G protein salpha 60A


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: Gs, which activates adenylyl cyclase; Gi, which inhibits adenylyl cyclase; Gq, 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 Gt and Gi 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 Gi, the exact residues of the receptor that are critical for recognizing the C terminus of Galphai/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 Galphat 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 Galphat that contact rhodopsin. Arg-310 located at the alpha4-beta6 loop of Galphat 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 5HT1B serotonin receptor with Galphai1 as well as in receptor-catalyzed Gi 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 Gs, Gi, and Gq 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 Galphao2beta1gamma3, whereas inhibition by M4 muscarinic receptors is mediated by Galphao1beta1gamma4. The elimination of Galphao 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 Galphai2 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 Galphaq/11beta1/4gamma4 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 Gq-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, Gq 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 insect G alpha-like proteins

The Drosophila concertina(cta) gene encodes a G alpha-like protein. The CTA protein is only 35-44% identical to any of the previously characterized Drosophila G alpha proteins. CTA is more similar to mouse proteins G alpha 12 and G alpha13. CTA is necessary to coordinate cell shape changes during gastrulation. Mutant embryos fail to undergo proper gastrulation. The central region of the ventral zone fails to undergo the rapid transition to a groove. Some cells constrict their apices and move their nuclei basally, but this process seems poorly coordinated. Patches of cells that have failed to undergo apical constriction are left amid cells that have changed shape appropriately. An abnormal furrow is ultimately formed in many mutant embryos, but it lacks the depth and length of the furrow in wild type. At the posterior end, mutants fail to constrict enough cells to form the shallow cup in which the pole cells sit. Formation of the cephalic fold remains normal. In spite of the gastrulation defect, mutants have normal polarity. Cuticles have normal denticle belts and other cuticular features. The cuticle has holes at the anterior and posterior ends, presumably because of improper morphogenetic movements. While removal of maternal cta produces these developmental defects, zygotic elimination has no effect on viability. In early embryos, highly abundant amounts of messenger RNA are found throughout the cytoplasm. Upon blastoderm formation the level of messenger RNA decreases and it remains low throughout gastrulation until the extended germband stage. Then a higher level of CTA mRNA accumulates in the mesoderm, presumably from zygotic expression. In the ovary, mRNA first appears in the germarium. Throughout oogenesis, the accumulation of messenger is restricted to the germline and is observed in both the nurse cells and the oocyte. It is thought that cta acts through the cytoskeleton, perhaps through modification of actin polymerization (Parks, 1991)

The folded gastrulation (fog) gene is required during Drosophila gastrulation for two morphogenetic movements: formation of the ventral furrow and invagination of the posterior midgut primordium. fog coordinates cell shape changes during these invaginations by inducing apical constriction of cells in spatially and temporally defined manners. fog is expressed in the invagination primordia in a pattern that precisely prefigures the pattern of later constrictions. Overexpression of fog in the dorsoanterior region of the embryo induces ectopic constrictions, indicating localization of fog transcripts may define domains of cell shape changes. fog encodes a novel protein with a putative signal sequence but no potential transmembrane domains. It has been suggested that fog functions as a secreted signal that activates the G protein alpha subunit encoded by concertina in neighboring cells. It seems that cell-cell communication ensures the rapid, orderly progression of constriction initiations from the middle of invagination primordia out toward the margins (Costa, 1994).

Two Drosophila G oalpha proteins have been identified that are produced by three distinct transcripts from a single G oalpha gene. The two G oalpha proteins are produced by use of alternate first exons and differ in 7 of 21 N-terminal amino acids. The G oalpha transcripts are only expressed at low levels in adults, primarily in the abdomen. The highest levels of expression occur in embryos and pupae. At least three distinct G oalpha transcripts are found in adult flies. A 6.0 kb transcript is specific to adult heads; a 3.5 kb transript is specific to adult bodies, and a 4.2 kb transcript is found in both heads and bodies. These transcripts are each differentially expressed during development. G oalpha transcripts are seen in ovaries. During embryogenesis, after completion of germband retracted, elevated levels of G salpha and G oalpha are first detected in the forming neuropil of the brain and ventral ganglion. This pattern persists for the duration of embryogenesis and through adult life (Wolfgang, 1991).

A single Drosophila G ialpha gene produces two transcripts which result in the production of a single G ialpha protein. G ialpha transcripts are present in ovaries. G ialpha distribution throughout embryogenesis is markedly different from that observed for G salpha and G oalpha. G ialpha derived from maternal mRNA is present in early embryos. High levels of granular G ialpha reactivity is detected in three distinct and successive patterns in cleavage stage embryos. Gi alpha-reactive granules are either uniformly distributed about the periphery of the embryo, organized into longitudinal rows in the periphery and partially localized to the posterior pole, or densely packed and entirely localized to the posterior pole. All three stages occur prior to nuclear migration to the periphery of the embryo. By the end of the blastoderm, the granules can no longer be detected. Thus, very early in development, G ialpha is rapidly restricted to the posterior end of the embryo where it persists only through the blastoderm stage. G ialpha immunoreactivity reappears at stage 14 in prospective cardioblast cells at the leading edge of the advancing mesoderm during dorsal closure. These cells will form the tube of the dorsal vessel or heart. Staining is not detected in pericardial cells. G ialpha staining is also seen in the chordotonal organs. The ventral ganglion of stage 17 embryos contains 11 pairs of large dorsal midline cells exhibiting G ialpha staining. Additionally, in each abdominal hemiganglion, 1 or 2 additional lateral cells stain. A thoracic nerve also shows consistent high levels of staining in late embryos. Additionally a number of unidentified cell bodies in the brain and subesophageal ganglion are stained. Staining in fibers, but not the cell bodies in the ventral ganglion, can be detected throughout larval life (Wolfgang, 1991).

Rho GTPases play an important role in diverse biological processes, such as actin cytoskeleton organization, gene transcription, cell cycle progression and adhesion. They are required during early Drosophila development for proper execution of morphogenetic movements of individual cells and groups of cells important for the formation of the embryonic body plan. Loss-of-function mutations, isolated in the Drosophila Rho1 gene during a genetic screen for maternal-effect mutations, allow for an investigation of the specific roles Rho1 plays in the context of the developing organism. Rho1 is required for many early events: loss of Rho1 function results in both maternal and embryonic phenotypes. Embryos homozygous for the Rho1 mutation exhibit a characteristic zygotic phenotype, which includes severe defects in head involution and imperfect dorsal closure. Two phenotypes are associated with reduction of maternal Rho1 activity: the actin cytoskeleton is disrupted in egg chambers, especially in the ring canals; embryos display patterning defects as a result of improper maintenance of segmentation gene expression. Despite showing imperfect dorsal closure, Rho1 does not activate downstream genes or interact genetically with members of the JNK signaling pathway. The JNK pathway is used by Rho1 relatives Rac and Cdc42 for proper dorsal closure. Consistent with its roles in regulating actin cytoskeletal organization, Rho1 interacts genetically and physically with the Drosophila formin homolog, cappuccino. Rho1 interacts both genetically and physically with concertina, a Galpha protein involved in cell shape changes during gastrulation (Magie, 1999).

Genetic interactions of Rho1 were detected with two mutations: cappuccino (capu; formin homolog) and concertina (cta; Ga protein). Egg chambers from mothers heterozygous for Rho1 (Rho1/+) exhibit normal actin morphology. Egg chambers from mothers trans-heterozygous for Rho1 and capu (Rho1;capu/+) or cta (Rho1:cta/+) exhibit disruptions of the ovarian actin cytoskeleton, similar to those in females with reduced maternal Rho1 activity. While capu has been shown to affect actin integrity during oogenesis, similar studies have not been reported for cta. Phallodin staining was examined in egg chambers from homozygous cta mothers and similar, albeit weaker, defects are found in the actin cytoskeleton. The interaction of Rho1 with capu is more severe than with cta: the subsequent embryos from the Rho1/cta interaction survive, whereas the embryos from Rho1/capu are inviable and exhibit severe patterning defects. An in vitro binding assay was used to examine the interaction specificity between the Rho1 and Cta or Capu proteins. Rho1 fused to glutathione S-transferase (GST-Rho1) was expressed in bacteria and immobilized on glutathione-Sepharose beads. Rho1 was then tested for its ability to bind 35 S-labeled full-length Cta or Capu proteins. Consistent with the observed genetic interactions, Rho1 specifically pulls down full-length Cta or Capu. Interestingly, Capu preferentially interacts with GTP-bound Rho1, whereas Cta interacts equally with GTP- or GDP-bound Rho1 (Magie, 1999).

The numerous seemingly distinct biological responses of the Rho GTPase suggests that its activation must be both temporally and spatially regulated. Part of this regulation is likely to come from interaction of Rho with different GEFs. The mechanisms that lead to activation of Rho family proteins by extracellular signals are thought to be similar to those of Ras: they are mediated by GEFs linked to heterotrimeric G protein coupled membrane receptors. A large family of RhoGEFs have been identified in mammalian systems, some of which are specific for a particular family member (e.g., Lbc for Rho; Tiam1 for Rac), while other GEFs act on all members. Three RhoGEFs have been identified in Drosophila, but little is known about their specificity. No mutations corresponding to DRhoGEF1 have been reported. DRhoGEF2 has been shown to affect many of the morphogenetic movements associated with gastrulation and suppress genetic phenotypes associated with overexpression of wild type or constitutively active Rho. However, while both Rho1 and DRhoGEF2 loss-of-function mutations affect gastrulation, their phenotypes are very different: no trans-heterozygous genetic interactions between DRhoGEF2 and loss-of-function Rho1 mutations have been detected. The third Drosophila RhoGEF, Pebble, does interact genetically with Rho1 loss-of-function mutations. Since the identified RhoGEFs do not have completely overlapping phenotypes with Rho1 loss-of-function mutations, it is likely that additional RhoGEFs exist. Similarly, since phenotypes associated with loss-of-function mutations in Rac, Cdc42 and RhoL have not yet been reported, the specificity of the existing RhoGEFs is not yet known (Magie, 1999 and references therein).

Work in fibroblasts suggests a role for subunits of the heterotrimeric Galpha proteins (G12 and G13) in Rho-mediated signaling. While the exact link between the G proteins and Rho family proteins has not been described, a physical interaction between specific RhoGEFs and Galpha proteins was recently reported. GEFs are thought to act immediately upstream of Rho family proteins. concertina is a Galpha-like G protein that is important in transducing signals necessary to appropriately organize cell shape changes during Drosophila gastrulation. Ectopic expression studies utilizing dominant-negative Rho1 have led to the implication of concertina in the cell shape changes leading to proper ventral furrow formation; this is consistent with studies showing disruption of Drosophila cellularization after microinjection of the botulinum C3 exoenzyme Rho-specific inhibitor. While Rho1 loss-of-function mutations do not show the same severe cellularization or gastrulation phenotypes of DRhoGEF2, Rho1 does interact both genetically and physically with cta, suggesting that Rho1 is likely to be a downstream effector of the Cta Galpha protein in the ovary. Interestingly, Cta interacts equally with the GTP- and GDP-bound forms of Rho1 and may form a complex including GEFs. Proper oogenesis and morphogenesis in Drosophila are dependent on Rho1 activity. Because these are complicated developmental processes involving multiple cellular events, it is expected that a large number of genes are involved in regulating and executing them. To understand the biochemical mechanisms through which Rho family proteins regulate the organization of the actin cytoskeleton, gene transcription, and their other associated activities, identification of regulatory factors and cellular targets is essential. Drosophila offers a genetically amenable system in which to systematically identify components of the Rho pathway required for the proper execution of these events. Future genetic screens with loss-of-function Rho1 mutations should also help in identification of regulators and effectors, an important step in describing the pathways through which Rho acts in the organism (Magie, 1999 and references therein).

Another G protein alpha subunit, G falpha, is expressed in the developing midgut and transiently in the amnioserosa. G falpha has a overall sequence identity of 30-38% with vertebrate and Drosophila G alpha proteins (Quan, 1993).

Ecdysteroidogenesis in the prothoracic glands of the tobacco hornworm Manduca sexta is stimulated by the cerebral neuropeptide prothoracicotropic hormone (PTTH: see Bombyx and Manduca prothoracicotropic hormone). PTTH-stimulated cAMP synthesis and ecdysone secretion are dependent on the presence of extracellular calcium, suggesting that PTTH enhances calcium entry into the cytosol. Such entry into the cytosol might involve the opening of a plasma membrane calcium channel, or a mechanism dependent on prior inositol triphosphate (IP3)-mediated release of intracellularly stored calcium. In pupal prothoracic glands, PTTH does not increase IP3 or other inositol phosphates over the course of times ranging from seconds up to 30 min, even in the presence of lithium. However, the L-type calcium channel antagonist nitrendipine completely prevents PTTH-stimulated ecdysone synthesis. A 41 kDa G-protein in prothoracic glands is ADP-ribosylated by pertussis toxin. However, PTTH-stimulated ecdysone synthesis is unaffected by prior exposure to pertussis toxin, indicating that the 41 kDa protein is not involved in the acute stimulation of steroidogenesis. By contrast, cholera toxin has a stimulatory effect on ecdysone secretion, suggesting the involvement of a Gs-like protein. Based on the absence of PTTH-stimulated inositol phosphate formation in pupal prothoracic glands, it is suggested that calcium mobilization may occur through the opening of a calcium channel, possibly regulated by Gs (Girgenrath, 1996).

A network of stimulatory and inhibitory Galpha-subunits regulates olfaction in Caenorhabditis elegans

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).

Somatic cAMP signaling regulates MSP-dependent oocyte growth and meiotic maturation in C. elegans

Soma-germline interactions control fertility at many levels, including stem cell proliferation, meiosis and gametogenesis, yet the nature of these fundamental signaling mechanisms and their potential evolutionary conservation are incompletely understood. In C. elegans, a sperm-sensing mechanism regulates oocyte meiotic maturation and ovulation, tightly coordinating sperm availability and fertilization. Sperm release the major sperm protein (MSP) signal to trigger meiotic resumption (meiotic maturation) and to promote contraction of the follicle-like gonadal sheath cells that surround oocytes. Using genetic mosaic analysis, it was shown that all known MSP-dependent meiotic maturation events in the germline require G{alpha}s-adenylate cyclase signaling in the gonadal sheath cells. The MSP hormone promotes the sustained actomyosin-dependent cytoplasmic streaming that drives oocyte growth. Furthermore, it was demonstrated that efficient oocyte production and cytoplasmic streaming require G{alpha}s-adenylate cyclase signaling in the gonadal sheath cells, thereby providing a somatic mechanism that coordinates oocyte growth and meiotic maturation with sperm availability. Genetic evidence is presented that MSP and G{alpha}s-adenylate cyclase signaling regulate oocyte growth and meiotic maturation in part by antagonizing gap-junctional communication between sheath cells and oocytes. In the absence of MSP or G{alpha}s-adenylate cyclase signaling, MSP binding sites are enriched and appear clustered on sheath cells. These results are discussed in the context of a model in which the sheath cells function as the major initial sensor of MSP, potentially via multiple classes of G-protein-coupled receptors. These findings highlight a remarkable similarity between the regulation of meiotic resumption by soma-germline interactions in C. elegans and mammals, in which oocytes arrest during meiotic prophase I and then resume meiosis in response to hormonal signaling: for example, luteinizing hormone (Govindan, 2009).

MSP and GLP-1/Notch signaling coordinately regulate actomyosin-dependent cytoplasmic streaming and oocyte growth in C. elegans

Fertility depends on germline stem cell proliferation, meiosis and gametogenesis, yet how these key transitions are coordinated is unclear. In C. elegans, this study shows that GLP-1/Notch signaling functions in the germline to modulate oocyte growth when sperm are available for fertilization and the major sperm protein (MSP) hormone is present. Reduction-of-function mutations in glp-1 cause oocytes to grow abnormally large when MSP is present and G{alpha}s-adenylate cyclase signaling in the gonadal sheath cells is active. By contrast, gain-of-function glp-1 mutations lead to the production of small oocytes. Surprisingly, proper oocyte growth depends on distal tip cell signaling involving the redundant function of GLP-1 ligands LAG-2 and APX-1. GLP-1 signaling also affects two cellular oocyte growth processes, actomyosin-dependent cytoplasmic streaming and oocyte cellularization. glp-1 reduction-of-function mutants exhibit elevated rates of cytoplasmic streaming and delayed cellularization. GLP-1 signaling in oocyte growth depends in part on the downstream function of the FBF-1/2 PUF RNA-binding proteins. Furthermore, abnormal oocyte growth in glp-1 mutants, but not the inappropriate differentiation of germline stem cells, requires the function of the cell death pathway. The data support a model in which GLP-1 function in MSP-dependent oocyte growth is separable from its role in the proliferation versus meiotic entry decision. Thus, two major germline signaling centers, distal GLP-1 activation and proximal MSP signaling, coordinate several spatially and temporally distinct processes by which germline stem cells differentiate into functional oocytes (Nadarajan, 2009).

Deep conservation of genes required for both Drosophila melanogaster and Caenorhabditis elegans sleep includes a role for dopaminergic signaling

Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. This prediction is tested directly in this study. During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. Twenty-six genes required for Drosophila melanogaster sleep were identified. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, a conserved genetic pathway was delineated by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. The comprehensive analysis presented in this study suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry (Singh, 2014).

G protein alternative splicing

The GNAS1 gene encodes the alpha subunit of the G protein Gs, which couples receptor binding by several hormones to activation of adenylate cyclase. Null mutations of GNAS1 cause pseudohypoparathyroidism (PHP) type Ia, in which hormone resistance occurs in association with a characteristic osteodystrophy. The observation that PHP Ia almost always is inherited maternally has led to the suggestion that GNAS1 may be an imprinted gene. Although Gsalpha expression (directed by the promoter upstream of exon 1) is biallelic, GNAS1 is indeed imprinted in a promoter-specific fashion. Parthenogenetic lymphocyte DNA was used to screen by restriction landmark genomic scanning for loci showing differential methylation between paternal and maternal alleles. This screen identified a region that is found to be methylated exclusively on a maternal allele and is located approximately 35 kb upstream of GNAS1 exon 1. This region contains three novel exons that are spliced into alternative GNAS1 mRNA species, including one exon that encodes the human homolog of the large G protein XLalphas. Transcription of these novel mRNAs is exclusively from the paternal allele in all tissues examined. The differential imprinting of separate protein products of GNAS1 therefore may contribute to the anomalous inheritance of PHP Ia (Hayward, 1998).

G protein interactions

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism forcompartmentalization of cAMP-dependent protein kinase (see Drosophila 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. Another novel cDNA isolated from that screen has been cloned and characterized. 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 identified the full length D-AKAP2. It is composed of 372 amino acids, including 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 (Huang, 1997).

Heterotrimeric G proteins transduce signals from cell surface receptors to modulate the activity of cellular effectors. Src, the product of the first characterized proto-oncogene and the first identified protein tyrosine kinase, plays a critical role in the signal transduction of G protein–coupled receptors. However, the mechanism of biochemical regulation of Src by G proteins is not known. Galphas and Galphai, but neither Galphaq, Galpha12 nor Gbetagamma, directly stimulate the kinase activity of downregulated c-Src. Galphas and Galphai similarly modulate Hck, another member of Src-family tyrosine kinases. Galphas and Galphai bind to the catalytic domain and change the conformation of Src, leading to increased accessibility of the active site to substrates. These data demonstrate that the Src family tyrosine kinases are direct effectors of G proteins (Ma, 2000).

Evidence is presented that activated Galphas and Galphai, but neither Galphaq, Galpha12, nor Gbetagamma, directly regulate Src-family tyrosine kinases by activating the downregulated, C-terminal phosphorylated kinase. In Src-family tyrosine kinase knockout SYF cells, GalphasQ227L and GalphaiQ205L induced tyrosine phosphorylation of many of cellular proteins is severely reduced, suggesting that Src-family tyrosine kinases are the major mediator of Galphas- and Galphai-induced protein tyrosine phosphorylation in cells. Furthermore, the switch II region of Galphas is involved in interacting with Src. In general, G proteins, including heterotrimeric and Ras-family G proteins, use their effector domains to contact the downstream effectors. For Ras, the switch I region is a core effector domain essential for all effector interactions. The switch II region of Galphas and Galphai has been shown to be important in interacting with their common effector adenylyl cyclase. These data suggest that Galphas uses some residues such as I235 in the switch II region, and likely residues in other regions, to interact with the catalytic domain of Src. These studies significantly advance understanding of an important aspect of the cross-talk between G protein-coupled receptors and tyrosine kinases. Furthermore, these experiments provide a possible mechanism of action by which the gip2 mutant of Galphai and the gsp mutant of Galphas induce oncogenicity (Ma, 2000).

There are previous reports indicating that some biological effects of Galphas and Galphai could not be explained by their opposing effects on adenylyl cyclases. Both activated mutants (GTPase deficient) of Galphas and Galphai are oncogenes, found in certain human tumors. Both Galphas and Galphai can transform cells and activate the mitogen-activated protein kinase (MAPK) pathways. Activated Galphai subunits induce a transformed phenotype in fibroblasts independent of inhibition of adenylyl cyclase. Also, Galphas and Galphai regulate adipogenesis in mouse 3T3-L1 cells and stem cell differentiation of F9 teratocarcinoma cells into primitive endoderm, which could not be explained by changes in intracellular cAMP. Furthermore, inhibition of magnesium uptake in S49 cells by isoproterenol or prostaglandin E1 has been shown to be Galphas-dependent, but cAMP and PKA-independent. Similarly, in differentiating wing epithelial cells of Drosophila, activation of Galphas leads to formation of wing blisters. This pathway has been genetically demonstrated to be independent of PKA. Recently, engagement of beta-adrenergic receptors initiate a Galphas-dependent, PKA-independent pathway leading to apoptosis in S49 cells. These reports, together with the observations presented here, clearly indicate that Galphas and Galphai can signal through novel transduction pathways, in addition to the classically defined cAMP second messenger system. The relative contribution of these different effector systems to the physiology of G proteins in organisms remains to be addressed (Ma, 2000).

c-Src kinase activity can be modulated by either tyrosine phosphorylation or conformational changes. Neither Galphas nor Galphai change the Tyr527 phosphorylation state, suggesting that autodephosphorylation of Tyr527 is not the regulatory mechanism used by G proteins. Galphas and Galphai do increase the autophosphorylation of Tyr416. Binding data showing that Galphas and Galphai interact with the catalytic domain of Src suggest that a conformational change model is likely. Indeed, a phosphotyrosine binding experiment has shown that G protein stimulation causes the release of phospho-Tyr527 from the SH2 domain, making it available for interacting with other proteins. In a similar manner, activation of Hck kinase activity by the Nef protein of human immunodeficiency virus-1 (HIV-1), comes about through a conformational change, which occurs in the absence of dephosphorylation of the tail Tyr527. Enzymatic kinetic measurement reveals that the major effect of Galphas and Galphai is to decrease the Km for the peptide substrate. These data suggest that G protein binding changes the conformation of c-Src and allows the peptide substrate easier access to the active site. This model is consistent with the structural data of Src-family tyrosine kinases. In the downregulated state, two intramolecular interactions stabilize the restrained conformation of the kinase domain. The activation loop forms an alpha helix that packs between the upper and lower lobes of the catalytic domain, thus blocking the peptide substrate binding site. In the active state, the activation loop swings away from the entrance of the catalytic cleft, allowing access of the substrate to the active site. This activation mode has also been observed in the activation of cyclin-dependent kinase (cdk). Cyclin binding to the catalytic domain repositions cdk's activation loop and permits access of substrates to the active site. It is proposed that G protein binding to the catalytic domain modulates the position and conformation of the activation loop, as well as other elements in the catalytic domain. This could lead to relief of steric hindrance at the entrance to the catalytic cleft; increased accessibility of the active site to substrates, and exposure of the side chain of Tyr416, making it a better substrate for autophosphorylation (the Tyr416 hydroxyl group is buried in the catalytic cleft in the downregulated form) and thus increased kinase activity. Regardless of the detailed chemical mechanism, these data demonstrate that c-Src and Hck are novel direct effectors of Galphas and Galphai proteins (Ma, 2000).

G proteins and Ca2+ signaling

The alpha subunit of the stimulatory heterotrimeric G protein (Gsalpha) is critical for the beta-adrenergic receptor activation of the cAMP messenger system. However, the role of Gsalpha in regulating cardiac Ca2+ channel activity remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac Gsalpha were used to assess the role of Gsalpha on the whole-cell Ca2+ currents (ICa). Cardiac myocytes from transgenic mice had a 490% higher peak ICa, when compared with those of either wild-type controls or Gsalpha-nonexpressing littermates. The effect of Gsalpha overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTPgammaS-activated Gsalpha. This effect is not mediated by protein kinase A activation, since intracellular perfusion with a protein kinase A inhibitor renders the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated Gsalpha. The data indicate that Gsalpha overexpression is associated with a constitutive enhancement of ICa which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase (Lader, 1998).

G protein salphas target adenylyl cyclase

G protein salpha 60A Evolutionary homologs part 2/2

G protein salpha 60A: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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