Copper and zinc inhibit Galphas function: a nucleotide-free state of Galphas induced by Cu2+ and Zn2+

The stimulatory GTP-binding protein of adenylyl cyclase (AC) regulates hormone-stimulated production of cAMP. Here, we demonstrate that Cu(2+) and Zn(2+) inhibit the steady-state GTPase activity of the alpha subunit of GTP-binding protein (Galpha(s)) but do not alter its intrinsic GTPase activity. Cu(2+) and Zn(2+) decrease steady-state GTPase activity by inhibiting the binding of GTP to Galpha(s). Moreover, Cu(2+) and Zn(2+) increase GDP dissociation from Galpha(s) and render the G protein in a nucleotide-free state. However, these cations do not alter the dissociation of the guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) that is already bound to the Galpha(s). Because of their ability to inhibit GTPgammaS binding, preincubation of Cu(2+) or Zn(2+) with Galpha(s) does not permit GTPgammaS to activate Galpha(s) and stimulate AC activity. However, preincubation of Galpha(s) with GTPgammaS followed by addition of Cu(2+) or Zn(2+) did not alter the ability of Galpha(s) to stimulate AC activity. Interestingly, AlF(4)(-) partially restored the ability of Galpha(s), which had been preincubated with Cu(2+) or Zn(2+), to stimulate AC; AlF(4)(-) does not permit the re-association of unbound GDP with Galpha(s). Thus, the interaction of AlF(4)(-) with the nucleotide-free Galpha(s) is sufficient to activate AC. Using antibodies to the N and C termini of Galpha(s), we show that the Cu(2+) interaction site on the G protein is in the C terminus. We conclude that Cu(2+) and Zn(2+) generate a nucleotide-free state of Galpha(s) and that, in the absence of any nucleotide, the gamma-phosphate mimic of GTP, AlF(4)(-), alters Galpha(s) structure sufficiently to permit stimulation of AC activity. Moreover, our finding that isoproterenol-stimulated AC activity was more sensitive to inhibition by Cu(2+) and Zn(2+) as compared with forskolin-stimulated activity is consistent with Galpha(s) being a primary target of these cations in regulating the signaling from receptor to AC.     

Partial rescue of functional interactions of a nonpalmitoylated mutant of the G-protein G alpha s by fusion to the beta-adrenergic receptor

Most heterotrimeric G-protein alpha subunits are posttranslationally modified by palmitoylation, a reversible process that is dynamically regulated. We analyzed the effects of Galpha(s) palmitoylation for its intracellular distribution and ability to couple to the beta-adrenergic receptor (betaAR) and stimulate adenylyl cyclase. Subcellular fractionation and immunofluorescence microscopy of stably transfected cyc(-) cells, which lack endogenous Galpha(s), showed that wild-type Galpha(s) was predominantly localized at the plasma membrane, but the mutant C3A-Galpha(s), which does not incorporate [(3)H]palmitate, was mostly associated with intracellular membranes. In agreement with this mislocalization, C3A-Galpha(s) showed neither isoproterenol- or GTPgammaS-stimulated adenylyl cyclase activation nor GTPgammaS-sensitive high-affinity agonist binding, all of which were present in the wild-type Galpha(s) expressing cells. Fusion of C3A-Galpha(s) with the betaAR [betaAR-(C3A)Galpha(s)] partially rescued its ability to induce high-affinity agonist binding and to stimulate adenylyl cyclase activity after isoproterenol or GTPgammaS treatment. In comparison to results with the WT-Galpha(s) and betaAR (betaAR-Galpha(s)) fusion protein, the betaAR-(C3A)Galpha(s) fusion protein was about half as efficient at coupling to the receptor and effector. Chemical depalmitoylation by hydroxylamine of membranes expressing betaAR-Galpha(s) reduced the high-affinity agonist binding and adenylyl cyclase activation to a similar degree as that observed in betaAR-(C3A)Galpha(s) expressing membranes. Altogether, these findings indicate that palmitoylation ensured proper localization of Galpha(s) and facilitated bimolecular interactions of Galpha(s) with the betaAR and adenylyl cyclase.     

GTPgammaS regulation of a 12-transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) triphosphate (GTPgammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPgammaS stimulated the adenylyl cyclase activity of DdGCA(kqd) with properties similar to those of the wild-type enzyme (decrease of K(m) and increase of V(max)), demonstrating that GTPgammaS stimulation is independent of substrate specificity. Furthermore, GTPgammaS activation of DdGCA(kqd) is retained in several null mutants of Galpha and Gbeta proteins, indicating that GTPgammaS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.     

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.     

Pertussis toxin-insensitive activation of the heterotrimeric G-proteins Gi/Go by the NG108-15 G-protein activator

A ligand-independent activator of heterotrimeric brain G-protein was partially purified from detergent-solubilized extracts of the neuroblastoma-glioma cell hybrid NG108-15. The G-protein activator (NG108-15 G-protein activator (NG-GPA)) increased [(35)S]guanosine 5'-O-(thiotriphosphate) ([(35)S]GTPgammaS) to purified brain G-protein in a magnesium-dependent manner and promoted GDP dissociation from Galpha(o). The NG-GPA also increased GTPgammaS binding to purified, recombinant Galpha(i2), Galpha(i3), and Galpha(o), but minimally altered nucleotide binding to purified transducin. The NG-GPA increased GTPgammaS binding to membrane-bound G-proteins and inhibited basal, forskolin- and hormone-stimulated adenylyl cyclase activity in DDT(1)-MF-2 cell membranes. In contrast to G-protein coupled receptor-mediated activation of heterotrimeric G-proteins in DDT(1)-MF-2 cell membrane preparations, the action of the NG-GPA was not altered by treatment of the cells with pertussis toxin. ADP-ribosylation of purified brain G-protein also failed to alter the increase in GTPgammaS binding elicited by the NG-GPA. Thus, the NG-GPA acts in a manner distinct from that of a G-protein coupled receptor and other recently described receptor-independent activators of G-protein signaling. These data indicate the presence of unexpected regulatory domains on G(i)/G(o) proteins and suggest the existence of pertussis toxin-insensitive modes of signal input to G(i)/G(o) signaling systems.     

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.     

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.     

A docking site for G protein βγ subunits on the parathyroid hormone 1 receptor supports signaling through multiple pathways

The G protein-coupled receptor for PTH and PTH-related protein (PTH1R) signals via many intracellular pathways. The purpose of this work is to investigate a G protein binding site on an intracellular domain of the PTH1R. The carboxy-terminal, cytoplasmic tail of the PTH1R fused to glutathione-S-transferase interacts with Gi/o proteins in vitro. All three subunits of the heterotrimer interact with the receptor C-tail. Activation of the heterotrimeric complex with GTPgammaS has no effect on Gbetagamma interactions, but markedly disrupts binding of the Galphai/o subunits to the receptor tail, suggesting that direct Gbetagamma binding indirectly links Galpha subunits to this region of the receptor. Gbetagamma subunits alone bind the C-tail with an affinity that is comparable to the heterotrimeric G protein complex. G protein complexes consisting of Galphashis6-beta1gamma2 and Galphaqhis6-beta1gamma2 also interact with the PTH1R tail in vitro. The Gbetagamma interaction domain is located on the juxta-membrane region of the tail between amino acids 468 and 491. Mutations that disrupt Gbetagamma interactions block PTH signaling via phospholipase Cbeta/[Ca2+]i and MAPK and markedly reduce signaling via adenylyl cyclase/cAMP. Herein, we define a domain on the PTH1R that is capable of binding G protein heterotrimeric complexes via direct Gbetagamma interactions.     

Selective inhibition of heterotrimeric Gs signaling. Targeting the receptor-G protein interface using a peptide minigene encoding the Galpha(s) carboxyl terminus

The blockade of heptahelical receptor coupling to heterotrimeric G proteins by the expression of peptides derived from G protein Galpha subunits represents a novel means of simultaneously inhibiting signals arising from multiple receptors that share a common G protein pool. Here we examined the mechanism of action and functional consequences of expression of an 83-amino acid polypeptide derived from the carboxyl terminus of Galpha(s) (GsCT). In membranes prepared from GsCT-expressing cells, the peptide blocked high affinity agonist binding to beta(2) adrenergic receptors (AR) and inhibited beta(2)AR-induced [35S]GTPgammaS loading of Galpha(s). GsCT expression inhibited beta(2)AR- and dopamine D(1A) receptor-mediated cAMP production, without affecting the cellular response to cholera toxin or forskolin, indicating that the peptide inhibited receptor-G(s) coupling without impairing G protein or adenylyl cyclase function. [35S]GTPgammaS loading of Galpha(q/11) by alpha(1B)ARs and Galpha(i) by alpha(2A)ARs and G(q/11)- or G(i)-mediated phosphatidylinositol hydrolysis was unaffected, indicating that the inhibitory effects of GsCT were selective for G(s). We next employed the GsCT construct to examine the complex role of G(s) in regulation of the ERK mitogen-activated protein kinase cascade, where activation of the cAMP-dependent protein kinase (PKA) pathway reportedly produces both stimulatory and inhibitory effects on heptahelical receptor-mediated ERK activation. For the beta(2)AR in HEK-293 cells, where PKA activity is required for ERK activation, expression of GsCT caused a net inhibition of ERK activation. In contrast, alpha(2A)AR-mediated ERK activation in COS-7 cells was enhanced by GsCT expression, consistent with the relief of a downstream inhibitory effect of PKA. ERK activation by the G(q/11)-coupled alpha(1B)AR was unaffected by GsCT. These findings suggest that peptide G protein inhibitors can provide insights into the complex interplay between G protein pools in cellular regulation.     

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.     

Cytoplasmic domain of natriuretic peptide receptor C constitutes Gi activator sequences that inhibit adenylyl cyclase activity

We have recently demonstrated that a 37-amino acid peptide corresponding to the cytoplasmic domain of the natriuretic peptide receptor C (NPR-C) inhibited adenylyl cyclase activity via pertussis toxin (PT)-sensitive G(i) protein. In the present studies, we have used seven different peptide fragments of the cytoplasmic domain of the NPR-C receptor with complete, partial, or no G(i) activator sequence to examine their effects on adenylyl cyclase activity. The peptides used were KKYRITIERRNH (peptide 1), RRNHQEESNIGK (peptide 2), HRELREDSIRSH (peptide 3), RRNHQEESNIGKHRELR (peptide 4), QEESNIGK (peptide X), ITIERRNH (peptide Y), and ITIYKKRRNHRE (peptide Z). Peptides 1, 3, and 4 have complete G(i) activator sequences, whereas peptides 2 and Y have partial G(i) activator sequences with truncated carboxyl or amino terminus, respectively. Peptide X has no structural specificity, whereas peptide Z is the scrambled peptide control for peptide 1. Peptides 1, 3, and 4 inhibited adenylyl cyclase activity in a concentration-dependent manner with apparent K(i) between 0.1 and 1 nm; however, peptide 2 inhibited adenylyl cyclase activity with a higher K(i) of about 10 nm, and peptides X, Y, and Z were unable to inhibit adenylyl cyclase activity. The maximal inhibitions observed were between 30 and 40%. The inhibition of adenylyl cyclase activity by peptides 1-4 was absolutely dependent on the presence of guanine nucleotides and was completely attenuated by PT treatment. In addition, the stimulatory effects of isoproterenol, glucagon, and forskolin on adenylyl cyclase activity were inhibited to different degrees by these peptides. These results suggest that the small peptide fragments of the cytoplasmic domain of the NPR-C receptor containing 12 or 17 amino acids were sufficient to inhibit adenylyl cyclase activity through a PT-sensitive G(i) protein. The peptides having complete structural specificity of G(i) activator sequences at both amino and carboxyl termini were more potent to inhibit adenylyl cyclase activity as compared with the peptides having a truncated carboxyl terminus, whereas the truncation of the amino-terminal motif completely attenuates adenylyl cyclase inhibition.     

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.     

Palmitoylation participates in G protein coupled signal transduction by affecting its oligomerization

Much in vivo and in vitro evidence has shown that the alpha subunits of heterotrimeric GTP-binding proteins (G proteins) exist as oligomers in their base state and disaggregate when being activated. In this article, the influence of palmitoylation modification of Galpha(o) on its oligomerization was explored extensively. Galpha(o) protein was expressed and purified from Escherichia coli strain JM109 cotransformed with pQE60(Galpha(o)) and pBB131(N-myristoyltransferase). Non-denaturing gel electrophoresis analysis revealed that Galpha(o) existed to a small extent as monomers but mostly as oligomers including dimers, trimers, tetramers and pentamers which could disaggregate completely into monomers by GTPgammaS stimulation. Palmitoylated Galpha(o), on the other hand, only present as oligomers that were difficult to disaggregate into monomers. The effect of palmitoylation on oligomerization of Galpha(o) was further investigated by several other biochemical and biophysical methods including gel filtration chromatography, analytical ultracentrifugation and atomic force microscopy analysis. The results consistently demonstrated that palmitoylation facilitated oligomerization of the Galpha(o) protein. Autoradiography indicated that [(14)C]-palmitoylated Galpha(o) would in no case disaggregate into monomers after treatment with GTPgammaS. [(35)S]-GTPgammaS binding activity assay showed that palmitoylated Galpha(o) was saturated at only 7.8 nmol/mg compared to 21.8 nmol/mg for non-palmitoylated Galpha(o). Fluorescent quenching studies using BODIPY FL-GTPgammaS as a probe showed that the conformation of GTP-binding domain of Galpha(o) tended to become more compact after palmitoylation. These results implied that palmitoylation may regulate the GDP/GTP exchange of Galpha(o) by influencing the oligomerization state of Galpha(o) and thereby modulate the on-off switch of the G protein in G protein-coupled signal transduction.     

New insights into the role of conserved, essential residues in the GTP binding/GTP hydrolytic cycle of large G proteins

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.     

Gbetagamma activation site in adenylyl cyclase type II. Adenylyl cyclase type III is inhibited by Gbetagamma

The Gbetagamma complex of heterotrimeric G proteins is the most outstanding example for the divergent regulation of mammalian adenylyl cyclases. The heterodimeric Gbetagamma complex inhibits some isoforms, e.g. ACI, and stimulates the isoforms ACII, -IV, and -VII. Although former studies identified the QEHA region located in the C2 domain of ACII as an important interaction site for Gbetagamma, the determinant of the stimulatory effect of Gbetagamma has not been detected. Here, we identified the C1b domain as the stimulatory region using full-length adenylyl cyclase. The relevant Gbetagamma signal transfer motif in IIC1b was determined as MTRYLESWGAAKPFAHL (amino acids 493-509). Amino acids of this PFAHL motif were absolutely necessary for ACII to be stimulated by Gbetagamma, whereas they were dispensable for Galpha(s) or forskolin stimulation. The PFAHL motif is present in all three adenylyl cyclase isoforms that are activated by Gbetagamma but is absent in other adenylyl cyclase isoforms as well as other known effectors of Gbetagamma. The emerging concept of two contact sites on different molecule halves for effective regulation of adenylyl cyclase is discussed.     

Structural model of a complex between the heterotrimeric G protein, Gsalpha, and tubulin

A number of studies have demonstrated interplay between the cytoskeleton and G protein signaling. Many of these studies have determined a specific interaction between tubulin, the building block of microtubules, and G proteins. The alpha subunits of some heterotrimeric G proteins, including Gsalpha, have been shown to interact strongly with tubulin. Binding of Galpha to tubulin results in increased dynamicity of microtubules due to activation of GTPase of tubulin. Tubulin also activates Gsalpha via a direct transfer of GTP between these molecules. Structural insight into the interaction between tubulin and Gsalpha was required, and was determined, in this report, through biochemical and molecular docking techniques. Solid phase peptide arrays suggested that a portion of the amino terminus, alpha2-beta4 (the region between switch II and switch III) and alpha3-beta5 (just distal to the switch III region) domains of Gsalpha are important for interaction with tubulin. Molecular docking studies revealed the best-fit models based on the biochemical data, showing an interface between the two molecules that includes the adenylyl cyclase/Gbetagamma interaction regions of Gsalpha and the exchangeable nucleotide-binding site of tubulin. These structural models explain the ability of tubulin to facilitate GTP exchange on Galpha and the ability of Galpha to activate tubulin GTPase.     

An antibody directed against residues 100-119 within the alpha-helical domain of Galpha(s) defines a novel contact site for beta-adrenergic receptors.

A polyclonal antiserum that recognizes residues 100-119 within the alpha-helical domain of Galpha(s) (K-20) caused a dissociation of G(s) into its component subunits and activated a cholera toxin-sensitive high affinity GTPase. Consistently, the antibody mimicked the stimulatory effects of the beta-adrenergic agonist, isoproterenol, on adenylyl cyclase, which is mediated by Galpha(s), and its inhibitory action on NADPH-dependent H(2)O(2) generation, a Gbetagamma-mediated response. A peptide corresponding to the target sequence of K-20 not only neutralized the receptor-mimetic effects of the antibody but inhibited the whole spectrum of isoproterenol action as well, including its antagonistic effects on adenylyl cyclase and NADPH-dependent H(2)O(2) generation. By contrast, COOH-terminal anti-Galpha(s) selectively inhibited the stimulatory effect of isoproterenol on cAMP formation without affecting its inhibitory effect on NADPH-dependent H(2)O(2) generation. The data are consistent with the concept that beta-adrenergic receptors interact with multiple sites on Galpha(s) each playing a distinct role, and strongly suggest that antibody K-20 defines a novel contact site for beta-adrenergic receptors that localizes to the alpha-helical domain and is essential for eliciting the complete spectrum of beta-adrenergic responses.     

Isolation and characterization of constitutively active mutants of mammalian adenylyl cyclase

A genetic screen in Saccharomyces cerevisiae identified mutations in mammalian adenylyl cyclase that activate the enzyme in the absence of G(s)alpha. Thirteen of these mutant proteins were characterized biochemically in an assay system that depends on a mixture of the two cytosolic domains (C(1) and C(2)) of mammalian adenylyl cyclases. Three mutations, I1010M, K1014N, and P1015Q located in the beta4-beta5 loop of the C(2) domain of type II adenylyl cyclase, increase enzymatic activity in the absence of activators. The K1014N mutation displays both increased maximal activity and apparent affinity for the C(1) domain of type V adenylyl cyclase in the absence of activators of the enzyme. The increased affinity of the mutant C(2) domain of adenylyl cyclase for the wild type C(1) domain was exploited to isolate a complex containing VC(1), IIC(2), and G(s)alpha-guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) in the absence of forskolin and a complex of VC(1), IIC(2), forskolin, and P-site inhibitor in the absence of G(s)alpha-GTPgammaS. The isolation of these complexes should facilitate solution of crystal structures of low activity states of adenylyl cyclase and thus determination of the mechanism of activation of the enzyme by forskolin and G(s)alpha.     

A bifunctional Galphai/Galphas modulatory peptide that attenuates adenylyl cyclase activity

Signaling via G-protein coupled receptors is initiated by receptor-catalyzed nucleotide exchange on Galpha subunits normally bound to GDP and Gbetagamma. Activated Galpha . GTP then regulates effectors such as adenylyl cyclase. Except for Gbetagamma, no known regulators bind the adenylyl cyclase-stimulatory subunit Galphas in its GDP-bound state. We recently described a peptide, KB-752, that binds and enhances the nucleotide exchange rate of the adenylyl cyclase-inhibitory subunit Galpha(i). Herein, we report that KB-752 binds Galpha(s) . GDP yet slows its rate of nucleotide exchange. KB-752 inhibits GTPgammaS-stimulated adenylyl cyclase activity in cell membranes, reflecting its opposing effects on nucleotide exchange by Galpha(i) and Galpha(s).     

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.