Age increases cardiac Galpha(i2) expression,resulting in enhanced coupling to G protein-coupled receptors

Cardiac G protein-coupled receptors that function through stimulatory G protein Galpha(s), such as beta(1)- and beta(2)-adrenergic receptors (beta(1)ARs and beta(2)ARs), play a key role in cardiac contractility. Recent data indicate that several Galpha(s)-coupled receptors in heart also activate Galpha(i), including beta(2)ARs (but not beta(1)ARs). Coupling of cardiac beta(2)ARs to Galpha(i) inhibits adenylyl cyclase and opposes beta(1)AR-mediated apoptosis. Dual coupling of beta(2)AR to both Galpha(s) and Galpha(i) is likely to alter beta(2)AR function in disease, such as congestive heart failure in which Galpha(i) levels are increased. Indeed, heart failure is characterized by reduced responsiveness of betaARs. Cardiac betaAR-responsiveness is also decreased with aging. However, whether age increases cardiac Galpha(i) has been controversial, with some studies reporting an increase and others reporting no change. The present study examines Galpha(i) in left ventricular membranes from young and old Fisher 344 rats by employing a comprehensive battery of biochemical assays. Immunoblotting reveals significant increases with age in left ventricular Galpha(i2), but no changes in Galpha(i3), Galpha(o), Galpha(s), Gbeta(1), or Gbeta(2). Aging also increases ADP-ribosylation of pertussis toxin-sensitive G proteins. Consistent with these results, basal as well as receptor-mediated incorporation of photoaffinity label [(32)P]azidoanilido-GTP indicates higher amounts of Galpha(i2) in older left ventricular membranes. Moreover, both basal and receptor-mediated adenylyl cyclase activities are lower in left ventricular membranes from older rats, and disabling of Galpha(i) with pertussis toxin increases both basal and receptor-stimulated adenylyl cyclase activity. Finally, age produces small but significant increases in muscarinic potency for the inhibition of both beta(1)AR- and beta(2)AR-stimulated adenylyl cyclase activity. The present study establishes that Galpha(i2) increases with age and provides data indicating that this increase dampens adenylyl cyclase activity.     

Functional coupling with Galpha(q) and Galpha(i1) protein subunits promotes high-affinity agonist binding to the neurotensin receptor NTS-1 expressed in Escherichia coli

To analyze the coupling of Galpha subunits to the rat neurotensin receptor NTS-1 (NTR), fusion proteins were expressed in Escherichia coli with various Galpha subunits covalently linked to the receptor C-terminus. The presence of Galpha(q) or Galpha(i/q), in which the six C-terminal residues of Galpha(i1) were replaced with those from Galpha(q), increased the percentage of receptors in the agonist high-affinity state. This effect was less pronounced for wild-type Galpha(i1) and not observed for Galpha(i/s). Functional coupling of neurotensin receptor to Galpha was demonstrated by neurotensin-induced [(35)S]GTPgammaS binding for the Galpha(q), Galpha(i/q) and Galpha(i1) subunits, but not for Galpha(i/s). Our results extend previous findings of the dual coupling of NTR to pertussis toxin-sensitive and -insensitive G-proteins in Chinese hamster ovary cells with preference for the latter.     

Activation of Galpha (i) and subsequent uncoupling of receptor-Galpha(i) signaling by Pasteurella multocida toxin

Bacterial protein toxins are powerful tools for elucidating signaling mechanisms in eukaryotic cells. A number of bacterial protein toxins, e.g. cholera toxin, pertussis toxin (PTx), or Pasteurella multocida toxin (PMT), target heterotrimeric G proteins and have been used to stimulate or block specific signaling pathways or to demonstrate the contribution of their target proteins in cellular effects. PMT is a major virulence factor of P. multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. PMT modulates various signaling pathways, including phospholipase Cbeta and RhoA, by acting on the heterotrimeric G proteins Galpha(q) and Galpha(12/13), respectively. Here we report that PMT is a powerful activator of G(i) protein. We show that PMT decreases basal isoproterenol and forskolin-stimulated cAMP accumulation in intact Swiss 3T3 cells, inhibits adenylyl cyclase activity in cell membrane preparations, and enhances the inhibition of cAMP accumulation caused by lysophosphatidic acid via endothelial differentiation gene receptors. PMT-mediated inhibition of cAMP production is independent of toxin activation of Galpha(q) and/or Galpha(12/13). Although the effects of PMT are not inhibited by PTx, PMT blocks PTx-catalyzed ADP-ribosylation of G(i). PMT also inhibits steady-state GTPase activity and GTP binding of G(i) in Swiss 3T3 cell membranes stimulated by lysophosphatidic acid. The data indicate that PMT is a novel activator of G(i), modulating its GTPase activity and converting it into a PTx-insensitive state.     

AGS3 and signal integration by Galpha(s)- and Galpha(i)-coupled receptors: AGS3 blocks the sensitization of adenylyl cyclase following prolonged stimulation of a Galpha(i)-coupled receptor by influencing processing of Galpha(i)

AGS3-LONG and AGS3-SHORT contain G-protein regulatory motifs that interact with and stabilize the GDP-bound conformation of Galpha(i) > Galpha(o). AGS3 and related proteins may influence signal strength or duration as well as the adaptation of the signaling system associated with sustained stimulation. To address these issues, we determined the effect of AGS3 on the integration of stimulatory (Galpha(s)-mediated vasoactive intestinal peptide receptor) and inhibitory (Galpha(i)-mediated alpha(2)-adrenergic receptor (alpha(2)-AR)) signals to adenylyl cyclase in Chinese hamster ovary cells. AGS3-SHORT and AGS3-LONG did not alter the VIP-induced increase in cAMP or the inhibitory effect of alpha(2)-AR activation. System adaptation was addressed by determining the influence of AGS3 on the sensitization of adenylyl cyclase that occurs following prolonged activation of a Galpha(i)-coupled receptor. Incubation of cells with the alpha(2)-AR agonist UK14304 (1 microm) for 18 h resulted in a approximately 1.8-fold increase in the vasoactive intestinal peptide-induced activation of adenylyl cyclase, and this was associated with a decrease in membrane-associated Galpha(i3). Both effects were blocked by AGS3-SHORT. AGS3-SHORT also decreased the rate of Galpha(i3) decay. A mutant AGS3-SHORT incapable of binding G-protein was inactive. These data suggest that AGS3 and perhaps other G-protein regulatory motif-containing proteins increase the stability of Galpha(i) in the membrane, which influences the adaptation of the cell to prolonged activation of Galpha(i)-coupled receptors.     

Prostacyclin receptor-induced STAT3 phosphorylation in human erythroleukemia cells is mediated via Galpha(s) and Galpha(16) hybrid signaling

Human prostacyclin receptor (hIP) stimulates STAT3 via pertussis toxin-insensitive G proteins in human erythroleukemia (HEL) cells. Since hIP can utilize G(s) and G(q) proteins for signal transduction and that both G proteins can induce STAT3 phosphorylation and activation via complex signaling networks, we sought to determine if one of them is predominant in mediating the hIP signal. Stimulation of STAT3 Tyr(705) and Ser(727) phosphorylations by the IP-specific agonist, cicaprost, was sensitive to inhibition of protein kinase A, phospholipase Cbeta, protein kinase C, calmodulin-dependent protein kinase II and Janus kinase 2/3. Unlike Galpha(16)-mediated regulation of STAT3 in the same cells, cicaprost-induced STAT3 Tyr(705) phosphorylation was resistant to inhibition of Src and MEK while STAT3 Ser(727) phosphorylation distinctly required phosphatidylinositol-3 kinase. This unique inhibitor-sensitivity pattern of STAT3 phosphorylation was reproduced in HEL cells by stimulating the G(16)-coupled C5a receptor in the presence of dibutyryl-cAMP, suggesting that the change in inhibitor-sensitivity was due to activation of the G(s) pathway. This postulation was confirmed by expressing constitutively active Galpha(16)QL and Galpha(s)QL in human embryonic kidney 293 cells and the inhibitor-sensitivity of Galpha(16)QL-induced STAT3 phosphorylations could be converted by the mere presence of Galpha(s)QL to resemble that obtained with cicaprost in HEL cells. In addition, the restoration of the Galpha(16)-mediated inhibitor-sensitivity upon cicaprost induction in Galpha(s)-knocked down HEL cells again verified the pivotal role of G(s) signal. Taken together, our observations illustrate that co-stimulation of G(s) and G(q) can result in the fine-tuning of STAT3 activation status, and this may provide the basis for cell type-specific responses following activation of hIP.     

Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.     

Gbetagamma inhibits Galpha GTPase-activating proteins by inhibition of Galpha-GTP binding during stimulation by receptor

Gbetagamma subunits modulate several distinct molecular events involved with G protein signaling. In addition to regulating several effector proteins, Gbetagamma subunits help anchor Galpha subunits to the plasma membrane, promote interaction of Galpha with receptors, stabilize the binding of GDP to Galpha to suppress spurious activation, and provide membrane contact points for G protein-coupled receptor kinases. Gbetagamma subunits have also been shown to inhibit the activities of GTPase-activating proteins (GAPs), both phospholipase C (PLC)-betas and RGS proteins, when assayed in solution under single turnover conditions. We show here that Gbetagamma subunits inhibit G protein GAP activity during receptor-stimulated, steady-state GTPase turnover. GDP/GTP exchange catalyzed by receptor requires Gbetagamma in amounts approximately equimolar to Galpha, but GAP inhibition was observed with superstoichiometric Gbetagamma. The potency of inhibition varied with the GAP and the Galpha subunit, but half-maximal inhibition of the GAP activity of PLC-beta1 was observed with 5-10 nM Gbetagamma, which is at or below the concentrations of Gbetagamma needed for regulation of physiologically relevant effector proteins. The kinetics of GAP inhibition of both receptor-stimulated GTPase activity and single turnover, solution-based GAP assays suggested a competitive mechanism in which Gbetagamma competes with GAPs for binding to the activated, GTP-bound Galpha subunit. An N-terminal truncation mutant of PLC-beta1 that cannot be directly regulated by Gbetagamma remained sensitive to inhibition of its GAP activity, suggesting that the Gbetagamma binding site relevant for GAP inhibition is on the Galpha subunit rather than on the GAP. Using fluorescence resonance energy transfer between cyan or yellow fluorescent protein-labeled G protein subunits and Alexa532-labeled RGS4, we found that Gbetagamma directly competes with RGS4 for high-affinity binding to Galpha(i)-GDP-AlF4.     

Rapid kinetics of regulator of G-protein signaling (RGS)-mediated Galphai and Galphao deactivation. Galpha specificity of RGS4 AND RGS7

Regulator of G-protein signaling (RGS) proteins accelerate GTP hydrolysis by Galpha subunits speeding deactivation. Galpha deactivation kinetics mediated by RGS are too fast to be directly studied using conventional radiochemical methods. We describe a stopped-flow spectroscopic approach to visualize these rapid kinetics by measuring the intrinsic tryptophan fluorescence decrease of Galpha accompanying GTP hydrolysis and Galpha deactivation on the millisecond time scale. Basal k(cat) values for Galpha(o), Galpha(i1), and Galpha(i2) at 20 degrees C were similar (0.025-0.033 s(-1)). Glutathione S-transferase fusion proteins containing RGS4 and an RGS7 box domain (amino acids 305-453) enhanced the rate of Galpha deactivation in a manner linear with RGS concentration. RGS4-stimulated rates could be measured up to 5 s(-1) at 3 microm, giving a catalytic efficiency of 1.7-2.8 x 10(6) m(-1) s(-1) for all three Galpha subunits. In contrast, RGS7 showed catalytic efficiencies of 0.44, 0.10, and 0.02 x 10(6) m(-1) s(-1) toward Galpha(o), Galpha(i2), and Galpha(i1), respectively. Thus RGS7 is a weaker GTPase activating protein than RGS4 toward all Galpha subunits tested, but it is specific for Galpha(o) over Galpha(i1) or Galpha(i2). Furthermore, the specificity of RGS7 for Galpha(o) does not depend on N- or C-terminal extensions or a Gbeta(5) subunit but resides in the RGS domain itself.     

Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase

The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.     

MANT-substituted guanine nucleotides: a novel class of potent adenylyl cyclase inhibitors

Mammals express nine membranous adenylyl cyclase (AC) isoforms (AC1-AC9), but the precise functions of AC isoforms are still incompletely understood. This situation is at least partially due to the paucity of potent and isoenzyme-specific AC inhibitors. The original aim of our research was to develop a fluorescence assay for the stimulatory G-protein of AC, G(s). 2'(3')-O-(N-methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and were previously used for the fluorescence analysis of purified G(i)/G(o)-proteins. We studied the effects of MANT-guanosine 5'-[gamma-thio]triphosphate (MANT-GTPgammaS) and MANT-guanosine 5'-[beta,gamma-imido]triphosphate (MANT-GppNHp) on Galpha(s)- and Galpha(i)-mediated signaling. MANT-GTPgammaS and MANT-GppNHp had lower affinities for Galpha(s) and Galpha(i) than GTPgammaS and GppNHp. In contrast to guanosine 5'-[beta-thio]diphosphate, MANT-GTPgammaS noncompetitively inhibited GTPgammaS-stimulated AC in Galpha(s)-expressing Sf9 insect cell membranes. AC inhibition by MANT-GTPgammaS and MANT-GppNHp was not due to Galpha(s) inhibition since it was also observed in Galpha(s)-deficient S49 cyc(-) lymphoma cell membranes. Mn(2+) blocked Galpha(i)-mediated AC inhibition by GTPgammaS and GppNHp in S49 cyc(-) membranes but not AC inhibition by MANT-GTPgammaS and MANT-GppNHp. MANT-GTPgammaS and MANT-GppNHp competitively inhibited forskolin/Mn(2+)-stimulated AC in S49 cyc(-) membranes with K(i) values of 53 nM and 160 nM, respectively. Taken together, MANT-substituted guanine nucleotides constitute a novel class of potent competitive AC inhibitors. The availability of potent fluorescent AC inhibitors will help us study the kinetics of AC/nucleotide interactions as well as function, trafficking and localization of AC isoenzymes in intact cells. In future studies, we will examine the specificity of MANT-nucleotides for AC isoenzymes.     

Selective stabilization of the high affinity binding conformation of glucagon receptor by the long splice variant of Galpha(s)

To analyze functional differences in the interactions of the glucagon receptor (GR) with the two predominant splice variants of Galpha(s), GR was covalently linked to the short and the long forms Galpha(s)-S and Galpha(s)-L to produce the fusion proteins GR-Galpha(s)-S and GR-Galpha(s)-L. GR-Galpha(s)-S bound glucagon with an affinity similar to that of GR, while GR-Galpha(s)-L showed a 10-fold higher affinity for glucagon. In the presence of GTPgammaS, GR-Galpha(s)-L reverted to the low affinity glucagon binding conformation. Both GR-Galpha(s)-L and GR-Galpha(s)-S were constitutively active, causing elevated basal levels of cAMP even in the absence of glucagon. A mutant GR that failed to activate G(s) (G23D1R) was fused to Galpha(s)-L. G23D1R-Galpha(s)-L bound glucagon with high affinity, but failed to elevate cAMP levels, suggesting that the mechanisms of GR-mediated Galpha(s)-L activation and Galpha(s)-L-induced high affinity glucagon binding are independent. Both GR-Galpha(s)-S and GR-Galpha(s)-L bound the antagonist desHis(1)[Nle(9),Ala(11),Ala(16)]glucagon amide with affinities similar to GR. The antagonist displayed partial agonist activity with GR-Galpha(s)-L, but not with GR-Galpha(s)-S. Therefore, the partial agonist activity of the antagonist observed in intact cells appears to be due to GRs coupled to Galpha(s)-L. We conclude that Galpha(s)-S and Galpha(s)-L interact differently with GR and that specific coupling of GR to Galpha(s)-L may account for GTP-sensitive high affinity glucagon binding.     

Mastoparan inhibits beta-adrenoceptor-G(s) signaling by changing the localization of Galpha(s) in lipid rafts

Mastoparan, a wasp venom toxin, has various pharmacological activities, the mechanisms of which are still unknown. To clarify the action of mastoparan on G protein-coupled receptor-mediated signaling, we previously examined the effect of mastoparan on G(q)-mediated signaling and demonstrated that mastoparan binds to gangliosides causing a decrease in Galpha(q/11) content in lipid rafts, and resulting in the inhibition of G(q)-mediated phosphoinositide hydrolysis (Sugama et al., Mol. Pharmacol., 68, 1466, 2005). In the present study, we examined the effect of mastoparan on beta-adrenoceptor-G(s) signaling in 1321N1 human astrocytoma cells. Mastoparan inhibited isoproterenol-induced elevation of cyclic AMP in a concentration-dependent manner. Although mastoparan is known to be an activator of G(i), pertussis toxin only slightly attenuated mastoparan-induced inhibition of cyclic AMP elevation, suggesting that a major part of the inhibition of cyclic AMP elevation induced by mastoparan is not mediated by Galpha(i). By contrast, mastoparan-induced inhibition of cyclic AMP elevation was clearly attenuated by preincubation of the cells with ganglioside mixtures. Moreover, mastoparan changed the localization of Galpha(s) in lipid rafts without disrupting the structure of lipid rafts. Fluorescent staining analysis showed that mastoparan released GFP-Galpha(s) from plasma membranes into the cytosol. These results suggest that the mastoparan-induced suppression of cyclic AMP elevation is mainly caused by changing the localization of Galpha(s) in lipid rafts into a compartment in the cellular interior where it is not available to activate adenylyl cyclase.     

Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange

Heterotrimeric G proteins are molecular switches that regulate numerous signaling pathways involved in cellular physiology. This characteristic is achieved by the adoption of two principal states: an inactive, GDP bound state and an active, GTP bound state. Under basal conditions, G proteins exist in the inactive, GDP bound state; thus, nucleotide exchange is crucial to the onset of signaling. Despite our understanding of G protein signaling pathways, the mechanism of nucleotide exchange remains elusive. We employed phage display technology to identify nucleotide state-dependent Galpha binding peptides. Herein, we report a GDP-selective Galpha binding peptide, KB-752, that enhances spontaneous nucleotide exchange of Galpha(i) subunits. Structural determination of the Galpha(i1)/peptide complex reveals unique changes in the Galpha switch regions predicted to enhance nucleotide exchange by creating a GDP dissociation route. Our results cast light onto a potential mechanism by which Galpha subunits adopt a conformation suitable for nucleotide exchange.     

Targeted inactivation of Galpha(i) does not alter cardiac function or beta-adrenergic sensitivity

Inhibitory Galpha(i) protein increases in the myocardium during hypertrophy and has been associated with beta-adrenergic receptor (beta-AR) desensitization, contractile dysfunction, and progression of cardiac disease. The role of Galpha(i) proteins in mediating basal cardiac function and beta-AR response in nonpathological myocardium, however, is uncertain. Transgenic mice with targeted inactivation of Galpha(i2) or Galpha(i3) were examined for in vivo cardiac function with the use of conscious echocardiography and for ex vivo cardiac response to inotropic stimulation with the use of Langendorff blood-perfused isolated hearts and adult ventricular cardiomyocytes. Echocardiography revealed that percent fractional shortening and heart rate were similar among wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. Comparable baseline diastolic and contractile performance was also observed in isolated hearts and isolated ventricular myocytes from wild-type mice and mice lacking Galpha(i) proteins. Isoproterenol infusion enhanced diastolic and contractile performance to a similar degree in wild-type, Galpha(i2)-null, and Galpha(i3)-null mice. These data demonstrate no observable role for inhibitory G proteins in mediating basal cardiac function or sensitivity to beta-AR stimulation in nonpathological myocardium.     

Mammalian Ric-8A (synembryn) is a heterotrimeric Galpha protein guanine nucleotide exchange factor

The activation of heterotrimeric G proteins is accomplished primarily by the guanine nucleotide exchange activity of ligand-bound G protein-coupled receptors. The existence of nonreceptor guanine nucleotide exchange factors for G proteins has also been postulated. Yeast two-hybrid screens with Galpha(o) and Galpha(s) as baits were performed to identify binding partners of these proteins. Two mammalian homologs of the Caenorhabditis elegans protein Ric-8 were identified in these screens: Ric-8A (Ric-8/synembryn) and Ric-8B. Purification and biochemical characterization of recombinant Ric-8A revealed that it is a potent guanine nucleotide exchange factor for a subset of Galpha proteins including Galpha(q), Galpha(i1), and Galpha(o), but not Galpha(s). The mechanism of Ric-8A-mediated guanine nucleotide exchange was elucidated. Ric-8A interacts with GDP-bound Galpha proteins, stimulates release of GDP, and forms a stable nucleotide-free transition state complex with the Galpha protein; this complex dissociates upon binding of GTP to Galpha.     

Regulator of G protein signaling Z1 (RGSZ1) interacts with Galpha i subunits and regulates Galpha i-mediated cell signaling

Regulator of G protein signaling (RGS) proteins constitute a family of over 20 proteins that negatively regulate heterotrimeric G protein-coupled receptor signaling pathways by enhancing endogenous GTPase activities of G protein alpha subunits. RGSZ1, one of the RGS proteins specifically localized to the brain, has been cloned previously and described as a selective GTPase accelerating protein for Galpha(z) subunit. Here, we employed several methods to provide new evidence that RGSZ1 interacts not only with Galpha(z,) but also with Galpha(i), as supported by in vitro binding assays and functional studies. Using glutathione S-transferase fusion protein pull-down assays, glutathione S-transferase-RGSZ1 protein was shown to bind (35)S-labeled Galpha(i1) protein in an AlF(4)(-)dependent manner. The interaction between RGSZ1 and Galpha(i) was confirmed further by co-immunoprecipitation studies and yeast two-hybrid experiments using a quantitative luciferase reporter gene. Extending these observations to functional studies, RGSZ1 accelerated endogenous GTPase activity of Galpha(i1) in single-turnover GTPase assays. Human RGSZ1 functionally regulated GPA1 (a yeast Galpha(i)-like protein)-mediated yeast pheromone response when expressed in a SST2 (yeast RGS protein) knockout strain. In PC12 cells, transfected RGSZ1 blocked mitogen-activated protein kinase activity induced by UK14304, an alpha(2)-adrenergic receptor agonist. Furthermore, RGSZ1 attenuated D2 dopamine receptor agonist-induced serum response element reporter gene activity in Chinese hamster ovary cells. In summary, these data suggest that RGSZ1 serves as a GTPase accelerating protein for Galpha(i) and regulates Galpha(i)-mediated signaling, thus expanding the potential role of RGSZ1 in G protein-mediated cellular activities.     

The myristoylated amino terminus of Galpha(i)(1) plays a critical role in the structure and function of Galpha(i)(1) subunits in solution

To determine the role of the myristoylated amino terminus of Galpha in G protein activation, nine individual cysteine mutations along the myristoylated amino terminus of Galpha(i) were expressed in a functionally Cys-less background. Thiol reactive EPR and fluorescent probes were attached to each site as local reporters of mobility and conformational changes upon activation of Galpha(i)GDP by AlF(4)(-), as well as binding to Gbetagamma. EPR and steady state fluorescence anisotropy are consistent with a high degree of immobility for labeled residues in solution all along the amino terminus of myristoylated Galpha(i). This is in contrast to the high mobility of this region in nonmyristoylated Galpha(i) [Medkova, M., et al. (2002) Biochemistry 41, 9962-9972]. Steady state fluorescence measurements revealed pronounced increases in fluorescence upon activation for residues 14-17 and 21 located midway through the 30-amino acid stretch comprising the amino-terminal region. Collectively, the data suggest that myristoylation is an important structural determinant of the amino terminus of Galpha(i) proteins.     

Galpha(s) is palmitoylated at the N-terminal glycine

Covalent lipid attachments are essential co- and post-translational modifications for signalling proteins. Galpha(s), the alpha-subunit of the heterotrimeric G protein that activates adenylyl cyclase, is known to be palmitoylated at the third N-terminal amino acid, a cysteine. Palmitoylation is involved in anchoring Galpha(s) to the membrane by increasing its intrinsic hydrophobicity. We identified by mass spectrometry a second, functionally even more important, covalent modification. It consists of another palmitoyl residue attached to the preceding glycine (Gly(2)). Palmitoylation at this position has profound consequences for levels of signal transduction. It sensitizes the cell up to 200-fold for adenylyl cyclase-stimulating agents. The inhibitory inputs mediated by Galpha(i) are downregulated to <10%. Thereby, Gly(2)-palmitoylation of Galpha(s) relieves cellular stimulation at the level of adenylyl cyclase whereas it renders the inhibitory modulation via Galpha(i) more difficult.     

Stimulated D(1) dopamine receptors couple to multiple Galpha proteins in different brain regions

Previous studies have revealed that activation of rat striatal D(1) dopamine receptors stimulates both adenylyl cyclase and phospholipase C via G(s) and G(q), respectively. The differential distribution of these systems in brain supports the existence of distinct receptor systems. The present communication extends the study by examining other brain regions: hippocampus, amygdala, and frontal cortex. In membrane preparations of these brain regions, selective stimulation of D(1) dopamine receptors increases the hydrolysis of phosphatidylinositol/phosphatidylinositol 4,5-biphosphate. In these brain regions, D(1) dopamine receptors couple differentially to multiple Galpha protein subunits. Antisera against Galpha(q) blocks dopamine-stimulated PIP(2) hydrolysis in hippocampal and in striatal membranes. The binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(i) was enhanced in all brain regions. Dopamine also increased the binding of [(35)S]GTPgammaS or [alpha-(32)P]GTP to Galpha(q) in these brain regions: hippocampus = amygdala > frontal cortex. However, dopamine-stimulated binding of [(35)S]GTPgammaS to Galphas only in the frontal cortex and striatum. This differential coupling profile in the brain regions was not related to a differential regional distribution of the Galpha proteins. Dopamine induced increases in GTPgammaS binding to Galpha(s) and Galpha(q) was blocked by the D(1) antagonist SCH23390 but not by D(2) receptor antagonist l-sulpiride, suggesting that D(1) dopamine receptors couple to both Galpha(s) and Galpha(q) proteins. Co-immunoprecipitation of Galpha proteins with receptor-binding sites indicate that in the frontal cortex, D(1) dopamine-binding sites are associated with both Galpha(s) and Galpha(q) and, in hippocampus or amygdala, D(1) dopamine receptors couple solely to Galpha(q). The results indicate that in addition to the D(1)/G(s)/adenylyl cyclase system, brain D(1)-like dopamine receptor sites activate phospholipase C through Galpha(q) protein.