaling in insulation preclude an unambiguous interpretation of genetic-interaction experiments and thus the linking of moody to Gi/Go/loco by genetic means. Double-mutant combinations between moody and loco were generated using genomic mutants as well as RNAi, with very complex results: in moody loco genomic double mutants, the insulation defect is worse than that of loco alone, while in moody loco RNAi double mutants the insulation defect is similar to that of moody alone. This strong suppression of loco by moody is also observed in the survival and motor behavior of the RNAi-treated animals. Thus the phenotype of the double-mutant combination is dependent on the remaining levels of moody and loco, with moody suppressing the loco phenotype when loco elimination is near complete (Schwabe, 2005).

To understand how the GPCR signaling components effect insulation at the cellular level, the distribution of different markers in the surface glia was examined under moody and loco loss-of-function conditions and under glial overexpression of Gi-wt. To rule out cell fating and migration defects, the presence and position of the surface glia were determined using the panglial nuclear marker Repo. In all three mutant situations, the full complement of surface glia is present at the surface of the nerve cord, with the positioning of nuclei slightly more variable than in wt (Schwabe, 2005).

In the three mutants, the SJ marker Nrg-GFP still localizes to the lateral membrane compartment, but the label is of variable intensity and sometimes absent, indicating that the integrity of the normally continuous circumferential SJ belt is compromised. Notably, the size and shape of the surface glia are also very irregular. While qualitatively similar, the phenotypic defects are more severe in loco and under Gi-wt overexpression than in moody, in line with the results of functional assays. When examining the three mutants with the actin marker GFP-Moesin, it was found that the cortical actin cytoskeleton is disrupted in varying degrees, ranging from a thinning to complete absence of marker, comparable to the effects observed with Nrg-GFP. However, GFP-positive fibrous structures are present within the cells, indicating that the abnormalities are largely restricted to the cell cortex. The microtubule organization, as judged by tau-GFP marker expression, appears normal in the mutants. The light-microscopic evaluation thus demonstrates that, in the GPCR signaling mutants, the surface glia are positioned correctly and capable of forming a contiguous epithelial sheet as well as septate junctions. Instead, the defects occur at a finer scale—abnormally variable cell shapes and sizes, and irregular distribution of cortical actin and SJ material (Schwabe, 2005).

The changes in cell shape and actin distribution that were observed in the three mutants might simply be a secondary consequence of abnormalities in the SJ belt; to test this possibility, how a loss of the SJ affects the morphology and the actin cytoskeleton of the surface glia was examined. SJ components are interdependent for the formation and localization of the septa, and lack of a single component, such as Nrg, leads to nearly complete loss of the junction and severe insulation defects. In Nrg mutants, the surface glial cell shape and cortical actin distribution show only mild abnormalities. Thus, in contrast to the GPCR signaling mutants, the complete removal of the SJ causes only weak cytoskeletal defects, strongly arguing against an indirect effect. It is concluded that GPCR signaling most likely functions by regulating the cortical actin cytoskeleton of the surface glia, which in turn affects the positioning of SJ material along the lateral membrane (Schwabe, 2005).

More detailed insight into the nature of the defects in GPCR signaling mutants is afforded by electron microscopy. The surface glia in nerve cords of first-instar wild-type and mutant larvae were examined. Initially, dye penetration into the nerve cord was tested using ruthenium red. In wild-type, the dye diffuses only superficially into the surface glial layer, while in moody and loco mutants the dye penetrates deep into the nerve cord, in concordance with light-microscopic data. Tissue organization and SJ morphology were examined under regular fixation in randomly selected transverse sections. It has been reported that the surface glial sheath is discontinuous in loco mutant nerve cords, but this analysis was carried out at 16 hr of development, i.e., at a time when, even in wild-type, SJs are not yet established and the nerve cord is not sealed. In contrast to these findings, in the current study it was observed that, in loco as well as moody mutants, the glial sheath is in fact contiguous at the end of embryonic development. The ultrastructure of individual septa and their spacing also appear normal, indicating that moody and loco do not affect septa formation per se. However, the global organization of the junctions within the glial sheath appears perturbed: in wild-type, the surface glia form deep interdigitations, and the SJs are extended, well-organized structures that retain orientation in the same plane over long distances. In moody and loco mutants, the SJs are much less organized; they are significantly shorter in length and do not form long planar extents as in wild-type (Schwabe, 2005).

Taken together, the light- and electron-microscopic evaluations of the GPCR signaling mutants both show defects in the organization of the surface glial epithelium. The reduction in SJ length is consonant with the variability and local disappearance of the Nrg-GFP marker. Since the sealing capacity of the junction is thought to be a function of its length, the reduction in mean SJ length in the mutants provides a compelling explanation for the observed insulation defect (Schwabe, 2005).

Therefore, in addition to a reduction of the insulating SJs, this analysis of the GPCR signaling mutants revealed irregular cell shape and size, as well as weaker and variable accumulation of cortical actin in the surface glia. These data suggest that the primary defect in the mutants lies with a failure to stabilize the cortical actin, whose proper distribution is required for the complex extended morphology of the glia, which then affects SJ formation as a secondary consequence. Several lines of evidence exclude the reverse chain of causality, that is, a primary SJ defect resulting in destabilization of cortical actin and cell-shape change. Surface glia coalesce into a contiguous sheath and show strong accumulation of cortical actin before SJ material accumulates and sealing is completed. In the GPCR signaling mutants, there is misdistribution of SJ material along the cell perimeter, but the junctions do form. Finally, the GPCR signaling mutants show cell-shape and cortical actin defects that are much more severe than those observed in the near complete absence of SJ (Schwabe, 2005).

Compared to the columnar epithelia of the ectoderm and the trachea, the surface glial sheath is very thin. Compensating for their lack in height, surface glia form deep “tongue-and-groove” interdigitations with their neighbors. This increases the length of the intercellular membrane juxtaposition and thus of the SJ, which ultimately determines the tightness of the seal. It is proposed that the surface glial interdigitations are the principal target of regulation by GPCR signaling. In GPCR signaling mutants, a loss of cortical actin leads to diminished interdigitation and thus to a shortening of the SJ, resulting in greater permeability of the seal. This model integrates all the observations made at the light- and electron-microscopic levels (Schwabe, 2005).

he RGS gene loco is essential for male reproductive system differentiation in Drosophila

The loco gene encodes several different isoforms of a regulator of G-protein signalling. These different isoforms of LOCO are part of a pathway enabling cells to respond to external signals. LOCO is known to be required at various developmental stages including neuroblast division, glial cell formation and oogenesis. Less is known about LOCO and its involvement in male development. Therefore to gain further insight into the role of LOCO in development a genetic screen was carried out and males with reduced fertility were analyzed. A number of lethal loco mutants and four semi-lethal lines were identified, that generate males with reduced fertility. A fifth loco transcript was identified; it is differentially expressed in developing pupae. The expression pattern of all loco transcripts were identified during pupal development in the adult testes, both in wild type and loco mutant strains. In addition it was also shown that there are various G-protein alpha subunits expressed in the testis all of which may be potential binding partners of LOCO. It is proposed that the male sterility in the new loco mutants results from a failure of accurate morphogenesis of the adult reproductive system during metamorphosis, and it is proposed that this is due to a loss of expression of loco c3. Thus, it is concluded that specific isoforms of loco are required for the differentiation of the male gonad and genital disc (McGurk, 2008).

A number of homozygous lethal mutant lines of loco were isolated. These lines die at a variety of developmental stages, however, among them four lines were able to generate some homozygous adult males, which were semi-sterile. It is suggested that this is most likely to be due to a failure of the correct morphogenesis of the testis and reproductive organ derivatives of the larval gonad. This adds another role to the wide range of developmental decisions that are known to be dependent upon loco. It has been shown that loco mutants die as embryos showing abnormalities in the contacts between glial cells. Previous studies illustrated that there is a requirement for loco in cytoplasmic dumping from the nurse cells to the oocyte and that loco is required for correct patterning of the eggshell and embryo. There is also a large maternal supply of loco in the embryo probably explaining why the embryos die so late in embryogenesis. Finally, it was shown more recently that loco contributes to asymmetric cell division of neuroblasts. These findings suggest that G-protein signalling may be important at wide variety of developmental stages in Drosophila (McGurk, 2008)..

This study describes the expression of a fifth transcript, loco c5. The expression of loco c1, loco c2, loco c3, and loco c5 in the wild-type testis and developing pupae is described, and it was shown that there is developmental regulation of loco c5 expression during morphogenesis. In addition it was shown that several G proteins are expressed in the male gonads and are therefore potential binding partners for the various LOCO isoforms. It is possible that the protein isoforms, expressing different conserved domains, will have different binding specificities and preferences for different G-proteins. The G protein Gαi (G-oα65A) binds to loco c2 and it is also co-expressed with loco in a variety of cell types. This study has shown by PCR that other Gα subunits are expressed (Goα47A, Gα49B, and Gα73B) in the testis and thus there is potential for LOCO to interact with other Gα subunits (McGurk, 2008). The analysis of the final morphology of the adult reproductive system in all of the flies analysed, strongly suggests that there is a failure in male gonad and genital morphogenesis. It is possible that loco c3 expression could be the underlying reason for this phenotype. However the variability in testes morphology between flies may hint that there is some level of redundancy between the loco transcripts. Thus, while loco is clearly essential, a lack of or reduction of loco c3 expression does not cause a complete failure of gonad and genital differentiation. The loco mutants that were isolated still express several loco transcripts, so further mutants will be needed which disrupt different transcripts or sets of transcripts to discover the role of loco and G-protein signalling in spermatogenesis and to further investigate it in imaginal discs and in the somatic cells of the gonad (McGurk, 2008).

EVOLUTIONARY HOMOLOGS

Yeast and Invertebrate RGS protein homologs

A novel member of the recently identified family of regulators of heterotrimeric G protein signalling (RGS) in the yeast Saccharomyces cerevisiae has been characterized. The YOR107w/RGS2 gene was isolated as a multi-copy suppressor of glucose-induced loss of heat resistance in stationary phase cells. The N-terminal half of the Rgs2 protein consists of a typical RGS domain. Deletion of Rgs2 enhances, while its overexpression reduces, the glucose-induced accumulation of cAMP. Overexpression of RGS2 generates phenotypes consistent with low activity of cAMP-dependent protein kinase A (PKA), such as enhanced accumulation of trehalose and glycogen, enhanced heat resistance and elevated expression of STRE-controlled genes. Deletion of RGS2 causes the opposite phenotypes. Rgs2 functions as a negative regulator of glucose-induced cAMP signaling through direct GTPase activation of the Gs-alpha protein Gpa2. Rgs2 and Gpa2 constitute the second cognate RGS-G-alpha protein pair identified in yeast, in addition to the mating pheromone pathway regulators Sst2 and Gpa1. Moreover, Rgs2 and Sst2 exert specific, non-overlapping functions, and deletion mutants in Rgs2 and Sst2 are complemented to some extent by different mammalian RGS proteins (Versele, 1999).

The frequencies of certain periodic behaviors of the nematode C. elegans are regulated in a dose-dependent manner by the activity of the gene egl-10, a regulator of G protein signaling. These behaviors are modulated oppositely by the activity of the G protein alpha subunit gene goa-1, suggesting that egl-10 may regulate a G protein signaling pathway in a dose-dependent fashion. Loss-of-function alleles of goa-1 strongly increase the frequency of egg-laying behavior and also increase the frequencies of locomotory body bends and other behaviors. Loss-of-function alleles of egl-1 severely reduce or abolish egg-laying behavior and initiate body bends less frequently. egl-10 encodes a protein similar to Sst2p, a negative regulator of G protein signaling in yeast. EGL-10 protein is localized in neural processes, where it may function in neurotransmitter signaling. Two previously known and 13 newly identified mammalian genes have similarity to egl-10 and SST2; it is proposed that members of this family regulate many G protein signaling pathways (Koelle, 1996).

Asymmetric divisions are crucial for generating cell diversity; they rely on coupling between polarity cues and spindle positioning, but how this coupling is achieved is poorly understood. In one-cell stage Caenorhabditis elegans embryos, polarity cues set by the PAR proteins mediate asymmetric spindle positioning by governing an imbalance of net pulling forces acting on spindle poles. The GoLoco-containing proteins GPR-1 and GPR-2, as well as the Galpha subunits GOA-1 and GPA-16, are essential for generation of proper pulling forces. GPR-1/2 interacts with guanosine diphosphate-bound GOA-1 and were enriched on the posterior cortex in a par-3- and par-2-dependent manner. Thus, the extent of net pulling forces may depend on cortical Galpha activity, which is regulated by anterior-posterior polarity cues through GPR-1/2 (Colombo, 2003).

A Drosophila gene encoding a homolog of the regulator of G-protein signaling (RGS) protein family has been identified. This gene (dRGS7) is expressed in neurons of the embryo and adult fly and is predicted to encode a 428-amino acid protein with >55% overall amino acid sequence identity with the vertebrate protein RGS7 and the C. elegans EGL-10. The dRGS7 protein is 50% conserved in the C-terminal RGS domain with RGS7 and EGL-10 but, remarkably, displays much greater conservation with the N-terminal regions of these proteins. This finding implies a conserved function for these homologs from divergent species involving domains outside the RGS domain. The dRGS7 protein also has a domain of similarity with Dishevelled and pleckstrin, raising the possibility that these proteins interact with common signaling components (Elmore, 1998).

To elucidate the cellular role of the heterotrimeric G protein G_o, a molecular genetic approach has been taken in Caenorhabditis elegans. A screen was carried out for suppressors of activated GOA-1 (G_oalpha) that do not simply decrease GOA-1 expression. Mutations were found in only two genes, sag-1 and eat-16. Animals defective in either gene display a hyperactive phenotype similar to that of goa-1 loss-of-function mutants. Double-mutant analysis indicates that both sag-1 and eat-16 act downstream of, or parallel to, G_oalpha and negatively regulate EGL-30 (G_qalpha) signaling. eat-16 encodes a regulator of G protein signaling (RGS) most similar to the mammalian RGS7 and RGS9 proteins and can inhibit endogenous mammalian G_q/G₁₁ in COS-7 cells. Animals defective in both sag-1 and eat-16 are not viable, but reducing function in egl-30 restores viability, indicating that the lethality of the eat-16; sag-1 double mutant is due to excessive G_qalpha activity. Analysis of these mutations indicates that the G_o and G_q pathways function antagonistically in C. elegans, and that G_oalpha negatively regulates the G_q pathway, possibly via EAT-16 or SAG-1. It is proposed that a major cellular role of G_o is to antagonize signaling by G_q (Hajdu-Cronin, 1999).

These results are consistent with a model in which a network of G protein pathways within cells can affect behavior by both positive and negative cross talk. Although synergistic effects between G_i/o and G_q pathways are known, the results presented here indicate negative regulation of G_qalpha or its downstream targets by G_oalpha. That G_o and G_q function antagonistically in some way was implied from the opposite phenotypes of goa-1 and egl-30 mutations (Brundage, 1996). The isolation and analysis of GOA-1 suppressors involved in G_qalpha signaling support the model that G_oalpha functions to modulate behavior by down-regulating the G_q pathway in C. elegans and perhaps in other species as well. These results are analogous to the stimulatory and inhibitory effects of G_s and G_i on adenylyl cyclase, raising the possibility that G protein subunits that act antagonistically are more universal than previously thought (Hajdu-Cronin, 1999 and references).

Heterotrimeric G proteins promote microtubule forces that position mitotic spindles during asymmetric cell division in C. elegans embryos. While all previously studied G protein functions require activation by seven-transmembrane receptors, this function appears to be receptor independent. Mutating a regulator of G protein signaling, RGS-7, results in hyperasymmetric spindle movements due to decreased force on one spindle pole. RGS-7 is localized at the cell cortex, and its effects require two redundant Galpha_o-related G proteins and their nonreceptor activators RIC-8 (see Drosophila Ric-8) and GPR-1/2. Using recombinant proteins, it was found that RIC-8 stimulates GTP binding by Galpha_o and that the RGS domain of RGS-7 stimulates GTP hydrolysis by Galpha_o, demonstrating that Galpha_o passes through the GTP bound state during its activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 instead appears to promote G protein function asymmetrically in the cell, perhaps acting as a G protein effector (Hess, 2004).

The heterotrimeric G proteins that control C. elegans spindle movements operate via an activation/inactivation cycle different from the signal transduction G protein cycle. Two redundant Gα_o-related Gα proteins, GOA-1 and GPA-16, along with the Gβ subunit GPB-1 and the Gγ subunit GPC-2, are required for proper spindle movements in C. elegans embryos. Activation of these G proteins is thought to be receptor independent, since (1) it occurs in the one-cell C. elegans zygote, which is encased by an impermeable egg shell, so that no source of an extracellular ligand is obvious, and (2) a set of nontransmembrane proteins have been identified that appear to activate the G proteins in lieu of transmembrane receptor(s). Removal of any of these activators results in spindle movement defects similar to those in embryos lacking the Gα proteins. The activators include the 97% identical GPR-1 and GPR-2 proteins, which contain a GPR/GoLoco motif that binds GOA-1 in its GDP bound form. The involvement of Gα_o and GPR/GoLoco proteins in mitotic spindle control appears to be evolutionarily conserved, since the GPR/GoLoco motif protein PINS acts with a Gα_i/o protein to control asymmetric neuroblast divisions in Drosophila, the mammalian GPR/GoLoco protein LGN regulates mitotic spindle organization, and the mammalian Gα_o protein is found associated with the mitotic spindle in cultured cells. In C. elegans, GPR-1/2 proteins form a complex with the coiled-coil protein LIN-5, which localizes GPR-1/2 to the cell cortex and mitotic spindle. An additional nonreceptor activator that controls C. elegans centrosome movements is RIC-8, whose mammalian ortholog Ric-8A was recently shown to act in vitro as a guanine nucleotide exchange factor for G proteins including Gα_o (Hess, 2004).

Fundamental issues regarding the mechanism of asymmetric spindle positioning remain unresolved: (1) all models propose that asymmetric microtubule forces are generated by greater G protein activity at the posterior than at the anterior pole of the zygote, but it remains unclear how such asymmetric G protein activity is generated; (2) alternative models have been proposed in which either a Gα·GDP/GPR complex or Gα·GTP is the active G protein species that promotes microtubule forces, but it remains to be established which of these species are actually generated and active; (3) the mechanism by which an active G protein controls microtubule forces is unknown (Hess, 2004).

This study shows that an RGS protein, RGS-7, controls asymmetric movements of the mitotic spindle. RGS-7 affects force on the anterior but not the posterior spindle pole, suggesting that it is a source of asymmetric G protein function. In vitro, RIC-8 promotes GTP binding by Gα_o, while RGS-7 acts as a Gα_o GTPase activator, demonstrating that Gα_o is present in its GTP bound form as part of its receptor-independent activity cycle. While GTPase activators typically inactivate G proteins, RGS-7 apparently promotes G protein function. RGS-7 could serve dual roles as both a Gα_o inactivator and a Gα_o effector so that its net function is to promote microtubule force (Hess, 2004).

Signal transduction through heterotrimeric G proteins is critical for sensory response across species. Regulator of G protein signaling (RGS) proteins are negative regulators of signal transduction. This study describes a role for C. elegans RGS-3 in the regulation of sensory behaviors. rgs-3 mutant animals fail to respond to intense sensory stimuli but respond normally to low concentrations of specific odorants. Loss of RGS-3 leads to aberrantly increased G protein-coupled calcium signaling but decreased synaptic output, ultimately leading to behavioral defects. Thus, rgs-3 responses are restored by decreasing G protein-coupled signal transduction, either genetically or by exogenous dopamine, by expressing a calcium-binding protein to buffer calcium levels in sensory neurons or by enhancing glutamatergic synaptic transmission from sensory neurons. Therefore, while RGS proteins generally act to downregulate signaling, loss of a specific RGS protein in sensory neurons can lead to defective responses to external stimuli (Ferkey, 2007).

RGS12 and RGS14: RGS proteins with the highest level of homology to Drosophila Loco

Two novel rat regulators of G-protein signaling (RGS) cDNAs were cloned using a degenerate PCR strategy. The rRgs12 and rRgs14 cDNAs encode predicted polypeptides of 1387 and 544 amino acids, respectively. The human ortholog of rRgs12 was identified by alignment of cosmid sequences in the database that map the human RGS12 gene to chromosome 4p16.3. Furthermore, human ESTs with high homology to rRgs14 that map to human chromosome 5qter were identified. Northern blot analysis indicates that rRgs14 is expressed at high levels in brain, lung, and spleen, whereas rRgs12 is expressed at high levels in brain and lung and lower levels in testis, heart, and spleen. Analysis of the predicted rRGS12 and rRGS14 polypeptides indicates that they are closely related and possess regions of homology outside of the conserved RGS domain. Conserved regions in RGS12 were identified which are similar to protein domains found in mouse rhophilin and coiled-coil proteins, suggesting possible interactions with ras-like G-proteins (Snow, 1997).

G protein alpha interaction with RGS proteins

Using the yeast two-hybrid system, a human protein GAIP (G Alpha Interacting Protein) has been isolated that specifically interacts with the heterotrimeric GTP-binding protein G alpha i3. Interaction was verified by specific binding of in vitro-translated G alpha i3 with a GAIP-glutathione S-transferase fusion protein. GAIP is a small protein (217 amino acids, 24 kDa) that contains two potential phosphorylation sites for protein kinase C and seven for casein kinase 2. GAIP shows high homology to two previously identified human proteins, GOS8 and 1R20; two Caenorhabditis elegans proteins, CO5B5.7 and C29H12.3, and the FLBA gene product in Aspergillus nidulans -- all of unknown function. Significant homology was also found to the SST2 gene product in Saccharomyces cerevisiae that is known to interact with a yeast G alpha subunit (Gpa1). A highly conserved core domain of 125 amino acids characterizes this family of proteins. Analysis of deletion mutants demonstrates that the core domain is the site of GAIP's interaction with G alpha i3. GAIP is likely to be an early inducible phosphoprotein, because in its 3'-untranslated region its cDNA contains the TTTTGT sequence characteristic of early response genes. By Northern analysis GAIP's 1.6-kb mRNA is most abundant in lung, heart, placenta, and liver and is very low in brain, skeletal muscle, pancreas, and kidney. GAIP appears to interact exclusively with G alpha i3, since it did not interact with G alpha i2 and G alpha q. The fact that GAIP and Sst2 interact with G alpha subunits and share a common domain suggests that other members of the GAIP family also interact with G alpha subunits through the 125-amino-acid core domain (De Vries, 1995).

Palmitoylation inhibits by more than 90 percent the response of the alpha subunit of the guanine nucleotide-binding protein Gz to the guanosine triphosphatase (GTPase)-accelerating activity of Gz GAP, a Gz-selective member of the regulators of G-protein signaling (RGS) protein family of GTPase-activating proteins (GAPs). Palmitoylation both decreases the affinity of Gz GAP for the GTP-bound form of Galphaz by at least 90 percent and decreases the maximum rate of GTP hydrolysis. Inhibition is reversed by removal of the palmitoyl group. Palmitoylation of Galphaz also inhibits its response to the GAP activity of Galpha-interacting protein (GAIP), another RGS protein, and palmitoylation of Galphai1 inhibits its response to RGS4. The extent of inhibition of Gz GAP, GAIP, RGS4, and RGS10 correlates roughly with their intrinsic GAP activities for the Galpha target used in the assay. Reversible palmitoylation is thus a major determinant of Gz deactivation after its stimulation by receptors, and may be a general mechanism for prolonging or potentiating G-protein signaling (Tu, 1997).

Protein regulators of G protein signaling (RGS proteins) were discovered as negative regulators of heterotrimeric G protein-mediated signal transduction in yeast and worms. Experiments with purified recombinant proteins in vitro have established that RGS proteins accelerate the GTPase activity of certain G protein alpha subunits (the reaction responsible for their deactivation); these subunits can also act as effector antagonists. Either of two such RGS proteins, RGS4 or GAIP, attenuate signal transduction mediated by endogenous receptors, G proteins, and effectors when stably expressed as tagged proteins in transfected mammalian cells. The pattern of selectivity observed in vivo is similar to that seen in vitro. RGS4 and GAIP both attenuate Gi-mediated inhibition of cAMP synthesis. RGS4 is more effective than GAIP in blocking Gq-mediated activation of phospholipase Cbeta (C. Huang, 1997).

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals, probably by acting as GTPase-activating proteins for Galphai subunits. To find out whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, a comparison was undertaken of the activation of mitogen-activated protein kinase (MAPK) by (1) a Gq/11-coupled receptor [the bombesin receptor (BR)], and (2) a Gi-coupled receptor (the D2 dopamine receptor). RGS and these receptors were transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocks MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibits activation of MAPK by both receptors, causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibits BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibits inositol phosphate synthesis activated by the addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein (Yan, 1997).

Regulators of heterotrimeric G protein signaling (RGS) proteins are GTPase-activating proteins (GAPs) that accelerate GTP hydrolysis by Gq and Gi alpha subunits, thus attenuating signaling. Mechanisms that provide more precise regulatory specificity have been elusive. An N-terminal domain of RGS4 discriminates among receptor signaling complexes coupled via Gq. Accordingly, deletion of the N-terminal domain of RGS4 eliminates receptor selectivity and reduces potency by 10(4)-fold. Receptor selectivity and potency of inhibition are partially restored when the RGS4 box is added together with an N-terminal peptide. In vitro reconstitution experiments also indicate that sequences flanking the RGS4 box are essential for high potency GAP activity. Thus, RGS4 regulates Gq class signaling by the combined action of two domains: 1) the RGS box accelerates GTP hydrolysis by Galphaq and 2) the N terminus conveys high affinity and receptor-selective inhibition. These activities are each required for receptor selectivity and high potency inhibition of receptor-coupled Gq signaling (Zeng, 1998).

An investigation was carried out of the function and the mechanism of action of RGS3. An 80-kDa protein has been identified as RGS3 by immunoprecipitation and immunoblotting with anti-RGS3 antibodies in a human mesangial cell line (HMC) stably transfected with RGS3 cDNA. RGS3 binds to aluminum fluoride-activated Galpha11 and to a lesser extent to Galphai3. This binding is mediated by the RGS domain of RGS3. A role of RGS3 in postreceptor signaling was demonstrated by decreased calcium responses and mitogen-activated protein (MAP) kinase activity induced by endothelin-1 in HMC stably overexpressing RGS3. Depletion of endogenous RGS3 by transfection of antisense RGS3 cDNA in NIH 3T3 cells results in enhanced MAP kinase activation induced by endothelin-1. RGS3 has a unique cytosolic localization. Activation of G proteins by AlF4-, NaF, or endothelin-1 results in redistribution of RGS3 from cytosol to the plasma membrane. Agonist-induced translocation of RGS3 occurs by a dual mechanism involving both C-terminal (RGS domain) and N-terminal regions of RGS3. Thus, coexpression of RGS3 with a constitutively active mutant of Galpha11 (Galpha11-QL) results in the binding of RGS3 (but not of its N-terminal fragment) to the membrane fraction and in its interaction with Galpha11-QL in vitro without any stimuli. However, both full-length RGS3 and its N-terminal domain translocate to the plasma membrane upon stimulation of intact cells with endothelin-1. The effect of endothelin-1 is mimicked by calcium ionophore A23187, suggesting the importance of Ca2+ in the mechanism of redistribution of RGS3. These data indicate that RGS3 inhibits G protein-coupled receptor signaling by a complex mechanism involving its translocation to the membrane in addition to its established function as a GTPase-activating protein (Dulin, 1999).

Regulators of G protein signaling (RGS) modulate G protein activity by functioning as GTPase-activating proteins (GAPs) for alpha-subunits of heterotrimeric G proteins. RGS14 regulates G protein nucleotide exchange and hydrolysis by acting as a GAP through its RGS domain and as a guanine nucleotide dissociation inhibitor (GDI) through its GoLoco motif. RGS14 exerts GDI activity on Galpha_i1, but not Galpha_o. Selective interactions are mediated by contacts between the alphaA and alphaB helices of the Galpha_i1 helical domain and the GoLoco C terminus. Three isoforms of Galpha_i exist in mammalian cells. This study asked whether all three isoforms were subject to RGS14 GDI activity. RGS14 inhibits guanine nucleotide exchange on Galpha_i1 and Galpha_i3, but not Galpha_i2. Galpha_i2 could be rendered sensitive to RGS14 GDI activity by replacement of residues within the alpha-helical domain. In addition to the contact residues in the alphaA and alphaB helices previously identified, it was found that the alphaA/alphaB and alphaB/alphaC loops are important determinants of Galpha_i selectivity. The striking selectivity observed for RGS14 GDI activity in vitro points to Galpha_i1 and Galpha_i3 as the likely targets of RGS14-GoLoco regulation in vivo (Mittal, 2004).

RGS proteins stimulate the intrinsic GTPase activity of the a subunits of heterotrimeric G-proteins, and thereby negatively regulate G-protein-coupled receptor signalling. RGS14 stimulates the GTPase activities of Ga_o and Ga_i subunits through its N-terminal RGS domain, and down-modulates signalling from receptors coupled to G_i. It also contains a central domain that binds active Rap proteins, as well as a C-terminal GoLoco/G-protein regulatory motif that has been shown to act in vitro as a GDP-dissociation inhibitor for Ga_i. In order to elucidate the respective contributions of the three functional domains of RGS14 to its ability to regulate G_i signalling, RGS14 mutants invalidated in each of its domains, as well as truncated molecules, were generated and their effects on G_i signalling were assessed via the bg pathway in a stable cell line ectopically expressing the G_i-coupled M2 muscarinic acetylcholine receptor (HEK-m2). The RGS and GoLoco domains of RGS14 are independently able to inhibit signalling downstream of G_i. Targeting of the isolated GoLoco domain to membranes, by myristoylation/palmitoylation or Rap binding, enhances its inhibitory activity on G_i signalling. Finally, in the context of the full RGS14 molecule, the RGS and GoLoco domains co-operate to confer maximal activity on RGS14. It is therefore proposed that RGS14 combines the inhibition of G_i activation or coupling to receptors via its GoLoco domain with stimulation of the GTPase activity of Ga_i-GTP via its RGS domain to negatively regulate signalling downstream of G_i (Traver, 2004).

Heterotrimeric G-proteins bind to cell-surface receptors and are integral in transmission of signals from outside the cell. Upon activation of the Galpha subunit by binding of GTP, the Galpha and Gbetagamma subunits dissociate and interact with effector proteins for signal transduction. Regulatory proteins with the 19-amino-acid GoLoco motif can bind to Galpha subunits and maintain G-protein subunit dissociation in the absence of Galpha activation. This study describes the structural determinants of GoLoco activity as revealed by the crystal structure of Galpha(i1) GDP bound to the GoLoco region of the RGS protein RGS14. Key contacts are described between the GoLoco motif and Galpha protein, including the extension of GoLoco's highly conserved Asp/Glu-Gln-Arg triad into the nucleotide-binding pocket of Galpha to make direct contact with the GDP alpha- and beta-phosphates. The structural organization of the GoLoco Galpha(i1) complex, when combined with supporting data from domain-swapping experiments, suggests that the Galpha all-helical domain and GoLoco-region carboxy-terminal residues control the specificity of GoLoco Galpha interactions (Kimple, 2002).

G protein beta interaction with RGS proteins

Regulators of G protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) toward the alpha subunits of heterotrimeric, signal-transducing G proteins. RGS11 contains a G protein gamma subunit-like (GGL) domain between its Dishevelled/Egl-10/Pleckstrin and RGS domains. GGL domains are also found in RGS6, RGS7, RGS9, and the Caenorhabditis elegans protein EGL-10. Coexpression of RGS11 with different Gbeta subunits reveals specific interaction between RGS11 and Gbeta5. The expression of mRNA for RGS11 and Gbeta5 in human tissues overlaps. The Gbeta5/RGS11 heterodimer acts as a GAP on Galphao, apparently selectively. RGS proteins that contain GGL domains appear to act as GAPs for Galpha proteins and form complexes with specific Gbeta subunits, adding to the combinatorial complexity of G protein-mediated signaling pathways (Snow, 1998b).

The G protein beta subunit Gbeta5 deviates significantly from the other four members of Gbeta-subunit family in amino acid sequence and subcellular localization. To detect the protein targets of Gbeta5 in vivo, a native Gbeta5 protein complex was isolated from the retinal cytosolic fraction and the protein tightly associated with Gbeta5 was identified as the regulator of G protein signaling (RGS) protein, RGS7. Complexes of Gbeta5 with RGS proteins can be formed in vitro from the recombinant proteins. The reconstituted Gbeta5-RGS dimers are similar to the native retinal complex in their behavior on gel-filtration and cation-exchange chromatographies and can be immunoprecipitated with either anti-Gbeta5 or anti-RGS7 antibodies. The specific Gbeta5-RGS7 interaction is determined by a distinct domain in RGS that has a striking homology to Ggamma subunits. Deletion of this domain prevents the RGS7-Gbeta5 binding, although the interaction with Galpha is retained. Substitution of the Ggamma-like domain of RGS7 with a portion of Ggamma1 changes its binding specificity from Gbeta5 to Gbeta1. The interaction of Gbeta5 with RGS7 blocks the binding of RGS7 to the Galpha subunit Galphao, indicating that Gbeta5 is a specific RGS inhibitor (Levay, 1999).

RGS14 is a mitotic spindle protein essential from the first division of the mammalian zygote

Heterotrimeric G protein alpha subunits, RGS proteins, and GoLoco motif proteins have been implicated in the control of mitotic spindle dynamics in C. elegans and D. melanogaster. RGS14 is expressed by the mouse embryonic genome immediately prior to the first mitosis, where it colocalizes with the anastral mitotic apparatus of the mouse zygote. Loss of Rgs14 expression in the mouse zygote results in cytofragmentation and failure to progress to the 2-cell stage. RGS14 is found in all tissues and segregates to the nucleus in interphase and to the mitotic spindle and centrioles during mitosis. Alteration of RGS14 levels in exponentially proliferating cells leads to cell growth arrest. These results indicate that RGS14 is one of the earliest essential products of the mammalian embryonic genome yet described and has a general role in mitosis (Martin-McCaffrey, 2004).

RGS protein expression and function in neurons and the brain

A clone of the regulator of G-protein signaling, RGS9, was isolated from a rat striatum-minus-cerebellum-minus-hippocampus subtracted library generated by directional tag polymerase chain reaction subtraction. The full-length cDNA clone encodes a 444 amino acid protein containing a 118 amino acid RGS domain, which corresponds to an evolutionarily conserved domain that is present in all members of the RGS family of proteins. Outside of the homology domain, RGS9 shows more extended similarity to human RGS6 and RGS7, rat RGS12, and the C. elegans protein EGL-10. During embryonic and early postnatal stages of development, two RGS9 transcripts of approximately 1.4 Kb and 1.8 Kb are detected in whole brain. After postnatal day 10, accumulation of the larger transcript increases progressively (until adulthood) at the expense of the smaller transcript, which is undetectable in the adult. In adult rat brain, the 1.8-Kb RGS9 transcript is detected in the striatum but not in other brain regions or peripheral tissues. In situ hybridization in rat and mouse demonstrates that RGS9 mRNA is expressed predominantly in medium-sized, spiny neurons of the neostriatum and in neurons of the nucleus accumbens and olfactory tubercle. Relatively strong signals are also detected in some hypothalamic nuclei. Its selective expression suggests that RGS9 may play an important role in modulation of the complex signaling pathways of the basal ganglia (Thomas, 1998).

Regulators of G-protein signaling (RGS) proteins act as GTPase-activating proteins (GAPs) for alpha subunits of heterotrimeric G-proteins. Previous in situ hybridization analysis of mRNAs encoding RGS3-RGS11 reveal region-specific expression patterns in rat brain. RGS9 shows a particularly striking pattern of almost exclusive enrichment in striatum. In a parallel study, RGS9 cDNA (here referred to as RGS9-1) was cloned from retinal cDNA libraries, and the encoded protein was identified as a GAP for transducin (Galphat) in rod outer segments. A novel splice variant of RGS9, RGS9-2, encodes a striatal-specific isoform of the protein. RGS9-2 is 191 amino acids longer than the retinal isoform, has a unique 3' untranslated region, and is highly enriched in striatum, with much lower levels seen in other brain regions and no expression detectable in retina. RGS9-2 protein is restricted to striatal neuropil and absent in striatal terminal fields. The functional activity of RGS9-2 is supported by the finding that it, but not RGS9-1, dampens the Gi/o-coupled mu-opioid receptor response in vitro. Characterization of a bacterial artificial chromosome genomic clone of approximately 200 Kb indicates that these isoforms represent alternatively spliced mRNAs from a single gene and that the RGS domain, conserved among all known RGS members, is encoded over three distinct exons. The distinct C-terminal domains of RGS9-2 and RGS9-1 presumably contribute to unique regulatory properties in the neural and retinal cells in which these proteins are selectively expressed (Rahman, 1999).

Regulators of G protein signaling (RGS) proteins serve as potent GTPase-activating proteins for the heterotrimeric G proteins alphai/o and aq/11. This study describes the immunohistochemical distribution of RGS7 throughout the adult rat brain and its cellular colocalization with Galphaq/11, an important G protein-coupled receptor signal transducer for phospholipase Cbeta-mediated activity. In general, both RGS7 and Galphaq/11 display a heterogeneous and overlapping regional distribution. RGS7 immunoreactivity is observed in cortical layers I-VI, being most intense in the neuropil of layer I. In the hippocampal formation, RGS7 immunoreactivity is concentrated in the strata oriens, strata radiatum, mossy fibers, and polymorphic cells, with faint to nondetectable immunolabeling within the dentate gyrus granule cells and CA1-CA3 subfield pyramidal cells. Numerous diencephalic and brainstem nuclei also display dense RGS7 immunostaining. Dual immunofluorescence labeling studies with the two protein-specific antibodies indicate a cellular selectivity in the colocalization between RGS7 and Galphaq/11 within many discrete brain regions, such as the superficial cortical layer I, hilus area of the hippocampal formation, and cerebellar Golgi cells. To assess the ability of Galphaq/11-mediated signaling pathways to dynamically modulate RGS expression, primary cortical neuronal cultures were incubated with phorbol 12,13-dibutyrate, a selective protein kinase C activator. A time-dependent increase in levels of mRNA for RGS7, but not RGS4, is observed. These results provide novel information on the region- and cell-specific pattern of distribution of RGS7 with the transmembrane signal transducer, Galphaq/11. A possible RGS7-selective neuronal feedback adaptation on Galphaq/11-mediated pathway function is described which may play an important role in signaling specificity in the brain (Khawaja, 1999).

Long-term neuronal plasticity is known to be dependent on rapid de novo synthesis of mRNA and protein. Recent studies provide insight into the molecules involved in this response. mRNA encoding a member of the regulator of G-protein signaling (RGS) family, RGS2, is rapidly induced in neurons of the hippocampus, cortex, and striatum in response to stimuli that evoke plasticity. Although several members of the RGS family are expressed in brain with discrete neuronal localizations, RGS2 appears unique in that its expression is dynamically responsive to neuronal activity. In biochemical assays, RGS2 stimulates the GTPase activity of the alpha subunit of Gq and Gi1. The effect on Gi1 was observed only after reconstitution of the protein in phospholipid vesicles containing M2 muscarinic acetylcholine receptors. RGS2 also inhibits both Gq- and Gi-dependent responses in transfected cells. These studies suggest a novel mechanism linking neuronal activity and signal transduction (Ingi, 1998).

A novel splice variant of RGS 9 was isolated from a rat hypothalamus, human retina, and a human kidney (Wilm's) tumor. This variant, termed RGS 9L, differs from the retinal form (termed RGS 9S) in that it contains a 211- (rat) or 205- (human) amino acid proline-rich domain on the carboxyl terminus. The pattern of RGS 9 mRNA splicing is tissue specific, with striatum, hypothalamus and nucleus accumbens expressing RGS 9L, whereas retina and pineal express RGS 9S almost exclusively. This pattern of mRNA splicing seemed to be highly conserved between human and rodents, suggesting cell-specific differences in the function of these variants. Transient expression of RGS 9L augments basal and beta-adrenergic receptor-stimulated adenylyl cyclase activity while suppressing dopamine D2 receptor-mediated inhibition. Furthermore, RGS 9L expression greatly accelerates the decay of dopamine D2 receptor-induced GIRK current. These results indicate that RGS 9L inhibits heterotrimeric Gi function in vivo, probably by acting as a GTPase-activating protein. The human RGS 9 gene was localized to chromosome 17 q23-24 by radiation hybrid and fluorescent in situ hybridization analyses. The RGS 9 gene is within a previously defined locus for retinitis pigmentosa (RP 17), a disease that has been linked to genes in the rhodopsin/transducin/cGMP signaling pathway (Granneman, 1998).

The present study demonstrates that the regulator of G-protein-signaling protein type 4 (RGS4) is differentially regulated in the locus coeruleus (LC) and the paraventricular nucleus (PVN) of the hypothalamus by chronic stress and glucocorticoid treatments. Acute or chronic administration of corticosterone to adult rats decreases RGS4 mRNA levels in the PVN but increases these levels in the LC. Similarly, chronic unpredictable stress decreases RGS4 mRNA levels in the PVN but has a strong trend to increase these levels in the LC. Chronic stress also decreases RGS4 mRNA levels in the pituitary. The molecular mechanisms of RGS4 mRNA regulation were further investigated in vitro in the LC-like CATH.a cell line and the neuroendocrine AtT20 cell line using the synthetic corticosterone analog dexamethasone. Consistent with the findings in vivo, dexamethasone treatment causes a dose- and time-dependent decrease in RGS4 mRNA levels in AtT20 cells but a dose- and time-dependent increase in CATH.a cells. RGS4 mRNA regulation seen in these two cell lines seems to be attributable, at least in part, to opposite changes in mRNA stability. The differential regulation of RGS4 expression in the LC and in key relays of the hypothalamic-pituitary-adrenal axis could contribute to the brain's region-specific and long-term adaptations to stress (Ni, 1999).

RGS proteins and muscarinic receptors

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs (Hosey, 1999).

RGS proteins and monovalent cation channels

Transmembrane signal transduction via heterotrimeric G proteins is reported to be inhibited by RGS (regulators of G-protein signaling) proteins. These RGS proteins work by increasing the GTPase activity of G protein alpha-subunits (G alpha), thereby driving G proteins into their inactive GDP-bound form. However, it is not known how RGS proteins regulate the kinetics of physiological responses that depend on G proteins. The isolation of a full-length complementary DNA encoding a neural-tissue-specific RGS protein, RGS8, is reported and its function has been determined. RGS8 binds preferentially to the alpha-subunits G(alpha)o and G(alpha)i3 and it functions as a GTPase-activating protein (GAP). When co-expressed in Xenopus oocytes with a G-protein-coupled receptor and a G-protein-coupled inwardly rectifying K+ channel (GIRK1/2), RGS8 accelerates not only the turning off but also the turning on of the GIRK1/2 current upon receptor stimulation, without affecting the dose-response relationship. It is concluded that RGS8 accelerates the modulation of G-protein-coupled channels and is not just a simple negative regulator. This property of RGS8 may be crucial for the rapid regulation of neuronal excitability upon stimulation of G-protein-coupled receptors (Doupnik, 1997).

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. Heterologous expression of regulators of G protein signaling (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the signal transduction pathway from the native receptor to GIRK channels. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors are dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship is significantly affected by RGS expression, although the agonist-independent basal GIRK current is suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of slow postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues (Doupnik, 1997).

RGS proteins and Ca2+ signaling

Regulators of G protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits, thereby attenuating signaling. RGS4 is a GTPase-activating protein for Gi and Gq class alpha subunits. In the present study, knockouts of Gq class genes in mice were used to evaluate the potency and selectivity of RGS4 in modulating Ca2+ signaling transduced by different Gq-coupled receptors. RGS4 inhibits phospholipase C activity and Ca2+ signaling in a receptor-selective manner in both permeabilized cells and cells dialyzed with RGS4 through a patch pipette. Receptor-dependent inhibition of Ca2+ signaling by RGS4 is observed in acini prepared from the rat and mouse pancreas. The response of mouse pancreatic acini to carbachol is about 4- and 33-fold more sensitive to RGS4 than that of bombesin and cholecystokinin (CCK), respectively. RGS1 and RGS16 are also potent inhibitors of Gq-dependent Ca2+ signaling and act in a receptor-selective manner. RGS1 shows approximately 1000-fold higher potency in inhibiting carbachol than CCK-dependent signaling. RGS16 is as effective as RGS1 in inhibiting carbachol-dependent signaling but only partially inhibits the response to CCK. By contrast, RGS2 inhibits the response to carbachol and CCK with equal potency. The same pattern of receptor-selective inhibition by RGS4 is observed in acinar cells from wild type and several single and double Gq class knockout mice. Thus, these receptors appear to couple Gq class alpha subunit isotypes equally. Differences in receptor selectivity for the action of RGS proteins indicates that regulatory specificity is conferred by interaction of RGS proteins with receptor complexes (Xu, 1999).

Regulators of G protein signaling (RGS) proteins bind to the alpha subunits of certain heterotrimeric G proteins and greatly enhance their rate of GTP hydrolysis, thereby determining the time course of interactions among Galpha, Gbetagamma, and their effectors. Voltage-gated N-type Ca2+ channels mediate neurosecretion, and these Ca2+ channels are powerfully inhibited by G proteins. To determine whether RGS proteins could influence Ca2+ channel function, the activity was recorded for N-type Ca2+ channels coexpressed in human embryonic kidney (HEK293) cells with G protein-coupled muscarinic (m2) receptors and various RGS proteins. Coexpression of full-length RGS3T, RGS3, or RGS8 significantly attenuates the magnitude of receptor-mediated Ca2+ channel inhibition. In control cells expressing alpha1B, alpha2, and beta3 Ca2+ channel subunits and m2 receptors, carbachol (1 microM) inhibits whole-cell currents by approximately 80% compared with only approximately 55% inhibition in cells also expressing exogenous RGS protein. A similar effect is produced by expression of the conserved core domain of RGS8. The attenuation of Ca2+ current inhibition results primarily from a shift in the steady state dose-response relationship to higher agonist concentrations. The kinetics of Ca2+ channel inhibition are also modified by RGS. Thus, in cells expressing RGS3T, the decay of prepulse facilitation is slower, and recovery of Ca2+ channels from inhibition after agonist removal is faster than in control cells. The effects of RGS proteins on Ca2+ channel modulation can be explained by their ability to act as GTPase-accelerating proteins for some Galpha subunits. These results suggest that RGS proteins may play important roles in shaping the magnitude and kinetics of physiological events, such as neurosecretion, that involve G protein-modulated Ca2+ channels (Melliti, 1999).

The functional roles subserved by G(alpha)z, a G protein alpha subunit found predominantly in neuronal tissues, have remained largely undefined. G(alpha)z couples neurotransmitter receptors to N-type Ca2+ channels when transiently overexpressed in rat sympathetic neurons. The G(alpha)z-mediated inhibition is voltage dependent and PTX insensitive. Recovery from G(alpha)z-mediated inhibition is extremely slow but accelerated by coexpression with RGS proteins. G(alpha)z selectively interacts with a subset of receptors that ordinarily couple to N-type Ca2+ channels via PTX-sensitive Go/i proteins. In addition, G(alpha)z rescues the activation of heterologously expressed GIRK channels in PTX-treated neurons. These results suggest that G(alpha)z is capable of coupling receptors to ion channels and might underlie PTX-insensitive ion channel modulation observed in neurons under physiological and pathological conditions (Jeong, 1998).

Group I mGluRs heterologously expressed in sympathetic neurons inhibit calcium [I(Ca)] and M-type potassium [I(M)] currents. Treatment with pertussis toxin (PTX) reveals a voltage-dependent, PTX-sensitive component of I(Ca) inhibition and a voltage-independent, PTX-insensitive component. Coexpression of RGS2 occludes mGluR1a inhibition of I(M) and makes I(Ca) inhibition voltage-dependent in PTX-treated cells, presumably by blocking the effects of G alpha(q/11)-GTP. These data indicate that mGluR1a can couple to G(i/o) as well as G(q/11). In addition, voltage-independent I(Ca) inhibition proceeds through a G alpha(q/11)-GTP-mediated pathway, which can be occluded by expressing RGS2, leaving active the voltage-dependent G betagamma-mediated inhibition. These data may reveal a functional role for the upregulation of RGS2 expression in in vivo systems (Kammermeier, 1999).

Agonist-evoked [Ca²⁺]_i oscillations have been considered a biophysical phenomenon reflecting the regulation of the IP₃ receptor by [Ca²⁺]_i. [Ca²⁺]_i oscillations are a biochemical phenomenon emanating from regulation of Ca2+ signaling by the regulators of G protein signaling (RGS) proteins. [Ca²⁺]_i oscillations evoked by G protein-coupled receptors require the action of RGS proteins. Inhibition of endogenous RGS protein action disrupts agonist-evoked [Ca²⁺]_i oscillations by a stepwise conversion to a sustained response. Based on these findings and the effect of mutant RGS proteins and anti-RGS protein antibodies on Ca2+ signaling, it is proposed that RGS proteins within the G protein-coupled receptor complexes provide a biochemical control of [Ca²⁺]_i oscillations (Luo, 2001).

A possible model for the biochemical control of [Ca²⁺]_i oscillation by RGS proteins is based on the present work and on the regulation of RGS protein function by PIP₃ and Ca²⁺-calmodulin (CaM). In the resting state, RGS proteins in signaling complexes are active and exert tonic inhibition by converting all spontaneously generated alphaq·GTP to alphaq·GDP. The findings that (1) all recombinant RGS proteins that interact with Galphaq tested to date are potent inhibitors of Ca²⁺ signaling when infused into cells and (2) activation of IP₃-dependent signaling by the K/Q mutant, and activation of Ca²⁺ signaling by the anti-RGS protein Abs show that RGS proteins are indeed active in resting cells to exert tonic inhibition of Ca²⁺ signaling. The agonist-activated receptor simulates the GDP/GTP exchange reaction to increase the rate of generation and the steady-state concentration of alpha·GTP. At this stage, the rate of alpha·GTP generation is faster than the rate of RGS protein-assisted GTP hydrolysis, resulting in the activation of PLCbeta, hydrolysis of PIP₂ to generate IP₃, and initiation of Ca²⁺ (step 1). In the first of the four steps described here, the activated receptor may not only activate Galpha, but may also stabilize the inactive state of RGS proteins by promoting the formation of PIP₃. The rate and extent of Galpha·GTP production and inhibition of RGS protein GAP activity is a function of agonist concentration. The Galphaq·PLCbeta complex continues to produce IP₃ and release Ca²⁺ until all stores exposed to IP₃ are depleted. At low concentration of agonist, only the stores in the vicinity of signaling complexes are exposed to IP₃. At increasing agonist concentration and, thus, IP₃ production, an increased fraction of the stores is discharged by IP₃ (Luo, 2001).

At the end of Ca²⁺ release and at the peak of [Ca²⁺]_i increase, high [Ca²⁺]_i in the vicinity of the IP₃ pore inhibits the channel to reduce the Ca²⁺ permeability of the stores' membrane. To terminate the stimulated state, the activity of RGS proteins has to be restored. This can be by formation of sufficient [Ca²⁺-CaM] to relieve the inhibition of RGS protein GAP activity by PIP₃ (step 2). This will lead to binding of the Ca²⁺/CaM/RGS protein to alphaq·GTP, acceleration of GTP hydrolysis, and inhibition of PLCbeta and IP₃ production. The reduction in [IP₃] together with reduced IP₃R channel activity, reloads the stores with Ca²⁺ (step 3). It is proposed that subsequent dissociation of Ca²⁺-CaM from RGS proteins stabilizes the RGS protein conformation that binds PIP₃ (or another inhibitor) to block RGS GAP activity (step 4). An important feature of the transition between step 3 and step 4 is that Galphaq cannot activate PLCbeta until Ca²⁺-CaM dissociates from RGS proteins. This provides a plausible mechanism for regulation of oscillation frequency. For many calmodulin-regulated proteins, regulation of enzymatic activity by calmodulin shows a hysteresis behavior. Upon Ca²⁺ increase, binding of calmodulin to target proteins and their activation is fast, whereas upon reduction of Ca²⁺, dissociation of Ca²⁺-CaM and termination of the active state is slow. Such behavior may also be a feature of the interaction of Ca²⁺-CaM with RGS proteins and determines the duration of the delay between [Ca²⁺]_i spikes. At low agonist concentrations, the rates of Galphaq activation and, possibly, dissociation of Ca²⁺-CaM from RGS proteins are slow, resulting in a low frequency oscillation in [IP₃] and [Ca²⁺]_i. Increasing agonist concentration can increase [Ca²⁺]_i oscillation frequency by increasing the rates of Galphaq activation and/or dissociation of Ca²⁺-CaM from RGS proteins. At very high agonist concentration, the rate of Galphaq activation is maximal, and RGS proteins remain in the inactive, PIP₃-bound state. This would, of course, result in a sustained response (Luo, 2001).

Biochemical control of Ca²⁺ oscillations by RGS proteins can explain several features of the oscillations. (1) The mode of activation of Galphaq by the receptor and inhibition of Galphaq by Ca²⁺-CaM bound RGS proteins can generate the receptor-specific patterns of [Ca²⁺]_i spiking observed in many cell types. In fact, RGS proteins differentially interact with the muscarinic, CCK, and bombesin receptors in pancreatic acini. (2) The model can explain how several receptors respond to increasing agonist concentration with increased oscillation frequency without affecting spike amplitude. Increased rates of Galphaq activation of the same signaling complexes will result in constant amplitude but increased oscillations frequency. (3) In many cells, including pancreatic acinar cells, the shape of individual Ca²⁺ spikes is receptor specific. The shape of individual [Ca²⁺]_i spikes can be determined by the rates of Galphaq activation and periodic activation and inactivation of RGS GAP activity. (4) Slow dissociation of Ca²⁺-CaM from RGS proteins can explain oscillations in [IP₃] and maintain low [IP₃] between [Ca²⁺]_i spikes. Reduction in [IP₃] between Ca²⁺ spikes can explain how Ca²⁺ release remains inactive for long periods of time between the spikes (Luo, 2001).

It is important to note that the biochemical (by RGS proteins) and biophysical (by Ca²⁺-dependent regulation of IP₃R) regulation of [Ca²⁺]_i oscillations are not mutually exclusive. Rather, it is likely that both regulatory events determine the final shape of the oscillations. However, the results suggest that the primary oscillator is the receptor·Galphaq·RGS protein complex. Regulation of IP₃R channel activity and, probably, the Ca²⁺ pumps is necessary to aid the receptor complex in controlling the oscillations. For example, inhibition of the IP₃R channel by high local Ca²⁺ will start reuptake of Ca²⁺ into the store by the SERCA pumps prior to complete reduction in [IP₃]. By placing the oscillator at the receptor complex, the receptor governs regulation of [Ca²⁺]_i oscillations. Furthermore, in this manner the oscillations can be precisely controlled by the state of occupancy of the receptor with ligands. In other words, the mode of occupancy of the receptor with agonist will determine the type of signal transduced into the cell interior (Luo, 2001).

L7/Pcp-2 is a GoLoco domain protein encoded by a Purkinje cell dendritic mRNA. Although biochemical interactions of GoLoco proteins with Galpha_o and Galpha_i are well documented, little is known about effector function modulation resulting from these interactions. The P-type Ca2+ channels might be physiological effectors of L7 because (1) they are the major voltage-dependent Ca2+ channels (VDCC) that modulate Purkinje cell output and (2) they are regulated by G_i/o proteins. As a first step towards validating this hypothesis and to further understand the possible physiological effect of L7 protein and its two isoforms, Ca(v)2.1 channels and kappa-opioid receptors (KORs) have been coexpressed with varying amounts of L7A or L7B in Xenopus oocytes and ionic currents were measured by two-electrode voltage clamping. Without receptor activation L7 did not alter the Ca2+ channel activity. With tonic and weak activation of the receptors, however, the Ca2+ channels were inhibited by 40%-50%. This inhibition was enhanced by low, but dampened by high, expression levels of L7A and L7B and differences were observed between the two isoforms. The enhancing effect of L7 was occluded by overexpression of Gbetagamma, whereas the disinhibition was antagonized by overexpression of Galpha_o. It is proposed that L7 differentially affects the Galpha and Gbetagamma arms of receptor-induced G_i/o signaling in a concentration-dependent manner, through which it increases the dynamic range of regulation of P/Q-type Ca2+ channels by G_i/o protein-coupled receptors. This provides a framework for designing further experiments to determine how dendritic local fluctuations in L7 protein levels might influence signal processing in Purkinje cells (Kinoshita-Kawada, 2004).

RGS proteins and dopamine receptors

Of all partners involved in G-protein coupled receptor (GPCR) signalling, the regulator of G-protein signalling (RGS) proteins are the only ones showing fast gene expression changes after various stimuli. These expression changes can offer feedback regulation to GPCR signalling since RGS accelerate the return of G-proteins to their inactive form and exert regulatory functions on intracellular effectors. However, it is not yet known which RGS regulates which receptor transduction pathways in the brain. To start to answer this question, the influence was studied of specific agonists and antagonists of the dopamine D1 and D2 receptors on the gene expression of the five most abundant RGS in the striatum: RGS2, RGS4, RGS8, RGS9 and RGS10. Only changes in RGS2 and RGS4 mRNA levels were observed. The D1 agonist SKF82958 and D2 antagonist haloperidol causes an up-regulation of RGS2. The D1 antagonist SCH23390 and D2 agonist quinpirole caused a down-regulation of RGS2 and an up-regulation of RGS4. D1 and D2 receptors exert opposite effects on RGS2 expression, as they do on cAMP levels, suggesting a cAMP-mediated transcription of RGS2. This was confirmed by the unique induction of RGS2 (+ 111.1%) observed in the periventricular zone of the striatum after intracerebroventricular injection of forskolin. RGS4 is up-regulated only when RGS2 is down-regulated. This suggests that both RGS exert distinct functions. Considering the coupling of D1 and D2 receptors to the intracellular effector adenylate cyclase 5 (AC5) through their respective Galpha subunits in the striatum, these data suggest that RGS2 regulates the D1/Galphaolf/AC5 pathway and RGS4 the D2/Galphao/AC5 pathway (Taymans, 2003).

Regulators of G protein signaling (RGS) modulate heterotrimeric G proteins in part by serving as GTPase-activating proteins for Galpha subunits. A role was examined for RGS9-2, an RGS subtype highly enriched in striatum, in modulating dopamine D2 receptor function. Viral-mediated overexpression of RGS9-2 in rat nucleus accumbens (ventral striatum) reduces locomotor responses to cocaine (an indirect dopamine agonist) and to D2 but not to D1 receptor agonists. Conversely, RGS9 knockout mice show heightened locomotor and rewarding responses to cocaine and related psychostimulants. In vitro expression of RGS9-2 in Xenopus oocytes accelerates the off-kinetics of D2 receptor-induced GIRK currents, consistent with the in vivo data. Finally, chronic cocaine exposure increases RGS9-2 levels in nucleus accumbens. Together, these data demonstrate a functional interaction between RGS9-2 and D2 receptor signaling and the behavioral actions of psychostimulants and suggest that psychostimulant induction of RGS9-2 represents a compensatory adaptation that diminishes drug responsiveness (Rahman, 2003).

Regulator of G protein signaling (RGS) proteins negatively regulate receptor-mediated second messenger responses by enhancing the GTPase activity of Galpha subunits. A receptor-specific role for an RGS protein is described at the level of an individual brain neuron. RGS9-2 and Gbeta(5) mRNA and protein complexes were detected in striatal cholinergic and gamma-aminobutyric acidergic neurons. Dialysis of cholinergic neurons with RGS9 constructs enhances basal Ca(2+) channel currents and reduces D(2) dopamine receptor modulation of Cav2.2 channels. These constructs did not alter M(2) muscarinic receptor modulation of Cav2.2 currents in the same neuron. The noncatalytic DEP-GGL domain of RGS9 antagonizes endogenous RGS9-2 activity, enhancing D(2) receptor modulation of Ca(2+) currents. In vitro, RGS9 constructs accelerate GTPase activity, in agreement with electrophysiological measurements, and do so more effectively at Go than Gi. These results implicate RGS9-2 as a specific regulator of dopamine receptor-mediated signaling in the striatum and identify a role for GAP activity modulation by the DEP-GGL domain (Cabrera-Vera, 2004).

RGS proteins and vesicular trafficking

A mammalian protein called GIPC (for GAIP interacting protein, C terminus), which has a central PDZ domain and a C-terminal acyl carrier protein (ACP) domain. The PDZ domain of GIPC specifically interacts with RGS-GAIP, a GTPase-activating protein (GAP) for Galphai subunits recently localized on clathrin-coated vesicles. Analysis of deletion mutants indicates that the PDZ domain of GIPC specifically interacts with the C terminus of GAIP (11 amino acids) in the yeast two-hybrid system and glutathione S-transferase (GST)-GIPC pull-down assays, but GIPC does not interact with other members of the RGS (regulators of G protein signaling) family tested. This finding is in keeping with the fact that the C terminus of GAIP is unique and possesses a modified C-terminal PDZ-binding motif (SEA). By immunoblotting of membrane fractions prepared from HeLa cells, it was found that there are two pools of GIPC: a soluble or cytosolic pool (70%) and a membrane-associated pool (30%). By immunofluorescence, endogenous and GFP-tagged GIPC show both a diffuse and punctate cytoplasmic distribution in HeLa cells reflecting, respectively, the existence of soluble and membrane-associated pools. By immunoelectron microscopy the membrane pool of GIPC has been seen to be associated with clusters of vesicles located near the plasma membrane. These data provide direct evidence that the C terminus of a RGS protein is involved in interactions specific for a given RGS protein and implicates GAIP in the regulation of additional functions, besides its GAP activity. The location of GIPC together with its binding to GAIP suggest that GAIP and GIPC may be components of a G protein-coupled signaling complex involved in the regulation of vesicular trafficking. The presence of an ACP domain suggests a putative function for GIPC in the acylation of vesicle-bound proteins (De Vries, 1998).

Proteins of the regulators of G protein signaling (RGS) family bind to Galpha subunits to downregulate their signaling in a variety of systems. Galpha-interacting protein (GAIP) is a mammalian RGS protein that shows high affinity for the activated state of Galphai-3, a protein known to regulate post-Golgi trafficking of secreted proteins in kidney epithelial cells. This study aimed to localize GAIP in epithelial cells and to investigate its potential role in the regulation of membrane trafficking. LLC-PK1 cells were stably transfected with a c-myc-tagged GAIP cDNA. In the transfected and untransfected cells, GAIP is found in the cytosol and on cell membranes. Membrane-bound GAIP is localized on budding vesicles around Golgi stacks. When an in vitro assay is used to generate vesicles from isolated rat liver and Madin-Darby canine kidney cell Golgi membranes, GAIP is found to be concentrated in fractions of newly budded Golgi vesicles. Finally, the constitutive trafficking and secretion of sulfated proteoglycans was measured in cell lines overexpressing GAIP. Evidence is found for GAIP regulation of secretory trafficking upstream of the trans-Golgi network but not in post-Golgi secretion. The location and functional effects of GAIP overlap only partially with those of Galphai-3 and suggest multiple roles for GAIP in epithelial cells (Wylie, 1999).

RGS proteins and Wnt signaling

RGS family members are GTPase activating proteins (GAPs) that antagonize signaling by heterotrimeric G proteins. Injection of Xenopus embryos with RNA encoding rat RGS4 (rRGS4), a GAP for Gi and Gq, results in shortened trunks and decreased skeletal muscle. This phenotype is nearly identical to the effect of injection of either frzb or dominant negative Xwnt-8. Injection of human RGS2, which selectively deactivates Gq, has similar effects. rRGS4 inhibits the ability of early Xwnt-8 but not Xdsh misexpression to cause axis duplication. This effect is distinct from axin family members that contain RGS-like domains but act downstream of Xdsh. Two Xenopus RGS4 homologs have been identified, one of which, Xrgs4a, is expressed as a Spemann organizer component. Injection of Xenopus embryos with Xrgs4a also results in shortened trunks and decreased skeletal muscle. These results suggest that RGS proteins modulate Xwnt-8 signaling by attenuating the function of a G protein (Wu, 2000).

The importance of a G protein in early pattern formation raises the question of the identity of G protein effectors. One possibility is that an embryonic G protein directly activates PLC-beta or indirectly activates PLC-gamma, enzymes that generate IP3 and diacylglycerol. Previous work has demonstrated that IP3 levels rise sharply at the 64-cell stage of Xenopus embryogenesis and that they remain elevated for several hours. Diacylglycerol production can lead to protein kinase C activation, which, in turn, can phosphorylate and inactivate GSK-3beta. Indeed, wingless-induced inactivation of GSK-3beta in murine fibroblasts is sensitive to pharmacological inhibitors of PKC. A second possibility is that a G protein activates phosphatidylinositol-3 kinase, an enzyme that generates phosphatidylinositol-3,4,5-phosphate (PIP3) and other phosphorylated lipid products. The levels of PIP3 in early embryos have not been previously determined. PIP3 is an activator of protein kinase B (Akt), which can phosphorylate and inactivate GSK-3beta. In these ways, it is possible to hypothesize how activation of a G protein might lead to inactivation of GSK-3beta and stabilization of beta-catenin (Wu, 2000).

RGS proteins and visual signal transduction

The intrinsic GTPase activity of transducin (the Galpha subunit involved in visual transduction) controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory gamma-subunit of PDE (Pgamma), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. A human homolog of murine retinal specific RGS (hRGSr) was expressed in Escherichia coli and its role in the regulation of transducin GTPase activity was investigated. As with other RGS proteins, hRGSr interacts preferentially with a transitional conformation of the transducin alpha-subunit, while its binding to GtalphaGTPgammaS or GtalphaGDP is weak. hRGSr and Pgamma do not compete for the interaction with the transducin alpha-subunit. The affinity of the Pgamma interaction with transducin alpha-subunit is modestly enhanced by the addition of hRGSr. In a single turnover assay, hRGSr accelerates the GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold. Addition of Pgamma to the reconstituted system reduces the GTPase level accelerated by hRGSr. The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr are elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 microM rhodopsin these rates are elevated even further. These data suggest that the effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade (Natochin, 1997).

The rod outer segment phototransduction GAP (GTPase-accelerating protein) has been identified as RGS9, a member of the RGS family of G alpha GAPs. RGS9 mRNA expression is specific for photoreceptor cells, and RGS9 protein colocalizes with other phototransduction components to photoreceptor outer segment membranes. The RGS domain of RGS9 accelerates GTP hydrolysis by the visual G protein transducin, and this acceleration is enhanced by the gamma subunit of the phototransduction effector cGMP phosphodiesterase. These unique properties of RGS9 match those of the rod outer segment GAP and implicate it as a key element in the recovery phase of visual transduction (He, 1998).

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein alpha subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein beta subunit (Gbeta5L). RGS9 and Gbeta5L also form a complex when coexpressed in cell culture. These data are consistent with the recent observation that several RGS proteins, including RGS9, contain a G protein gamma-subunit like domain that can mediate RGS protein association with Gbeta5. An example of such a complex is reported whose cellular localization and function are clearly defined (Makino, 1999).

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Another unique property of RGS9 is reported: high specificity for Galphat. The core (RGS) domain of RGS9 stimulates Galphat GTPase activity 10-fold and Galphai1 GTPase activity only 2-fold at a concentration of 10 muM. Using chimeric Galphat/Galphai1 subunits it has been demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 are well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments are 185 nM and 2 muM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. This is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9 (Skiba, 1999).

RGS proteins, leukocytes and immunocompetent cells

Serpentine Galphai-linked receptors support rapid adhesion and directed migration of leukocytes and other cell types. The intracellular mechanisms mediating and regulating chemoattractant-directed adhesion and locomotion are only now beginning to be explored. Little is known about the GTPase activity of the Galphai proteins involved in adhesion and chemotaxis, or the significance of their regulation to these responses. Using transiently transfected lymphoid cells as a model system, it has been shown that expression of RGS1, RGS3, and RGS4 inhibits chemoattractant-induced migration. In contrast, RGS2, a regulator of Galphaq activity, has no effect on cell migration to any chemoattractant. RGS1, RGS3, and RGS4 also reduce rapid chemoattractant-triggered adhesion, although the proadhesive response appears quantitatively less sensitive to RGS action than chemotaxis. The results suggest that the duration of the Galphai signal may be a particularly important parameter in the chemotactic responses of leukocytes, and demonstrate the potential for RGS family members to regulate cellular adhesive and migratory behaviors (Bowman, 1998).

The newly recognized regulators of G protein signaling (RGS) attenuate heterotrimeric G protein signaling pathways. An IL-2-induced gene was cloned from human T cells, cytokine-responsive gene 1, which encodes a member of the RGS family, RGS16. The RGS16 protein binds Gialpha and Gqalpha proteins present in T cells, and inhibits Gi- and Gq-mediated signaling pathways. By comparison, the mitogen-induced RGS2 inhibits Gq but not Gi signaling. Moreover, the two RGS genes exhibit marked differences in expression patterns. The IL-2-induced expression of the RGS16 gene in T cells is suppressed by elevated cAMP, whereas the RGS2 gene shows a reciprocal pattern of regulation by these stimuli. Because the mitogen and cytokine receptors that trigger expression of RGS2 and RGS16 in T cells do not activate heterotrimeric G proteins, these RGS proteins and the G proteins that they regulate may play a heretofore unrecognized role in T cell functional responses to Ag and cytokine activation (Beadling, 1999).

Miscellaneous RGS protein interactions and functions

Regulator of G-protein signaling (RGS) proteins increase the intrinsic guanosine triphosphatase (GTPase) activity of G-protein alpha subunits in vitro, but how specific G-protein-coupled receptor systems are targeted for down-regulation by RGS proteins remains uncharacterized. The GTPase specificity of RGS12 is described and four alternatively spliced forms of human RGS12 mRNA are identified. Two RGS12 isoforms of 6.3 and 5.7 kilobases (kb), encoding both an N-terminal PDZ (PSD-95/Dlg/ZO-1) domain and the RGS domain, are expressed in most tissues, with highest levels observed in testis, ovary, spleen, cerebellum, and caudate nucleus. The 5.7-kb isoform has an alternative 3' end encoding a putative C-terminal PDZ domain docking site. Two smaller isoforms (3.1 and 3.7 kb), which lack the PDZ domain and encode the RGS domain with and without the alternative 3' end, respectively, are most abundantly expressed in brain, kidney, thymus, and prostate. In vitro biochemical assays indicate that RGS12 is a GTPase-activating protein for Gi class alpha subunits. Biochemical and interaction trap experiments suggest that the RGS12 N terminus acts as a classical PDZ domain, binding selectively to C-terminal (A/S)-T-X-(L/V) motifs as found within both the interleukin-8 receptor B (CXCR2) and the alternative 3' exon form of RGS12. The presence of an alternatively spliced PDZ domain within RGS12 suggests a mechanism by which RGS proteins may target specific G-protein-coupled receptor systems for desensitization (Snow, 1998a).

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. The cloning and characterization of another novel cDNA isolated from that screen is reported. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library has identified the full length D-AKAP2. It is composed of 372 amino acids, which includes the R binding fragment, residues 333-372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIalpha and RIIalpha. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Galpha protein, thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase (L. Huang, 1997).

Identification of a new family of proteins (RGS proteins) that function as negative regulators of G protein signaling has sparked new understanding of desensitization of this signaling process. Recent studies with several mammalian RGS proteins has delineated their ability to interact with and function as GTPase-activating proteins specifically for G proteins in the Gi family. The functional activity of RGS3 and a truncated form of RGS3 were investaged on G protein-coupled receptor-mediated activation of adenylyl cyclase, phosphoinositide phospholipase C, and mitogen-activated protein kinase in intact cells. Polymerase chain reaction and 5'-rapid amplification of cDNA ends analyses reveals the tissue-specific expression of a short form of the RGS3 transcript that encodes the approximate carboxyl-terminal half of RGS3. This truncated form of RGS3 (RGS3T) has been shown to function as a negative regulator of pheromone signaling in yeast. Baby hamster kidney cells transiently transfected with RGS3T cDNA exhibit a pronounced impairment in platelet-activating factor receptor-stimulated inositol phosphate production, a pertussis toxin-insensitive response. Similarly, calcitonin gene-related peptide receptor-stimulated increases in intracellular cAMP and pituitary adenylate-cyclase activating polypeptide receptor-stimulated increases in both cAMP and inositol phosphates are reduced significantly in RGS3T transfectants compared with vector-transfected control cells. In contrast, baby hamster kidney cells transfected with the full-length RGS3 cDNA show no impairment in cAMP and inositol phosphate production mediated by these G protein-coupled receptors. However, lysophosphatidic acid receptor-stimulated phosphorylation of endogenous ERK1 and ERK2 is impaired markedly in both RGS3 and RGS3T transfectants, demonstrating the functional ability of both RGS forms to modulate Gi-mediated signaling. These results provide the first evidence for regulatory effects of an RGS protein on Gs- and Gq-mediated signaling in intact cells and document that the carboxyl-terminal region of RGS3 comprises the structural domain for this activity (Chatterjee, 1997).

Transcriptional regulation of RGS proteins

Heterotrimeric G proteins transduce multiple growth-factor-receptor-initiated and intracellular signals that may lead to activation of the mitogen-activated or stress-activated protein kinases. A novel p53 target gene (A28-RGS14) is reported that is induced in response to genotoxic stress and encodes a novel member of a family of regulators of G protein signaling (RGS) proteins with proposed GTPase-activating protein activity. Overexpression of A28-RGS14p protein inhibits both Gi- and Gq-coupled growth-factor-receptor-mediated activation of the mitogen-activated protein kinase signaling pathway in mammalian cells. Thus, through the induction of A28-RGS14, p53 may regulate cellular sensitivity to growth and/or survival factors acting through G protein-coupled receptor pathways (Buckbinder, 1997).

RGS proteins and stress

RGS14 possesses an N-terminal RGS domain, two Raf-like Ras-binding domains, and a GoLoco motif, which has GDP dissociation inhibitor activity. This study shows that unique among the known mammalian RGS proteins, RGS14 localizes in centrosomes. Its first Ras-binding domain is sufficient to target RGS14 to centrosomes. RGS14 also shuttles between the cytoplasm and nucleus, and its nuclear export depends on the CRM-1 nuclear export receptor. Mutation of a nuclear export signal or treatment with leptomycin B causes nuclear accumulation of RGS14 and its association with promyelocytic leukemia protein nuclear bodies. Furthermore, a point mutant defective in nuclear export fails to target to centrosomes, suggesting that nuclear cytoplasmic shuttling is necessary for its proper localization. Mild heat stress, but not proteotoxic or transcription-linked stresses, re-localizes the RGS14 from the cytoplasm to promyelocytic leukemia nuclear bodies. Expression of RGS14, but not point mutants that disrupt the functional activity of its RGS domain or GoLoco motif, enhances the reporter gene activity. The multifunctional domains and the dynamic subcellular localization of RGS14 implicate it in a diverse set of cellular processes including centrosome and nuclear functions and stress-induced signaling pathways.

Activator of G protein signaling 3: a gatekeeper of cocaine sensitization and drug seeking

Chronic cocaine administration reduces G protein signaling efficacy. The expression of AGS3, which binds to GialphaGDP and inhibits GDP dissociation, is upregulated in the prefrontal cortex (PFC) during late withdrawal from repeated cocaine administration. Increased AGS3 is mimicked in the PFC of drug-naive rats by microinjecting a peptide containing the Giα binding domain (GPR) of AGS3 fused to the cell permeability domain of HIV-Tat. Infusion of Tat-GPR mimicked the phenotype of chronic cocaine-treated rats by manifesting sensitized locomotor behavior and drug seeking and by increasing glutamate transmission in nucleus accumbens. By preventing cocaine withdrawal-induced AGS3 expression with antisense oligonucleotides, signaling through Giα was normalized, and both cocaine-induced relapse to drug seeking and locomotor sensitization were prevented. When antisense oligonucleotide infusion was discontinued, drug seeking and sensitization were restored. It is proposed that AGS3 gates the expression of cocaine-induced plasticity by regulating G protein signaling in the PFC (Bowers, 2004).

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date revised: 20 December 2024

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