G protein oalpha 47A

EVOLUTIONARY HOMOLOGS part 1/3

G protein signaling

A large number of hormones, neurotransmitters, chemokines, local mediators, and sensory stimuli exert their effects on cells and organisms by binding to G protein-coupled receptors. More than a thousand such receptors are known, and more are being discovered all the time. Heterotrimeric G proteins transduce ligand binding to these receptors into intracellular responses, which underlie physiological responses of tissues and organisms. G proteins play important roles in determining the specificity and temporal characteristics of the cellular responses to signals. They are made up of alpha, beta, and gamma subunits, and although there are many gene products encoding each subunit (20 alpha, 6 beta, and 12 gamma gene products are known), four main classes of G proteins can be distinguished: G_s, which activates adenylyl cyclase; G_i, which inhibits adenylyl cyclase; G_q, which activates phospholipase C; and G₁₂ and G₁₃, of unknown function (Hamm, 1998 and references).

G proteins are inactive in the GDP-bound, heterotrimeric state and are activated by receptor-catalyzed guanine nucleotide exchange, resulting in GTP binding to the alpha subunit. GTP binding leads to dissociation of Galpha·GTP from Gbetagamma subunits and activation of downstream effectors by both Galpha·GTP and free Gbetagamma subunits. G protein deactivation is rate-limiting for turnoff of the cellular response and occurs when the Galpha subunit hydrolyzes GTP to GDP. The recent resolution of crystal structures of heterotrimeric G proteins in inactive and active conformations provides a structural framework for understanding their role as conformational switches in signaling pathways. As more and more novel pathways that use G proteins emerge, recognition of the diversity of regulatory mechanisms of G protein signaling is also increasing (Hamm, 1998 and references).

Galpha subunits contain two domains: a domain involved in binding and hydrolyzing GTP (the G domain), which is structurally identical to the superfamily of GTPases (including small G proteins and elongation factors) and a unique helical domain, which buries the GTP in the core of the protein. The beta subunit of heterotrimeric G proteins has a 7-membered beta-propeller structure based on its 7 WD-40 repeats. The gamma subunit interacts with beta through an N-terminal coiled coil and then all along the base of beta, making extensive contacts. The beta and gamma subunits form a functional unit that is not dissociable except by denaturation. G protein activation by receptors leads to GTP binding on the Galpha subunit. The structural nature of the GTP-mediated switch on the Galpha subunit is a change in conformation of three flexible regions designated Switches I, II, and III to a well ordered, GTP-bound activated conformation, with lowered affinity for Gbetagamma. This leads to increased affinity of Galpha·GTP for effectors, subunit dissociation, and generation of free Gbetagamma that can activate a number of effectors (Hamm, 1998 and references).

G protein-coupled receptors have a common body plan with seven transmembrane helices; the intracellular loops that connect these helices form the G protein-binding domain. Although no high resolution structure of a G protein-coupled receptor has yet been determined, recently a low resolution electron diffraction structure of rhodopsin, a model G protein-coupled receptor, shows the position and orientation of the seven transmembrane alpha-helices. Both mutagenesis and biochemical experiments with a variety of G protein-coupled receptors suggest that receptor activation by ligand binding causes changes in the relative orientations of transmembrane helices 3 and 6. These changes then affect the conformation of G protein-interacting intracellular loops of the receptor and thus uncover previously masked G protein-binding sites. When an activated receptor interacts with a heterotrimeric G protein, it induces GDP release from the G protein. It is thought that the receptor contact sites on the G protein are distant from the GDP-binding pocket, so the receptor must work 'at a distance' to change the conformation of the protein. Because GDP is buried within the protein between the two domains of Galpha, this must necessarily involve changing some interdomain interactions. Upon GDP release and in the absence of GTP a stable complex between the activated receptor and the heterotrimer is formed. This so-called 'empty pocket' conformation is of great interest, but its structure is as yet unknown (Hamm, 1998 and references).

What are the regions on G proteins that contact receptors, and how does G protein activation occur? The conformation of the GDP-bound heterotrimeric G proteins G_t and G_i shows the overall shape of the GDP-bound heterotrimer and the residues on the surface that can interact with other proteins and provides the structural context for understanding a variety of biochemical and mutagenesis studies of receptor-interacting regions on G proteins. The N-terminal region of the alpha subunit and the C-terminal region of the gamma subunit are both sites of lipid modification. These lipidated regions are relatively close together in the heterotrimer, suggesting a site of membrane attachment. There is good evidence for receptor contact surfaces on all three subunits (Hamm, 1998 and references).

On the alpha subunit, the best characterized receptor contact region is at the C terminus. The last 7 amino acids of the alpha subunit are disordered in the heterotrimer crystal structures, and analysis of receptor-binding peptides selected from a combinatorial peptide library shows that these 7 residues are the most critical. Studies using chimeric Galpha subunits confirm that in fact the last 5 residues contribute importantly to specificity of receptor G protein interaction. Elegant mutagenesis studies have shown that the C terminus of the third intracellular loop of receptors binds to this C-terminal region on Galpha subunits. In the case of M₂ muscarinic receptor coupling to G_i, the exact residues of the receptor that are critical for recognizing the C terminus of Galpha_i/o have been elucidated (Val-385, Thr-386, Ile-389, and Leu-390) (Hamm, 1998 and references).

A larger region of the C-terminal region of Galpha subunits, as well as the N-terminal helix, has been implicated in receptor contact. Alanine-scanning mutagenesis of Galpha_t and analysis of residues conserved in subclasses of G protein alpha subunits both identify a number of residues in the C-terminal 50 amino acids of Galpha_t that contact rhodopsin. Arg-310 located at the alpha4-beta6 loop of Galpha_t is completely blocked from tryptic proteolysis in the presence of light-activated rhodopsin, suggesting that the alpha4-beta6 loop region contributes to receptor contact. The alpha4-beta6 loop has also been implicated in specific interaction of the 5HT_1B serotonin receptor with Galpha_i1 as well as in receptor-catalyzed G_i activation (Hamm, 1998 and references).

It is clear that the betagamma subunits of heterotrimeric G proteins enhance receptor interaction with alpha subunits. Single Ala mutations in residues of the beta subunit that contact the alpha subunit block receptor-mediated GTP/GDP exchange. This suggests that the beta subunit must hold the alpha subunit rigidly in place for GDP release to occur. Direct binding interactions between receptor and betagamma subunit have been reported. A cross-linking study has demonstrated that the C-terminal 60-amino acid region of Gbeta can be cross-linked to an alpha₂-adrenergic receptor peptide corresponding to the intracellular third loop of the receptor. In addition, the C-terminal region of the gamma subunit of G proteins has been shown to be involved in receptor coupling and specificity (Hamm, 1998 and references).

The major classes of Galphas, the G_s, G_i, and G_q families of alpha subunits, have well known cellular targets. More recently, yeast two-hybrid screening has uncovered new targets. GAIP, a Galpha-interacting protein and a member of the RGS family of GTPase-activating proteins, was first identified in this way; and recently two more putative alpha targets, nucleobindin and a novel LGN repeat protein, have been described. So far, no physiological role for the latter two Galpha targets has been determined (Hamm, 1998 and references).

The complexity of signal transduction events in cells that are receiving and processing multiple signals is the subject of intense research. Some of the key questions are: (1) how much specificity is encoded in the direct protein-protein interactions; (2) are there other levels of cellular organization that impart specificity; and (3) what are the mechanisms of cross-regulation resulting in the final integrated cellular response? It is well known that multiple receptors can converge on a single G protein, and in many cases a single receptor can activate more than one G protein and thereby modulate multiple intracellular signals. In other cases, it seems that interaction of a single receptor with a given G protein is regulated by a high degree of selectivity imparted by specific heterotrimers. A number of excellent reviews describing the determinants of specific receptor-G protein interaction have recently appeared. Earlier in vitro studies of receptor-G protein interaction were often characterized by high promiscuity of receptor-G protein interaction, but a number of recent studies demonstrate that some receptors discriminate even between related G proteins within the same family (Hamm, 1998 and references).

In situ there can sometimes be high specificity. How is it achieved? The most exquisite specificity of receptor coupling to intracellular pathways by G proteins in vivo has been demonstrated using antisense oligonucleotides to suppress translation of specific G protein subunits. This technique allows suppression of distinct components involved in the signal transduction pathway and examination of any subsequent impaired cellular responses. Inhibition of calcium channels by somatostatin receptors in the GH₃ cell is mediated by Galpha_o2beta₁gamma₃, whereas inhibition by M₄ muscarinic receptors is mediated by Galpha_o1beta₁gamma₄. The elimination of Galpha_o by antisense technique abolishes somatostatin, M₄ muscarinic, and D₂ dopamine receptor-mediated inhibition of calcium entry in rat pituitary GH₄C₁ cells. By contrast, depletion of Galpha_i2 selectively impairs receptor-mediated inhibition of cAMP accumulation in the same system. Another antisense study indicates that the M₁ muscarinic receptor utilizes a specific G protein complex composed of Galpha_q/11beta_1/4gamma₄ to activate phospholipase C. A recent study shows coupling of angiotensin II AT1A receptors to regulation of Ca²⁺ channels, calcium-induced calcium release channels, and Na⁺/H⁺ exchange is via alpha₁₃beta₁gamma₃. This level of specificity is not seen in vitro or in transfection studies using overexpressed proteins. This raises the question of how targeting proteins or other cellular mechanisms can achieve high specificity. A number of organizing and targeting proteins and cellular structures are candidates for a role in specifying protein interactions in G protein signaling cascades. Another potential regulator of G protein specificity is targeted inactivation of a G protein by a GTPase-activating protein. In yeast, Ste5 is a scaffold protein that organizes the MAP kinase sequential enzyme cascade and contributes to specificity and fidelity of signaling. No mammalian homolog of Ste5 has been found. A particularly interesting possible scaffold for G protein-coupled signal transduction molecules is the growing family of PDZ domain-containing proteins, so named for the three proteins that contain them: postsynaptic density protein 95 (P); Drosophila discs large tumor suppressor (D), and zona occludens protein (Z). An unusual PDZ domain containing protein in Drosophila photoreceptors, called InaD, has 5 PDZ domains, each of which binds different signaling molecules of the G_q-regulated visual cascade, including rhodopsin, PLCbeta, protein kinase C, and the transient receptor potential protein (Trp), a homolog of the calcium-induced calcium release channel. Notably, G_q was missing from the complex. Another unusual PDZ domain-containing protein, Homer (see Drosophila Homer), contains a single PDZ domain, which binds to certain G protein-coupled metabotropic glutamate receptors in the brain. Other scaffold proteins are characterized by having multiple conserved domains such as phosphotyrosine-recognizing Src homology 2 (SH2) domains, SH3 domains, pleckstrin homology domains, Dbl homology domains, and domains with enzymatic activities, particularly activity controlling the GTP binding state of small G proteins such as guanine nucleotide exchange and GTPase-activating protein activity. Future studies may reveal more scaffolding or clustering mechanisms that may greatly increase the specificity of in vivo signal transduction by heterotrimeric G proteins (Hamm, 1998 and references).

Other Drosophila G alpha proteins

A new Drosophila G alpha gene (dgq) is differentially spliced, yielding two putative proteins, both of which contain guanine nucleotide binding and hydrolysis domains and share 50% identity with transducins and other G proteins. These proteins represent a new class of G alpha subunits because they lack both high amino acid identity with other G alpha proteins and the pertussis toxin ADP ribosylation site. The Dgq mRNA is detected by RNA-RNA Northern hybridization in wild-type heads but not in wild-type bodies or in the mutant eyes absent heads. Tissue in situ hybridization detects dgq expression only in the retina and ocellus of the adult head, making it a prime candidate for encoding the Drosophila transducin analog, the G protein required for phototransduction (Lee, 1990).

G_o alpha proteins in other insects

The heterotrimeric G proteins are an extended family of guanyl nucleotide-binding proteins that serve essential functions in the mature nervous system but whose contributions to neuronal development remain poorly understood. The potential role of one specific G protein, Go(alpha), in the control of neuronal migration has been examined. During embryogenesis of the moth, Manduca sexta, an identified population of undifferentiated neurons (the EP cells) migrate along sets of visceral muscle bands to form part of the enteric nervous system. Go(alpha)-related proteins are present in the EP cells during migration. A clone containing the full-length coding domain for Go(alpha) was sequenced from a Manduca cDNA library; digoxigenin-labeled probes were then made from this clone and used to examine the developmental expression of the Go(alpha) gene during embryogenesis. Go(alpha)-specific transcripts can first be detected in the EP cells several hours before the onset of their migration. The level of Go(alpha) expression in all of the EP cells continues to increase during migration, but subsequently is down-regulated in a subset of the postmigratory neurons at the time of their terminal differentiation. This pattern of regulated expression is consistent with the distribution of Go(alpha)-related protein in the EP cells. A semi-intact culture preparation of staged embryos was used to investigate the effects of G protein-specific toxins on the migratory process. Intracellular injections of the wasp toxin mastoparan, a specific activator of Go(alpha)-and Gi(alpha)-related proteins, inhibits the migration of individual EP cells. Injections of pertussis toxin (an inhibitor of Goalpha and Gialpha) or cholera toxin (a selective activator of Gsalpha) had no effect on migration, although pertussis toxin treatments did cause a measurable increase in the subsequent outgrowth of axonal processes. However, co-injection of mastoparan with pertussis toxin blocks the inhibitory effects of mastoparan alone. These results suggest that Go(alpha)-coupled signaling events within the EP cells may down-regulate their migratory behavior, possibly in response to inhibitory cues that normally guide migration in the developing embryo (Horgan, 1995).

G_o function in C. elegans

To identify genes controlling volatile anesthetic (VA) action, existing Caenorhabditis elegans mutants were screened, and it was found that strains with a reduction in Go signaling are VA resistant. Loss-of-function mutants of the gene goa-1, which codes for the alpha-subunit of Go, have EC50s for the VA isoflurane of 1.7- to 2.4-fold that of wild type. Strains overexpressing egl-10, which codes for an RGS protein negatively regulating goa-1, are also isoflurane resistant. However, sensitivity to halothane, a structurally distinct VA, is differentially affected by Go pathway mutants. The RGS overexpressing strains, a goa-1 missense mutant found to carry a novel mutation near the GTP-binding domain, and eat-16(rf) mutants, which suppress goa-1(gf) mutations, are all halothane resistant; goa-1(null) mutants have wild-type sensitivities. Double mutant strains carrying mutations in both goa-1 and unc-64, which codes for a neuronal syntaxin previously found to regulate VA sensitivity, show that the syntaxin mutant phenotypes depend in part on goa-1 expression. Pharmacological assays using the cholinesterase inhibitor aldicarb suggest that VAs and GOA-1 similarly downregulate cholinergic neurotransmitter release in C. elegans. Thus, the mechanism of action of VAs in C. elegans is regulated by Goalpha, and presynaptic Goalpha-effectors are candidate VA molecular targets (van Swinderena, 2001).

The neurotransmitter serotonin functions as a permissive signal for embryonic and postembryonic neuronal migration in the nematode C. elegans. In serotonin-deficient mutants, the migrations of the ALM, BDU, SDQR, and AVM neurons were often foreshortened or misdirected, indicating a serotonin requirement for normal migration. Moreover, exogenous serotonin can restore motility to AVM neurons in serotonin-deficient mutants as well as induce AVM-like migrations in the normally nonmotile neuron PVM; this indicates that serotonin functions as a permissive cue to enable neuronal motility. The migration defects of serotonin-deficient mutants are mimicked by ablations of serotonergic neuroendocrine cells, implicating humoral release of serotonin in these processes. Mutants defective in G_q and G_o signaling, or in N-type voltage-gated calcium channels, show migration phenotypes similar to serotonin-deficient mutants, and these molecules appear to genetically function downstream of serotonin in the control of neuronal migration. Thus, serotonin is important for promoting directed neuronal migration in the developing C. elegans nervous system. It is hypothesized that serotonin may promote cell motility through G protein-dependent modulation of voltage-gated calcium channels in the migrating cell (Kindt, 2002).

Most serotonin receptors are coupled to G protein-mediated signaling pathways; thus, it was reasoned that serotonin's effects on neuronal migration might require the activity of one or more G proteins. The C. elegans genome contains 20 genes encoding distinct G protein α subunits; viable loss- or reduction-of-function alleles have been identified for 17 of these. Among these strains, mutants carrying loss-of-function mutations in the G_o homolog goa-1 and the G_q homolog egl-30 display the most penetrant (approximately 30% for AVM) misplacement phenotypes. The effects of goa-1 and egl-30, like serotonin, are specific to cell body migration, since the AVM axon enters the ventral cord in the correct location in mutant animals even when the cell body from which the axon originated is misplaced. Interestingly, whereas a gain-of function allele of egl-30 causes significant misplacement of all four neurons, a goa-1 gain-of-function allele shows no misplacement phenotype for AVM, SDQR, or BDU. Thus, G_o, like serotonin, appears to play a permissive rather than an instructive role in the migrations of these three neurons (Kindt, 2002).

Together, these data suggested that serotonin's effect on AVM, SDQR, and BDU cell migration might be mediated by a G_o-dependent signal transduction pathway. To further examine this possibility, the cell migration phenotypes of tph-1; goa-1 double mutants was analyzed. In a double mutant carrying loss-of-function mutations in both tph-1 and goa-1, it was observed that, in these animals, the migration defects (assayed with respect to AVM, SDQR, and BDU) shows the same penetrance as in the tph-1 single mutant, implying that GOA-1 affected the same aspect of the AVM/SDQR/BDU migration process as serotonin (Kindt, 2002).

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).

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).

Identification of multiple G_o alpha isoforms

A HIT (hamster insulin-secreting tumor) cell cDNA library constructed in lambda gt11 was screened with a Go-specific oligonucleotide probe and six recombinant phages were isolated. The inserts of these phages encode two forms of alpha o, called here alpha o1 and alpha o2. The deduced amino acid sequence of alpha o1 is identical in all of its 354 amino acids to that reported previously for rat and bovine alpha o; that of alpha o2, also of 354 amino acids, is identical to alpha o1 up to and including amino acid 248 and differs thereafter in 26 amino acids. At the nucleotide level, alpha o1 and alpha o2 are identical up to and including the second base of the codon that specifies amino acid 243 and differs thereafter in 88 nucleotides of the remaining open reading frame and has no similarity to alpha o1 in its 3'-untranslated region. It is proposed that alpha o1 and alpha o2 result as a consequence of alternative splicing of a single alpha o transcript. Northern analysis with specifically designed oligonucleotides indicates that both forms of alpha o are expressed in normal tissues, e.g. brain. After in vitro transcription and translation, the peptides encoded in the alpha o1 and alpha o2 cDNAs could be ADP-ribosylated by pertussis toxin in the presence of added beta gamma dimers (Hsu, 1990).

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms show distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrates with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go are recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i. This more abundant form, called Go A, most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go are recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells. Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that two structurally distinct forms of Go have been identified that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes (Padrell, 1991).

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry (McIntire, 1998a).

The structural differences between two major forms of the alpha subunit of the heterotrimeric G protein GO were found to be due to deamidation of either of two Asn residues near the C-terminus of the proteins, in a region involved in receptor recognition. GO is the most abundant heterotrimeric G protein in mammalian brain. Two forms of the protein, GOA and GOB, are known to be generated by alternative splicing of a single GOalpha gene. A third isoform, alphaOC, represents about 1/3 of the alphaO protein in brain and is related to alphaOA, from which it is thought to be generated by protein modification. Mass spectrometry and chemical derivatization of tryptic fragments of the proteins were used to localize the structural difference between alphaOA and alphaOC to a C-terminal peptide. Sequence analysis of a C-terminal chymotryptic fragment both by ion trap mass spectrometry and by Edman degradation identified Asn346 and Asn347 of alphaOA as alternative deamidation sites in alphaOC. These structural differences have immediate implications for G protein function, as they occur in a conformationally sensitive part of the protein involved in receptor recognition and activation. Since Asn347 is a conserved residue present in most G protein alpha subunits outside the alphas family, these observations may have general significance for many G proteins. Deamidation may be a component of a novel process for modifying or adapting cellular responses mediated by G proteins (McIntire, 1998b).

Galphao, the most abundant G protein in mammalian brain, occurs at least in two subforms, i.e., Galphao1 and Galphao2, derived by alternative splicing of the mRNA. A third Galphao1-related isoform, Galphao3, has been purified, representing about 30% of total Go in brain. Initial studies revealed distinct biochemical properties of Galphao3 as compared with other Galphao isoforms. In matrix-assisted laser desorption/ionization peptide mass mapping of gel-isolated Galphao1 and Galphao3, C-terminal peptides showed a difference of +1 Da for Galphao3. Nanoelectrospray tandem mass spectrometry sequencing revealed an Asp instead of an Asn at position 346 of Galphao3. Gel electrophoretic analysis of recombinant Galphao3 showed the same mobility as native Galphao3 but distinct to Galphao1. The conversion of 346Asn-->Asp changed the signaling properties, including the velocity of the basal guanine nucleotide-exchange reaction, which points to the involvement of the C terminus in basal guanosine 5'-[gamma-thio]triphosphate binding. No cDNA coding for Galphao3 was detected, suggesting an enzymatic deamidation of Galphao1 by a yet-unidentified activity. Therefore, Galpha heterogeneity is generated not only at the DNA or RNA levels, but also at the protein level. The relative amount of Galphao1 and Galphao3 differs from cell type to cell type, indicating an additional principle of G protein regulation (Exner, 1999).

From nematodes to humans, animals employ neuromodulators like serotonin to regulate behavioral patterns and states. In the nematode C. elegans, serotonin has been shown to act in a modulatory fashion to increase the rate and alter the temporal pattern of egg laying. Though many candidate effectors and regulators of serotonin have been identified in genetic studies, their effects on specific neurons and muscles in the egg-laying circuitry have been difficult to determine. Using the genetically encoded Ca2+ indicator cameleon, it was found that serotonin acts directly on the vulval muscles to increase the frequency of Ca2+ transients. In contrast, the spontaneous activity of the egg-laying motorneurons is silenced by serotonin. Mutations in G protein alpha subunit genes alter the responses of both vulval muscles and egg-laying neurons to serotonin; specifically, mutations in the Gqalpha homolog egl-30 block serotonin stimulation of vulval muscle Ca2+ transients, while mutations in the Goalpha homolog goa-1 prevent the silencing of motorneuron activity by serotonin. These data indicate that serotonin stimulates egg laying by directly modulating the functional state of the vulval muscles. In addition, serotonin inhibits the activity of the motorneurons that release it, providing a feedback regulatory effect that may contribute to serotonin adaptation (Shyn, 2003)

The two pairs of sensory neurons of C. elegans, AWA and AWC, that mediate odorant attraction, express six Galpha-subunits, suggesting that olfaction is regulated by a complex signaling network. This study describes the cellular localization and functions of the six olfactory Galpha-subunits: GPA-2, GPA-3, GPA-5, GPA-6, GPA-13, and ODR-3. All except GPA-6 localize to sensory cilia, suggesting a direct role in sensory transduction. GPA-2, GPA-3, GPA-5, and GPA-6 are also present in cell bodies and axons and GPA-5 specifically localizes to synaptic sites. Analysis of animals with single- to sixfold loss-of-function mutations shows that olfaction involves a balance between multiple stimulatory and inhibitory signals. ODR-3 constitutes the main stimulatory signal and is sufficient for the detection of odorants. GPA-3 forms a second stimulatory signal in the AWA and AWC neurons, also sufficient for odorant detection. In AWA, signaling is suppressed by GPA-5. In AWC, GPA-2 and GPA-13 negatively and positively regulate signaling, respectively. Finally, it is shown that only ODR-3 plays a role in cilia morphogenesis. Defects in this process are, however, independent of olfactory behavior. These findings reveal the existence of a complex signaling network that controls odorant detection by C. elegans (Lans, 2004).

The results show that olfactory signaling in C. elegans is more complex than initially realized. Five Galpha-subunits regulate the response of C. elegans to attractive odorants. ODR-3 constitutes the main stimulatory signal in the AWA and AWC cells. GPA-3 also provides a stimulatory signal in both neuron pairs. GPA-5 and GPA-2 have inhibitory functions in the AWA and the AWC cells, respectively, and GPA-13 has a minor stimulatory role in the AWC cells. Although the odorant concentrations used in this study are not all detected specifically by either AWA or AWC, it is highly likely that the observed regulation by Galpha-subunits occurs only in these cells. First of all, no other cells have been reported to be involved in odorant attraction in C. elegans. Second, GPA-5 is expressed only in AWA and faintly in ADL, whereas AWC is the only sensory amphid cell in which GPA-2 is expressed. Finally, AWA-specific expression of GPA-3 rescues AWA-mediated olfaction in a gpa-3 odr-3 background (Lans, 2004).

It cannot be excluded that other ubiquitously expressed Galpha-subunits, like goa-1, and gpa-7, also function in the AWA and AWC cells. However, on the basis of the data presented in this study, it is not likely that one of these is required for olfactory signaling. It is striking that signaling in AWA and AWC, the only cells required for odorant attraction, involves different Galpha-subunits. Interestingly, this correlates well with the existence of dissimilar downstream signaling pathways in the two neuron pairs. In AWA, signaling is reminiscent of Drosophila phototransduction, due to its dependence on TRP channel proteins. AWC signaling, in contrast, resembles that in the mammalian main olfactory epithelium, where all signals converge on a cyclic nucleotide-gated channel. Signaling in this system also involves a separate modulatory pathway and multiple Galpha-proteins (Lans, 2004).

Why would C. elegans need several Galpha-subunits with overlapping or antagonizing functions to modulate olfaction? It is hypothesized that such a balanced signaling network is necessary to allow adjustment of the response to an odorant when odorant concentrations or other environmental conditions change. The inhibitory effects of GPA-2 and GPA-5 are most obvious when the animals are exposed to high odorant concentrations. In these circumstances, desensitization or adaptation mechanisms may become activated. Following prolonged exposure to high odorant concentrations, C. elegans shows a diminished response to that odorant, but not to other odorants. This process, which is called adaptation, involves the unknown adp-1 gene, the TRPV channel subunit OSM-9, and the cGMP-dependent protein kinase EGL-4. Since odorant adaptation is triggered by calcium and cGMP levels, it seems likely that this response is regulated by stimulating and inhibiting Galpha-subunits. Furthermore, odorant-specific adaptation is modulated by other environmental cues, which are probably transduced by G-proteins. For example, the absence of food stimulates benzaldehyde adaptation, but not isoamyl alcohol adaptation. Similarly, the presence of food suppresses benzaldehyde adaptation. Although it is uncertain where the integration of signals like food availability and odorants occurs, multiple G-proteins within one cell might be essential to control the proper response of an animal to several simultaneous cues (Lans, 2004).

In addition, overlapping G-protein pathways could facilitate discrimination between odorants detected by the same neuron. The finding that two Galpha-subunits, ODR-3 and GPA-3, can mediate the detection of all odorants provides a basis for this idea. Inhibition of ODR-3 and GPA-3 signaling by GPA-2 and GPA-5 could also be a means for odorant discrimination. Both adaptation and discrimination assays could shed more light on the involvement of G-proteins in adaptation and discrimination (Lans, 2004).

Finally, another hint at the biological significance of the G-protein network could be provided by the finding that vulval induction by the Ras-mitogen-activated protein kinase (MAPK) pathway is negatively regulated by GPA-5 and the GPCR SRA-13, depending on food conditions. This indicates that olfactory signaling not only serves to direct movement toward food, but also may regulate developmental processes (Lans, 2004).

This genetic analysis of G-protein function in olfactory signaling shows that the Galpha-subunits have specific functions, despite their shared localization in the cell. This raises the question of how specificity is organized and maintained. First of all, specificity may be defined by specific interactions with receptors, Gßgamma-subunits, and effectors. It is likely that ODR-3 and GPA-3 are activated by the same receptors during odorant detection. GPA-2 and GPA-5 might inhibit signaling by competing for these receptors, but could also be activated by different receptors or in a receptor-independent fashion. Recently, GPA-5 and SRA-13 have been found to negatively regulate a Ras/MAPK pathway downstream of ODR-3 in the AWC cells. Although this could provide a mechanism through which ODR-3 and GPA-3 signaling is inhibited, it is unclear how GPA-5 could function in the AWC cells. Furthermore, it is striking that no effect of GPA-2 on the Ras/MAPK pathway has been observed (Lans, 2004).

Another determinant is provided by the ßgamma-dimer that associates with the Galpha-subunits. C. elegans has two Gß- and two Ggamma-subunits, GPB-1 and -2 and GPC-1 and -2. GPC-1 functions specifically in a subset of sensory cells, but not in AWA and AWC. Therefore, the ubiquitously expressed GPC-2 is the likely candidate to interact with the olfactory Galpha-subunits. In AWA and AWC, GPC-2 may dimerize with either GPB-1 or GPB-2, which are both widely expressed. Alternatives to GPC-2, however, could be the Ggamma-like domain containing RGS proteins EAT-16 and EGL-10, which can interact with GPB-2. Thus, together there are four different Gßgamma(-like) partners. It is difficult to test which of these partners functions in vivo because both gpb-1 and gpc-2 mutations are lethal (Lans, 2004).

A spatial separation of different pathways is a further means to confer specificity. Within the cilia, compartmentalization could separate the different Galpha-subunits, as has been suggested to insulate cGMP signaling during odorant discrimination and adaptation. Another method to provide specificity could be a temporal induction of G-protein signaling. For example, the translocation of the cGMP-dependent protein kinase EGL-4 to the nucleus of the AWC cells, following long-term adaptation to odorants, may induce the expression of genes necessary for adaptation that would otherwise interfere with olfactory signaling. It is becoming increasingly clear that most signaling pathways are part of complex, intracellular signaling networks. This characterization of the olfactory system of C. elegans provides the possibility of identifying the molecular mechanisms that regulate specificity in vivo (Lans, 2004).

Interaction of G_o alpha proteins with G beta subunits

continues: G protein oalpha 47A Evolutionary homologs part 2/3 | part 3/3 |

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