Control of embryonic spindle positioning and Galpha activity by C. elegans RIC-8

Asymmetric spindle positioning is of fundamental importance for generating cell diversity during development. In the C. elegans 1 cell embryo, spindle positioning has been shown to depend on heterotrimeric G protein signaling. Two Galpha subunits, GOA-1 and GPA-16 (hereafter Galpha), and receptor independent activators of G protein signaling GPR-1 and GPR-2 (GPR-1/2) are required for proper regulation of spindle positioning . However, it remains unclear whether Galpha regulates spindle positioning in its GDP or GTP bound form. Here, we investigate the role of RIC-8 in this pathway. RIC-8 was genetically shown to act in concert with goa-1 to regulate centrosome movements in C. elegans . Interestingly, mammalian RIC-8 was recently found to behave as a GEF for Galpha subunits in vitro . We show that reduction of function of ric-8 results in a 1 cell embryo phenotype very similar to the phenotype of embryos depleted of Galpha. RIC-8 is able to directly bind to GOA-1, preferentially to GOA-1-GDP, consistent with a GEF role. RIC-8 is localized at the embryo cortex, and its activity is essential for the asymmetric localization of GPR-1/2. We suggest that RIC-8 directly modulates Galpha activity and that Galpha-GTP is the signaling molecule regulating spindle positioning in the early embryo.     

RIC-8 is required for GPR-1/2-dependent Galpha function during asymmetric division of C. elegans embryos

Heterotrimeric G proteins are crucial for asymmetric cell division, but the mechanisms of signal activation remain poorly understood. Here, we establish that the evolutionarily conserved protein RIC-8 is required for proper asymmetric division of one-cell stage C. elegans embryos. Spindle severing experiments demonstrate that RIC-8 is required for generation of substantial pulling forces on astral microtubules. RIC-8 physically interacts with GOA-1 and GPA-16, two Galpha subunits that act in a partially redundant manner in one-cell stage embryos. RIC-8 preferentially binds to GDP bound GOA-1 and is a guanine nucleotide exchange factor (GEF) for GOA-1. Our analysis suggests that RIC-8 acts before the GoLoco protein GPR-1/2 in the sequence of events leading to Galpha activation. Furthermore, coimmunoprecipitation and in vivo epistasis demonstrate that inactivation of the Gbeta subunit GPB-1 alleviates the need for RIC-8 in one-cell stage embryos. Our findings suggest a mechanism in which RIC-8 favors generation of Galpha free from Gbetagamma and enables GPR-1/2 to mediate asymmetric cell division.     

LET-99 opposes Galpha/GPR signaling to generate asymmetry for spindle positioning in response to PAR and MES-1/SRC-1 signaling

G-protein signaling plays important roles in asymmetric cell division. In C. elegans embryos, homologs of receptor-independent G protein activators, GPR-1 and GPR-2 (GPR-1/2), function together with Galpha (GOA-1 and GPA-16) to generate asymmetric spindle pole elongation during divisions in the P lineage. Although Galpha is uniformly localized at the cell cortex, the cortical localization of GPR-1/2 is asymmetric in dividing P cells. In this report, we show that the asymmetry of GPR-1/2 localization depends on PAR-3 and its downstream intermediate LET-99. Furthermore, in addition to its involvement in spindle elongation, Galpha is required for the intrinsically programmed nuclear rotation event that orients the spindle in the one-cell. LET-99 functions antagonistically to the Galpha/GPR-1/2 signaling pathway, providing an explanation for how Galpha-dependent force is regulated asymmetrically by PAR polarity cues during both nuclear rotation and anaphase spindle elongation. In addition, Galpha and LET-99 are required for spindle orientation during the extrinsically polarized division of EMS cells. In this cell, both GPR-1/2 and LET-99 are asymmetrically localized in response to the MES-1/SRC-1 signaling pathway. Their localization patterns at the EMS/P2 cell boundary are complementary, suggesting that LET-99 and Galpha/GPR-1/2 signaling function in opposite ways during this cell division as well. These results provide insight into how polarity cues are transmitted into specific spindle positions in both extrinsic and intrinsic pathways of asymmetric cell division.     

Receptor-independent activators of heterotrimeric G-protein signaling pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.     

Genetic screens in yeast to identify mammalian nonreceptor modulators of G-protein signaling.

We describe genetic screens in Saccharomyces cerevisiae designed to identify mammalian nonreceptor modulators of G-protein signaling pathways. Strains lacking a pheromone-responsive G-protein coupled receptor and expressing a mammalian-yeast Galpha hybrid protein were made conditional for growth upon either pheromone pathway activation (activator screen) or pheromone pathway inactivation (inhibitor screen). Mammalian cDNAs that conferred plasmid-dependent growth under restrictive conditions were identified. One of the cDNAs identified from the activator screen, a human Ras-related G protein that we term AGS1 (for activator of G-protein signaling), appears to function by facilitating guanosine triphosphate (GTP) exchange on the heterotrimeric Galpha. A cDNA product identified from the inhibitor screen encodes a previously identified regulator of G-protein signaling, human RGS5.     

Structural basis of function in heterotrimeric G proteins

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein alpha subunit (Galpha) binds GDP and Gbetagamma. Receptors activate G proteins by catalyzing GTP for GDP exchange on Galpha, leading to a structural change in the Galpha(GTP) and Gbetagamma subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Galpha subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Galpha, how these changes may lead to subunit dissociation and allow Galpha and Gbetagamma to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor-G protein complex and how this interaction leads to GDP release from Galpha. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor-G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.     

The GAPs, GEFs, and GDIs of heterotrimeric G-protein alpha subunits

The heterotrimeric G-protein alpha subunit has long been considered a bimodal, GTP-hydrolyzing switch controlling the duration of signal transduction by seven-transmembrane domain (7TM) cell-surface receptors. In 1996, we and others identified a superfamily of "regulator of G-protein signaling" (RGS) proteins that accelerate the rate of GTP hydrolysis by Galpha subunits (dubbed GTPase-accelerating protein or "GAP" activity). This discovery resolved the paradox between the rapid physiological timing seen for 7TM receptor signal transduction in vivo and the slow rates of GTP hydrolysis exhibited by purified Galpha subunits in vitro. Here, we review more recent discoveries that have highlighted newly-appreciated roles for RGS proteins beyond mere negative regulators of 7TM signaling. These new roles include the RGS-box-containing, RhoA-specific guanine nucleotide exchange factors (RGS-RhoGEFs) that serve as Galpha effectors to couple 7TM and semaphorin receptor signaling to RhoA activation, the potential for RGS12 to serve as a nexus for signaling from tyrosine kinases and G-proteins of both the Galpha and Ras-superfamilies, the potential for R7-subfamily RGS proteins to couple Galpha subunits to 7TM receptors in the absence of conventional Gbetagamma dimers, and the potential for the conjoint 7TM/RGS-box Arabidopsis protein AtRGS1 to serve as a ligand-operated GAP for the plant Galpha AtGPA1. Moreover, we review the discovery of novel biochemical activities that also impinge on the guanine nucleotide binding and hydrolysis cycle of Galpha subunits: namely, the guanine nucleotide dissociation inhibitor (GDI) activity of the GoLoco motif-containing proteins and the 7TM receptor-independent guanine nucleotide exchange factor (GEF) activity of Ric8/synembryn. Discovery of these novel GAP, GDI, and GEF activities have helped to illuminate a new role for Galpha subunit GDP/GTP cycling required for microtubule force generation and mitotic spindle function in chromosomal segregation.     

Cortical localization of the Galpha protein GPA-16 requires RIC-8 function during C. elegans asymmetric cell division

Understanding of the mechanisms governing spindle positioning during asymmetric division remains incomplete. During unequal division of one-cell stage C. elegans embryos, the Galpha proteins GOA-1 and GPA-16 act in a partially redundant manner to generate pulling forces along astral microtubules. Previous work focused primarily on GOA-1, whereas the mechanisms by which GPA-16 participates in this process are not well understood. Here, we report that GPA-16 is present predominantly at the cortex of one-cell stage embryos. Using co-immunoprecipitation and surface plasmon resonance binding assays, we find that GPA-16 associates with RIC-8 and GPR-1/2, two proteins known to be required for pulling force generation. Using spindle severing as an assay for pulling forces, we demonstrate that inactivation of the Gbeta protein GPB-1 renders GPA-16 and GOA-1 entirely redundant. This suggests that the two Galpha proteins can activate the same pathway and that their dual presence is normally needed to counter Gbetagamma. Using nucleotide exchange assays, we establish that whereas GPR-1/2 acts as a guanine nucleotide dissociation inhibitor (GDI) for GPA-16, as it does for GOA-1, RIC-8 does not exhibit guanine nucleotide exchange factor (GEF) activity towards GPA-16, in contrast to its effect on GOA-1. We establish in addition that RIC-8 is required for cortical localization of GPA-16, whereas it is not required for that of GOA-1. Our analysis demonstrates that this requirement toward GPA-16 is distinct from the known function of RIC-8 in enabling interaction between Galpha proteins and GPR-1/2, thus providing novel insight into the mechanisms of asymmetric spindle positioning.     

Ric-8 controls Drosophila neural progenitor asymmetric division by regulating heterotrimeric G proteins

Asymmetric division of Drosophila neuroblasts (NBs) and the Caenorhabditis elegans zygote uses polarity cues provided by the Par proteins, as well as heterotrimeric G-protein-signalling that is activated by a receptor-independent mechanism mediated by GoLoco/GPR motif proteins. Another key component of this non-canonical G-protein activation mechanism is a non-receptor guanine nucleotide-exchange factor (GEF) for Galpha, RIC-8, which has recently been characterized in C. elegans and in mammals. We show here that the Drosophila Ric-8 homologue is required for asymmetric division of both NBs and pl cells. Ric-8 is necessary for membrane targeting of Galphai, Pins and Gbeta13F, presumably by regulating multiple Galpha subunit(s). Ric-8 forms an in vivo complex with Galphai and interacts preferentially with GDP-Galphai, which is consistent with Ric-8 acting as a GEF for Galphai. Comparisons of the phenotypes of Galphai, Ric-8, Gbeta13Fsingle and Ric-8;Gbeta13F double loss-of-function mutants indicate that, in NBs, Ric-8 positively regulates Gai activity. In addition, Gbetagamma acts to restrict Galphai (and GoLoco proteins) to the apical cortex, where Galphai (and Pins) can mediate asymmetric spindle geometry.     

Selective interaction of AGS3 with G-proteins and the influence of AGS3 on the activation state of G-proteins

AGS3 (activator of G-protein signaling 3) was isolated in a yeast-based functional screen for receptor-independent activators of heterotrimeric G-proteins. As an initial approach to define the role of AGS3 in mammalian signal processing, we defined the AGS3 subdomains involved in G-protein interaction, its selectivity for G-proteins, and its influence on the activation state of G-protein. Immunoblot analysis with AGS3 antisera indicated expression in rat brain, the neuronal-like cell lines PC12 and NG108-15, as well as the smooth muscle cell line DDT(1)-MF2. Immunofluorescence studies and confocal imaging indicated that AGS3 was predominantly cytoplasmic and enriched in microdomains of the cell. AGS3 coimmunoprecipitated with Galpha(i3) from cell and tissue lysates, indicating that a subpopulation of AGS3 and Galpha(i) exist as a complex in the cell. The coimmunoprecipitation of AGS3 and Galpha(i) was dependent upon the conformation of Galpha(i3) (GDP GTPgammaS (guanosine 5'-3-O-(thio)triphosphate)). The regions of AGS3 that bound Galpha(i) were localized to four amino acid repeats (G-protein regulatory motif (GPR)) in the carboxyl terminus (Pro(463)-Ser(650)), each of which were capable of binding Galpha(i). AGS3-GPR domains selectively interacted with Galpha(i) in tissue and cell lysates and with purified Galpha(i)/Galpha(t). Subsequent experiments with purified Galpha(i2) and Galpha(i3) indicated that the carboxyl-terminal region containing the four GPR motifs actually bound more than one Galpha(i) subunit at the same time. The AGS3-GPR domains effectively competed with Gbetagamma for binding to Galpha(t(GDP)) and blocked GTPgammaS binding to Galpha(i1). AGS3 and related proteins provide unexpected mechanisms for coordination of G-protein signaling pathways.     

A point mutation to Galphai selectively blocks GoLoco motif binding: direct evidence for Galpha.GoLoco complexes in mitotic spindle dynamics

Heterotrimeric G-protein Galpha subunits and GoLoco motif proteins are key members of a conserved set of regulatory proteins that influence invertebrate asymmetric cell division and vertebrate neuroepithelium and epithelial progenitor differentiation. GoLoco motif proteins bind selectively to the inhibitory subclass (Galphai) of Galpha subunits, and thus it is assumed that a Galphai.GoLoco motif protein complex plays a direct functional role in microtubule dynamics underlying spindle orientation and metaphase chromosomal segregation during cell division. To address this hypothesis directly, we rationally identified a point mutation to Galphai subunits that renders a selective loss-of-function for GoLoco motif binding, namely an asparagine-to-isoleucine substitution in the alphaD-alphaE loop of the Galpha helical domain. This GoLoco-insensitivity ("GLi") mutation prevented Galphai1 association with all human GoLoco motif proteins and abrogated interaction between the Caenorhabditis elegans Galpha subunit GOA-1 and the GPR-1 GoLoco motif. In contrast, the GLi mutation did not perturb any other biochemical or signaling properties of Galphai subunits, including nucleotide binding, intrinsic and RGS protein-accelerated GTP hydrolysis, and interactions with Gbetagamma dimers, adenylyl cyclase, and seven transmembrane-domain receptors. GoLoco insensitivity rendered Galphai subunits unable to recruit GoLoco motif proteins such as GPSM2/LGN and GPSM3 to the plasma membrane, and abrogated the exaggerated mitotic spindle rocking normally seen upon ectopic expression of wild type Galphai subunits in kidney epithelial cells. This GLi mutation should prove valuable in establishing the physiological roles of Galphai.GoLoco motif protein complexes in microtubule dynamics and spindle function during cell division as well as to delineate potential roles for GoLoco motifs in receptor-mediated signal transduction.     

Apoptotic signaling through the beta -adrenergic receptor. A new Gs effector pathway

Stimulation of beta-adrenergic receptor normally results in signaling by the heterotrimeric G protein G(s), leading to the activation of adenylyl cyclase, production of cAMP, and activation of cAMP-dependent protein kinase (PKA). Here we report that cell death of thymocytes can be induced after stimulation of beta-adrenergic receptor, or by addition of exogenous cAMP. Apoptotic cell death in both cases was observed with the appearance of terminal deoxynucleotidyl transferase-mediated UTP end labeling reactivity and the activation of caspase-3 in S49 T cells. Using thymocytes deficient in either Galpha(s) or PKA, we find that engagement of beta-adrenergic receptors initiated a Galpha(s)-dependent, PKA-independent pathway leading to apoptosis. This alternative pathway involves Src family tyrosine kinase Lck. Furthermore, we show that Lck protein kinase activity can be directly stimulated by purified Galpha(s). Our data reveal a new signaling pathway for Galpha(s), distinct from the classical PKA pathway, that accounts for the apoptotic action of beta-adrenergic receptors.     

D2 dopamine receptor activation of potassium channels is selectively decoupled by Galpha-specific GoLoco motif peptides

The GoLoco motif is a short polypeptide sequence found in G-protein signaling regulators such as regulator of G-protein signaling proteins type 12 and 14 and activator of G-protein signaling protein type 3. A unique property of the GoLoco motifs from these three proteins is their preferential interaction with guanosine diphosphate (GDP)-bound Galpha(i1), Galpha(i3) and, sometimes, Galpha(i2) subunits over Galpha(o) subunits. This interaction prevents both spontaneous guanine nucleotide release and reassociation of Galpha(i)-GDP with Gbetagamma. We utilized this property of the GoLoco motif to examine dopamine (D2 and D3) and somatostatin receptor coupling to G-protein-regulated inwardly rectifying potassium (GIRK) channels in mouse AtT20 cells. GoLoco motif peptides had no effect on either basal channel activity or the initial responses to agonists, suggesting that the GoLoco motif cannot disrupt pre-formed G-protein heterotrimers. GoLoco motif peptides did, however, interfere with human D2((short)) receptor coupling to GIRK channels as demonstrated by the progressively diminished responses after repeated agonist application. This behavior is consistent with some form of compartmentalization of D2 receptors and GIRK channels such that Gbetagamma subunits, freed by local receptor activation and prevented from reforming a heterotrimeric complex, are not functionally constrained within the receptor-channel complex and thus are unable to exert a persistent activating effect. In contrast, GoLoco motif peptides had no effect on either D3 or somatostatin coupling to GIRK channels. Our results suggest that GoLoco motif-based peptides will be useful tools in examining the specificity of G-protein-coupled receptor-effector coupling.     

Activation of heterotrimeric G-protein signaling by a ras-related protein. Implications for signal integration

Utilizing a functional screen in the yeast Saccharomyces cerevisiae we identified mammalian proteins that activate heterotrimeric G-protein signaling pathways in a receptor-independent fashion. One of the identified activators, termed AGS1 (for activator of G-protein signaling), is a human Ras-related G-protein that defines a distinct subgroup of the Ras superfamily. Expression of AGS1 in yeast and in mammalian cells results in specific activation of Galpha(i)/Galpha(o) heterotrimeric signaling pathways. In addition, the in vivo and in vitro properties of AGS1 are consistent with it functioning as a direct guanine nucleotide exchange factor for Galpha(i)/Galpha(o). AGS1 thus presents a unique mechanism for signal integration via heterotrimeric G-protein signaling pathways.     

Cooperation of Gq,Gi, and G12/13 in protein kinase D activation and phosphorylation induced by lysophosphatidic acid

To examine the contribution of different G-protein pathways to lysophosphatidic acid (LPA)-induced protein kinase D (PKD) activation, we tested the effect of LPA on PKD activity in murine embryonic cell lines deficient in Galpha(q/11) (Galpha(q/11) KO cells) or Galpha(12/13) (Galpha(12/13) KO cells) and used cells lacking rhodopsin kinase (RK cells) as a control. In RK and Galpha(12/13) KO cells, LPA induced PKD activation through a phospholipase C/protein kinase C pathway in a concentration-dependent fashion with maximal stimulation (6-fold for RK cells and 4-fold for Galpha(12/13) KO cells in autophosphorylation activity) achieved at 3 microm. In contrast, LPA did not induce any significant increase in PKD activity in Galpha(q/11) KO cells. However, LPA induced a significantly increased PKD activity when Galpha(q/11) KO cells were transfected with Galpha(q). LPA-induced PKD activation was modestly attenuated by prior exposure of RK cells to pertussis toxin (PTx) but abolished by the combination treatments of PTx and Clostridium difficile toxin B. Surprisingly, PTx alone strikingly inhibited LPA-induced PKD activation in a concentration-dependent fashion in Galpha(12/13) KO cells. Similar results were obtained when activation loop phosphorylation at Ser-744 was determined using an antibody that detects the phosphorylated state of this residue. Our results indicate that G(q) is necessary but not sufficient to mediate LPA-induced PKD activation. In addition to G(q), LPA requires additional G-protein pathways to elicit a maximal response with G(i) playing a critical role in Galpha(12/13) KO cells. We conclude that LPA induces PKD activation through G(q), G(i), and G(12) and propose that PKD activation is a point of convergence in the action of multiple G-protein pathways.     

Receptor-independent activators of heterotrimeric G-proteins

Heterotrimeric G-protein signalling systems are primarily activated via cell surface receptors possessing the seven membrane span motif. Several observations suggest the existence of other modes of input to such signalling systems either downstream of effectors or at the level of G-proteins themselves. Using a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae, we identified three proteins, AGS1-3 (for Activators of G-protein Signalling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1 defines a distinct member of the super family of ras related proteins. AGS2 is identical to mouse Tctex1, a protein that exists as a light chain component of the cytoplasmic motor protein dynein and subserves as yet undefined functions in cell signalling pathways. AGS3 possesses a series of tetratrico repeat motifs and a series of four amino acid repeats termed G-protein regulatory motifs. The GPR motifs are found in a number of proteins that interact with and regulate Galpha. Although each AGS protein activates G-protein signaling, they do so by different mechanisms within the context of the G-protein activation/deactivation cycle. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signalling pathways.     

RACK1 regulates specific functions of Gbetagamma

We showed previously that Gbetagamma interacts with Receptor for Activated C Kinase 1 (RACK1), a protein that not only binds activated protein kinase C (PKC) but also serves as an adaptor/scaffold for many signaling pathways. Here we report that RACK1 does not interact with Galpha subunits or heterotrimeric G proteins but binds free Gbetagamma subunits released from activated heterotrimeric G proteins following the activation of their cognate receptors in vivo. The association with Gbetagamma promotes the translocation of RACK1 from the cytosol to the membrane. Moreover, binding of RACK1 to Gbetagamma results in inhibition of Gbetagamma-mediated activation of phospholipase C beta2 and adenylyl cyclase II. However, RACK1 has no effect on other functions of Gbetagamma, such as activation of the mitogen-activated protein kinase signaling pathway or chemotaxis of HEK293 cells via the chemokine receptor CXCR2. Similarly, RACK1 does not affect signal transduction through the Galpha subunits of G(i), G(s), or G(q). Collectively, these findings suggest a role of RACK1 in regulating specific functions of Gbetagamma.     

Endogenous RGS protein action modulates mu-opioid signaling through Galphao. Effects on adenylyl cyclase,extracellular signal-regulated kinases,and intracellular calcium pathways

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins for the Galpha subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, we have prepared C6 cells stably expressing the mu-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive Galpha(o). We have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant Galpha(o) that is either RGS-insensitive or RGS-sensitive. The mu-receptor agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive Galpha(o). In contrast, mu-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GTPase-activating protein activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways.     

G蛋白信号调节因子的结构分类和功能

G蛋白信号调节因子是能够直接与激活的Gα亚基结合,显著刺激Gα亚基上的GTP酶活性,加速GTP水解,从而灭活或终止G蛋白信号的一组分子大小各异的多功能蛋白质家族。它们都共同拥有一个130个氨基酸的保守的RGS结构域,其功能是结合激活的Gα亚基,负调节G蛋白信号。许多RGS蛋白还拥有非RGS结构域,能够结合其它信号蛋白,从而整合和调节G蛋白信号之间以及G蛋白和其它信号系统之间的关系。