Leukemia-associated Rho guanine nucleotide exchange factor promotes G alpha q-coupled activation of RhoA

Leukemia-associated Rho guanine-nucleotide exchange factor (LARG) belongs to the subfamily of Dbl homology RhoGEF proteins (including p115 RhoGEF and PDZ-RhoGEF) that possess amino-terminal regulator of G protein signaling (RGS) boxes also found within GTPase-accelerating proteins (GAPs) for heterotrimeric G protein alpha subunits. p115 RhoGEF stimulates the intrinsic GTP hydrolysis activity of G alpha 12/13 subunits and acts as an effector for G13-coupled receptors by linking receptor activation to RhoA activation. The presence of RGS box and Dbl homology domains within LARG suggests this protein may also function as a GAP toward specific G alpha subunits and couple G alpha activation to RhoA-mediating signaling pathways. Unlike the RGS box of p115 RhoGEF, the RGS box of LARG interacts not only with G alpha 12 and G alpha 13 but also with G alpha q. In cellular coimmunoprecipitation studies, the LARG RGS box formed stable complexes with the transition state mimetic forms of G alpha q, G alpha 12, and G alpha 13. Expression of the LARG RGS box diminished the transforming activity of oncogenic G protein-coupled receptors (Mas, G2A, and m1-muscarinic cholinergic) coupled to G alpha q and G alpha 13. Activated G alpha q, as well as G alpha 12 and G alpha 13, cooperated with LARG and caused synergistic activation of RhoA, suggesting that all three G alpha subunits stimulate LARG-mediated activation of RhoA. Our findings suggest that the RhoA exchange factor LARG, unlike the related p115 RhoGEF and PDZ-RhoGEF proteins, can serve as an effector for Gq-coupled receptors, mediating their functional linkage to RhoA-dependent signaling pathways.     

Membrane interactions of G proteins and other related proteins

Guanine nucleotide-binding proteins, G proteins, propagate incoming messages from receptors to effector proteins. They switch from an inactive to active state by exchanging a GDP molecule for GTP, and they return to the inactive form by hydrolyzing GTP to GDP. Small monomeric G proteins, such as Ras, are involved in controlling cell proliferation, differentiation and apoptosis, and they interact with membranes through isoprenyl moieties, fatty acyl moieties, and electrostatic interactions. This protein-lipid binding facilitates productive encounters of Ras and Raf proteins in defined membrane regions, so that signals can subsequently proceed through MEK and ERK kinases, which constitute the canonical MAP kinase signaling cassette. On the other hand, heterotrimeric G proteins undergo co/post-translational modifications in the alpha (myristic and/or palmitic acid) and the gamma (farnesol or geranylgeraniol) subunits. These modifications not only assist the G protein to localize to the membrane but they also help distribute the heterotrimer (Galphabetagamma) and the subunits generated upon activation (Galpha and Gbetagamma) to appropriate membrane microdomains. These proteins transduce messages from ubiquitous serpentine receptors, which control important functions such as taste, vision, blood pressure, body weight, cell proliferation, mood, etc. Moreover, the exchange of GDP by GTP is triggered by nucleotide exchange factors. Membrane receptors that activate G proteins can be considered as such, but other cytosolic, membranal or amphitropic proteins can accelerate the rate of G protein exchange or even activate this process in the absence of receptor-mediated activation. These and other protein-protein interactions of G proteins with other signaling proteins are regulated by their lipid preferences. Thus, G protein-lipid interactions control the features of messages and cell physiology.     

Genetic and structural analysis of G protein alpha subunit regulatory domains.

Genetic and structural analysis of the alpha chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and beta gamma subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein alpha subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative alpha chains when expressed in different cell types. Organization of the effector activation, receptor and beta gamma contact domains is similar in the primary sequence of the different alpha subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein alpha chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.     

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.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.     

Expression and purification of functional G protein alpha subunits using a baculovirus expression system.

The alpha subunits of the heterotrimeric guanine nucleotide-binding proteins Gi1, Gi2, Gi3, G0, and Gs have been overexpressed in Sf9 cells using a baculovirus expression system. The Gi1 alpha, Gi2 alpha, Gi3 alpha, and G0 alpha have been purified to homogeneity from infected Spodoptera frugiperda (SF9) cells and characterized. Yields of up to 1.8 mg of purified recombinant G alpha have been obtained from 300-ml cultures of infected cells. The recombinant alpha subunits are myristoylated and are ADP-ribosylated by pertussis toxin only in the presence of beta gamma subunits. They bind guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) with low nM dissociation constants and stoichiometries of 0.8 mol/mol or greater. The rGi1 alpha, rGi2 alpha, and rGi3 alpha are capable of interacting with angiotensin II receptors based on their ability to restore high affinity angiotensin II binding in rat liver membranes shifted to a low affinity state with GTP gamma S.     

Promotion of the GTP-liganded state of the Go alpha protein by deletion of the C terminus.

G proteins are active as long as GTP is bound to the alpha subunit. Activation ends when GTP is cleaved to GDP that then stays bound to the active site. Agonist-liganded receptors allow formation of the active state by decreasing the affinity of alpha subunits for GDP allowing exchange of GDP for GTP. Since receptors interact with the C terminus of the alpha subunits, we tested whether deletion of the C terminus could mimic activation by receptors. Three deletions and one point mutation at the C terminus of alpha o were engineered in alpha o cDNA by the polymerase chain reaction, transcribed into RNA, and translated in a rabbit reticulocyte lysate. The ability of in vitro synthesized protein to bind guanine nucleotide was inferred from analysis of native tryptic cleavage patterns, while the ability of the proteins to associate with beta gamma was measured by sucrose density gradient centrifugation. Deletion of 14 amino acids, alpha oD[341], from the C terminus causes a large decrease in GDP affinity, with little or no change in guanosine 5'-3-O-(thio)triphosphate affinity. When GTP is present, alpha oD[341] remains in the activated conformation because exchange of GTP for GDP is rapid. Deletion of 10 amino acids, alpha oD[345], lowers GDP affinity, but less dramatically than in alpha oD[341]. Deletion of 5 amino acids, alpha oD[350], or mutation of Arg-349 to proline alpha oR[349P] has no detectable effects on GDP affinity. Deletion of up to 10 amino acids from the C terminus does not prevent formation of alpha beta gamma heterotrimers. We propose that the C terminus of the alpha subunit is a mobile region that blocks dissociation of GDP. Agonist-liganded receptors may move it aside to allow release of GDP, exchange for GTP, and activation of the alpha subunit.     

G protein alpha subunits activate tubulin GTPase and modulate microtubule polymerization dynamics

G proteins serve many functions involving the transfer of signals from cell surface receptors to intracellular effector molecules. Considerable evidence suggests that there is an interaction between G proteins and the cytoskeleton. In this report, G protein alpha subunits Gi1alpha, Gsalpha, and Goalpha are shown to activate the GTPase activity of tubulin, inhibit microtubule assembly, and accelerate microtubule dynamics. Gialpha inhibited polymerization of tubulin-GTP into microtubules by 80-90% in the absence of exogenous GTP. Addition of exogenous GTP, but not guanylylimidodiphosphate, which is resistant to hydrolysis, overcame the inhibition. Analysis of the dynamics of individual microtubules by video microscopy demonstrated that Gi1alpha increases the catastrophe frequency, the frequency of transition from growth to shortening. Thus, Galpha may play a role in modulating microtubule dynamic instability, providing a mechanism for the modification of the cytoskeleton by extracellular signals.     

Mechanisms of muscarinic receptor action on Go in reconstituted phospholipid vesicles

Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of protein) from bovine brain and the GTP-dependent regulatory protein, Go, were reconstituted with a lipid mixture of phosphatidylcholine and cholesterol. Essentially all of the receptors could interact with Go as evinced by increases in affinity for agonist as large as 800-fold. Both the alpha and beta gamma subunits of Go were required for this effect. Similarly, both subunits were required for the stimulation of guanine nucleotide exchange by agonists. This latter action of the receptor on Go was catalytic and potentiated markedly by prior treatment with dithiothreitol. Initially, agonist stimulation of association of GTP and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to Go was small and variable due to high basal rates. Prior addition of excess GDP inhibited the basal rate of exchange but allowed stimulation by agonists. Under these conditions, oxotremorine stimulated the rates of association of GTP gamma S up to 10-fold. This selective effect was not mimicked by GTP which inhibited both the basal and hormone-dependent rates. Direct examination of the association of GTP and GDP to Go demonstrated that agonist caused either stimulation or marked inhibition, respectively. These results indicate that receptors stimulate guanine nucleotide exchange on G proteins by both increasing the rates of dissociation of nucleotides and altering their relative affinities such that binding of GTP becomes highly favored over GDP. This would ensure the activation of G proteins by receptors in the presence of both nucleotides.     

A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go

A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 +/- 1.2, 24 +/- 5, and 34 +/- 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTP gamma S to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertussis toxin-sensitive G proteins.     

Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli

Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.     

Receptor activation of G proteins

G proteins are a highly conserved family of membrane-associated proteins composed of alpha, beta, and gamma subunits. The alpha subunit, which is unique for each G protein, binds GDP or GTP. Receptors such as those for beta- and alpha-adrenergic catecholamines, muscarinic agonists, and the retinal photoreceptor rhodopsin, catalyze the exchange of GDP for GTP binding to the alpha subunit of a specific G protein. G alpha.GTP regulates appropriate effector enzymes such as adenylyl cyclase or the cyclic GMP phosphodiesterase. The beta gamma-subunit complex of G proteins is required for efficient receptor-catalyzed alpha subunit guanine nucleotide exchange and also functions as an attenuator of alpha subunit activation of effector enzymes. Recent elucidation of both receptor and G protein primary sequence has allowed structural predictions and new experimental approaches to study the mechanism of receptor-catalyzed G protein regulation of specific effector systems and the control of cell function including metabolism, secretion, and growth.     

A G protein-gated K channel is activated via beta 2-adrenergic receptors and G beta gamma subunits in Xenopus oocytes

In many tissues, inwardly rectifying K channels are coupled to seven- helix receptors via the Gi/Go family of heterotrimeric G proteins. This activation proceeds at least partially via G beta gamma subunits. These experiments test the hypothesis that G beta gamma subunits activate the channel even if released from other classes of heterotrimeric G proteins. The G protein-gated K channel from rat atrium, KGA/GIRK1, was expressed in Xenopus oocytes with various receptors and G proteins. The beta 2-adrenergic receptor (beta 2AR), a Gs-linked receptor, activated large KGA currents when the alpha subunit, G alpha s, was also overexpressed. Although G alpha s augmented the coupling between beta 2AR and KGA, G alpha s also inhibited the basal, agonist-independent activity of KGA. KGA currents stimulated via beta 2AR activated, deactivated, and desensitized more slowly than currents stimulated via Gi/Go-linked receptors. There was partial occlusion between currents stimulated via beta 2AR and the m2 muscarinic receptor (a Gi/Go-linked receptor), indicating some convergence in the mechanism of activation by these two receptors. Although stimulation of beta 2AR also activates adenylyl cyclase and protein kinase A, activation of KGA via beta 2AR is not mediated by this second messenger pathway, because direct elevation of intracellular cAMP levels had no effect on KGA currents. Experiments with other coexpressed G protein alpha and beta gamma subunits showed that (a) a constitutively active G alpha s mutant did not suppress basal KGA currents and was only partially as effective as wild type G alpha s in coupling beta 2AR to KGA, and (b) beta gamma subunits increased basal KGA currents. These results reinforce present concepts that beta gamma subunits activate KGA, and also suggest that beta gamma subunits may provide a link between KGA and receptors not previously known to couple to inward rectifiers.     

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.     

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.     

Dominant-negative inhibition of pheromone receptor signaling by a single point mutation in the G protein alpha subunit

In yeast, two different constitutive mutants of the G protein alpha subunit have been reported. Gpa1(Q323L) cannot hydrolyze GTP and permanently activates the pheromone response pathway. Gpa1(N388D) was also proposed to lack GTPase activity, yet it has an inhibitory effect on pheromone responsiveness. We have characterized this inhibitory mutant (designated Galpha(ND)) and found that it binds GTP, interacts with G protein betagamma subunits, and exhibits full GTPase activity in vitro. Although pheromone leads to dissociation of the receptor from wild-type G protein, the same treatment promotes stable association of the receptor with Galpha(ND). We conclude that agonist binding to the receptor promotes the formation of a nondissociable complex with Galpha(ND), and in this manner prevents activation of the endogenous wild-type G protein. Dominant-negative mutants may be useful in matching specific receptors and their cognate G proteins and in determining mechanisms of G protein signaling specificity.     

Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha

We have reduced the GTPase activity of the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase, by introduction of point mutations analogous to those described in p21ras. Mutants G49V and Q227L differ from the wild type protein in the substitution of Val for Gly49 and Leu for Gln227, respectively (analogous to positions 12 and 61 in p21ras). Wild type and mutant proteins were synthesized in Escherichia coli, purified, and characterized. The rate constants for dissociation of GDP from G49V recombinant Gs alpha (rGs alpha) (0.47/min) and Q227L rGs alpha (0.23/min) differ by no more than 2-fold from that observed for the wild type protein (0.5/min). In marked contrast, the rate constants for hydrolysis of GTP by G49V rGs alpha (0.78/min) and Q227L rGs alpha (0.03-0.06/min) are 4-fold and roughly 100-fold slower than that for wild type rGs alpha (3.5/min). These reductions in the rate of hydrolysis of GTP result in significant fractional occupancy of these proteins by GTP in the presence of the nucleotide, 0.37 for G49V rGs alpha and 0.78 for Q227L rGs alpha, compared to 0.05 for wild type rGs alpha. When reconstituted with cyc- (Gs alpha-deficient) S49 cell membranes or purified adenylyl cyclase, both mutant proteins stimulate adenylyl cyclase activity in the presence of GTP to a much greater extent than does wild type Gs alpha; their maximal ability to activate the enzyme is largely unaltered. The fractional ability of a given Gs alpha polypeptide to active adenylyl cyclase in the presence of GTP correlates well with the fractinal occupancy of the protein by the nucleotide. The mutant subunits appear to interact normally with G protein beta gamma subunits, and their ability to activate adenylyl cyclase is enhanced by interaction with beta-adrenergic receptors. These results indicate that the structural analogy that has been inferred between the guanine nucleotide-binding domains of G proteins and the p21ras family is at least generally correct. They also provide confirmation of the kinetic model of G protein function and document mutations that permit the expression in vivo of constitutively activated G protein alpha subunits.     

Activation of extracellular signal-regulated kinase (ERK) and Akt by human serotonin 5-HT(1B) receptors in transfected BE(2)-C neuroblastoma cells is inhibited by RGS4

Regulator of G protein signaling (RGS) proteins are GTPase-activating proteins for heterotrimeric G proteins. One of the best-studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all G(i) and G(q) alpha subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)-C human neuroblastoma cells expressing human 5-HT(1B) receptors were used to demonstrate that RGS4 can inhibit the coupling of 5-HT(1B) receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal-regulated kinase. This activation was attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. As RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways.     

Interaction between cGMP-phosphodiesterase and transducin alpha-subunit in retinal rods. A cross-linking study.

Cross-linking of the different subunits of the retinal cGMP-phosphodiesterase (PDE) with its activator G alpha GTP gamma S (alpha subunit of the retinal G-protein transducin with GTP gamma S (guanosine 5'-O-(3-thiotriphosphate) bound) has been investigated using purified proteins, with a N-hydroxysuccinimide homobifunctional cross-linker, bis(sulfosuccinimidyl)suberate (BS3) and its cleavable analog 3,3'-dithiobis(sulfosuccinimidylpropionate) (DTSSP). Interaction of purified G-protein and PDE is achieved in the presence of lecithin vesicles, at protein concentrations sufficient for full PDE activation. Protein subunits linked with DTSSP are separated by cleavage of the disulfide bridge and identified by electrophoresis. Complexes of PDE alpha (PDE beta) with 1 and 2 molecules of activator G alpha GTP gamma S are observed, providing direct evidence for an interaction or at least a close proximity between 2 molecules of activator G alpha and each of the catalytic PDE subunits in the activated state of PDE. The results also reveal symmetrical roles of PDE alpha and PDE beta, with the existence of one site for PDE gamma and one site for G alpha on each catalytic subunit.     

G protein activation is prerequisite for functional coupling between Galpha/Gbetagamma and tubulin/microtubules

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. Interestingly, recent results suggest that G proteins also interact with microtubules and participate in cell division and differentiation. It has been shown earlier that both alpha and betagamma subunits of G proteins modulate microtubule assembly in vitro. Since G protein activation and subsequent dissociation of alpha and betagamma subunits are necessary for G proteins to participate in signaling processes, here we asked if similar activation is required for modulation of microtubule assembly by G proteins. We reconstituted Galphabetagamma heterotrimer from myristoylated-Galpha and prenylated-Gbetagamma, and found that the heterotrimer blocks Gi1alpha activation of tubulin GTPase and inhibits the ability of Gbeta1gamma2 to promote in vitro microtubule assembly. Results suggest that G protein activation is required for functional coupling between Galpha/Gbetagamma and tubulin/microtubules, and supports the notion that regulation of microtubules is an integral component of G protein mediated signaling.