Beta Gamma Dimers Research Articles

On the role of endogenous G-protein beta gamma subunits in N-type Ca2+ current inhibition by neurotransmitters in rat sympathetic neurones.

1. Using whole-cell and perforated-patch recordings, we have examined the part played by endogenous G-protein beta gamma subunits in neurotransmitter-mediated inhibition of N-type Ca2+ channel current (ICa) in dissociated rat superior cervical sympathetic neurones. 2. Expression of the C-terminus domain of beta-adrenergic receptor kinase 1 (beta ARK1), which contains the consensus motif (QXXER) for binding G beta gamma, reduced the fast (pertussis toxin (PTX)-sensitive) and voltage-dependent inhibition of ICa by noradrenaline and somatostatin, but not the slow (PTX-insensitive) and voltage-independent inhibition induced by angiotensin II. beta ARK1 peptide reduced GTP-gamma-S-induced voltage-dependent and PTX-sensitive inhibition of ICa but not GTP-gamma-S-mediated voltage-independent inhibition. 3. Overexpression of G beta 1 gamma 2, which mimicked the voltage-dependent inhibition by reducing ICa density and enhancing basal facilitation, occluded the voltage-dependent noradrenaline- and somatostatin-mediated inhibitions but not the inhibition mediated by angiotensin II. 4. Co-expression of the C-terminus of beta ARK1 with beta 1 and gamma 2 subunits prevented the effects of G beta gamma dimers on basal Ca2+ channel behaviour in a manner consistent with the sequestering of G beta gamma. 5. The expression of the C-terminus of beta ARK1 slowed down reinhibition kinetics of ICa following conditioning depolarizations and induced long-lasting facilitation by cumulatively sequestering beta gamma subunits. 6. Our findings identify endogenous G beta gamma as the mediator of the voltage-dependent, PTX-sensitive inhibition of ICa induced by both noradrenaline and somatostatin but not the voltage-independent. PTX-insensitive inhibition by angiotensin II. They also support the view that voltage-dependent inhibition results from a direct G beta gamma-Ca2+ channel interaction.

Characterization and purification from bovine neutrophils of a soluble guanine-nucleotide-binding protein that mediates isozyme-specific stimulation of phospholipase C beta2.

Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.

The Role of βγ and αγ Complexes in the Assembly of Human Fibrinogen

The role of alphagamma and betagamma dimers as intermediates in the assembly of fibrinogen was examined in cell fusion experiments using stably transfected baby hamster kidney cell lines expressing one or combinations of fibrinogen chains. Fibrinogen was readily formed and secreted into the culture media when cells co-expressing beta and gamma chains and generating betagamma complexes were fused with cells expressing only the alpha chain. Likewise, when cells co-expressing alpha and gamma chains and generating alphagamma complexes were fused with cells expressing only the beta chain, fibrinogen was also formed and secreted. The relative amounts of alphagamma or betagamma intermediates observed during fibrinogen biosynthesis were determined by the levels of the component chains; i.e. when the beta chain was limiting, the alphagamma dimer was the predominant intermediate; likewise, when the alpha chain was limiting, the betagamma complex was the predominant intermediate. The incorporation of preformed alphagamma and betagamma complexes into secreted fibrinogen did not require concurrent protein synthesis, as shown by experiments employing cycloheximide. These data strongly support the role of alphagamma and betagamma complexes as functional intermediates in the assembly of fibrinogen.

Guanine Nucleotide Exchange on Heterotrimeric Gi3 Protein Controls Autophagic Sequestration in HT-29 Cells

Recent results have shown that autophagic sequestration in the human colon cancer cell line HT-29 is controlled by the pertussis toxin-sensitive heterotrimeric Gi3 protein. Here we show that transfection of an antisense oligodeoxynucleotide to the alphai3-subunit markedly inhibits autophagic sequestration, whereas transfection of an antisense oligodeoxynucleotide to the alphai2-subunit does not change the rate of autophagy in HT-29 cells. Autophagic sequestration was arrested in cells transfected with a mutant of the alphai3-subunit (Q204L) that is restricted to the GTP-bound form. In Q204L-expressing cells, 3-methyladenine-sensitive degradation of long lived [14C]valine-labeled proteins was severely impaired and could not be stimulated by nutrient deprivation. Autophagy was also reduced when dissociation of the betagamma dimer from the GTP-bound alphai3-subunit was impaired in cells transfected with the G203A mutant. In contrast, a high rate of pertussis toxin-sensitive autophagy was observed in cells transfected with an alphai3-subunit mutant (S47N) which has an increased guanine nucleotide exchange rate and increased preference for GDP over GTP. Cells that express pertussis toxin-insensitive mutants of either wild-type alphai3-subunit (C351S) or S47N alphai3-subunit (S47N/C351S) exhibit a high rate of autophagy.

Functional Reconstitution in Situ of 5-Hydroxytryptamine2c (5HT2c) Receptors with αq and Inverse Agonism of 5HT2c Receptor Antagonists

Membranes prepared after infection of Sf9 cells with recombinant baculovirus containing the rat 5HT2c receptor DNA, but not after infection with wild-type virus, expressed high affinity binding sites for 125I-lysergic acid diethylamide and [3H]mesulergine. The receptor site density reached an optimum of 50-70 pmol/mg membrane protein at 60 h postinfection. Extraction of peripheral membrane proteins from the postnuclear membrane fraction with 6 M urea depleted GTPgammaS-binding 4-fold without decreasing 5HT2c receptor binding activity. Urea-extracted Sf9 membranes expressing the 5HT2c receptor catalyzed the activation of squid retinal alphaq but not bovine retinal alphat or bovine alphao/alphai. Productive interaction of 5HT2c receptors with squid alphaq was enhanced by the addition of betagamma dimers prepared from either bovine brain or bovine rod outer segment discs. While the addition of serotonin increased 5HT2c receptor-catalyzed GTPgammaS binding to alphaq, the unoccupied receptor was also catalytically active. The 5HT2c receptor antagonists, mesulergine, mianserin, and ketanserin competitively inhibited 5HT activation of the receptor with predicted rank-order affinities; and mianserin and ketanserin markedly inhibited basal 5HT2c receptor activity. Interestingly, this "inverse agonist" efficacy did not correlate with antagonist affinity for the 5HT2c receptor. Baculoviral expression of the 5HT2c receptor and urea extraction of postnuclear Sf9 cell membranes have provided a high density of in situ, uncoupled, G-protein-linked receptor useful for reconstitution with purified G-protein subunits. This has allowed for independent manipulation of receptor and G-protein chemical concentrations and has revealed that a G-protein-linked receptor can possess a significant basal catalytic activity and that antagonist compounds can act as inverse agonists of this basal activity at the level of receptor activation of G-proteins.

Role of the Prenyl Group on the G Protein γ Subunit in Coupling Trimeric G Proteins to A1 Adenosine Receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.

G Protein γ Subunits with Altered Prenylation Sequences Are Properly Modified When Expressed in Sf9 Cells

The gamma subunits of heterotrimeric G proteins undergo post-translational prenylation and carboxylmethylation after formation of the betagamma dimer, modifications that are essential for alpha-betagamma, betagamma-receptor, and betagamma-effector interactions. We have determined the specific prenyl group present on the beta1gamma1, beta1gamma2, and beta1gamma3 dimers purified from baculovirus-infected Sf9 cells by specific binding to G protein alpha subunits immobilized on agarose. These recombinant dimers undergo the same post-translational modifications determined for gamma1 and gamma2 isolated from mammalian tissues. Furthermore, infection of Sf9 cells with a recombinant baculovirus encoding an alteration of the gamma1 CaaX sequence (gamma1-S74L) resulted in geranylgeranylation of the resulting gamma1 subunit, and alteration of the gamma2 CaaX sequence to CAIS (gamma2-L71S) resulted in farnesylation. Both of these altered gamma subunits were able to associate stably with beta1, and the resulting betagamma dimer bound tightly to alpha-agarose and eluted specifically with aluminum fluoride. These results indicate that Sf9 insect cells properly process the CaaX motif in G protein gamma subunits and are a useful model system to study the role of prenylation in the protein-protein interactions in which the betagamma subunits participate.

Chronic Opioid Treatment Induces Adenylyl Cyclase V Superactivation: INVOLVEMENT OF Gβγ

It has been known for some time that chronic treatment of neuronal cells and tissues with opioids, contrary to their acute effect, leads to an increase in cAMP accumulation. This phenomenon, defined as adenylyl cyclase superactivation, has been implicated in opiate addiction, yet the mechanism by which it is induced remains unclear. Here, we show that this phenomenon can be reproduced and studied in COS-7 cells cotransfected with adenylyl cyclase type V and mu-opioid receptor cDNAs. These cells display acute opioid inhibition of adenylyl cyclase activity, whereas prolonged exposure to the mu-agonist morphine or [-Ala2, N-methyl-Phe4, Gly-ol5]enkephalin leads to a time-dependent superactivation of adenylyl cyclase. This superactivated state is reversible, because it is gradually lost following agonist withdrawal. Adenylyl cyclase superactivation can be prevented by pertussis toxin pretreatment, indicating the involvement of Gi/o proteins, or by cotransfection with the carboxyl terminus of beta-adrenergic receptor kinase or with alpha-transducin (scavengers of Gbetagamma dimers), indicating a role for the G protein betagamma dimers in adenylyl cyclase superactivation. However, contrary to several other Gbetagamma-dependent signal transduction mechanisms (e.g. the extracellular signal-regulated kinase 2/MAP kinase pathway), adenylyl cyclase superactivation is not affected by the Ras dominant negative mutant N17-Ras.

Nonselective and G betagamma-insensitive weaver K+ channels.

Homozygous weaver mice are profoundly ataxic because of the loss of granule cell neurons during cerebellar development. This granule cell loss appears to be caused by a genetic defect in the pore region (Gly156-->Ser) of the heterotrimeric guanine nucleotide-binding protein (G protein)-gated inwardly rectifying potassium (K+) channel subunit (GIRK2). A related subunit, GIRK1, associates with GIRK2 to constitute a neuronal G protein-gated inward rectifier K+ channel. The weaver allele of the GIRK2 subunit (wvGIRK2) caused loss of K+ selectivity when expressed either as wvGIRK2 homomultimers or as GIRK1-wvGIRK2 heteromultimers. The mutation also let to loss of sensitivity to G protein betagamma dimers. Expression of wvGIRK2 subunits let to increased cell death, presumably as a result of basal nonselective channel opening.

Inhibition of an inwardly rectifying K+ channel by G-protein α-subunits

Cholinergic muscarinic, serotonergic, opioid and several other G-protein-coupled neurotransmitter receptors activate inwardly rectifying K+ channels of the GIRK family, slowing the heartbeat and decreasing the excitability of neuronal cells. Inhibitory modulation of GIRKs by G-protein-coupled receptors may have important implications in cardiac and brain physiology. Previously G alpha and G beta gamma subunits of heterotrimeric G proteins have both been implicated in channel opening, but recent studies attribute this role primarily to the G beta gamma dimer that activates GIRKs in a membrane-delimited fashion, probably by direct binding to the channel protein. We report here that free GTP gamma S-activated G alpha i 1, but not G alpha i 2 or G alpha i 3, potently inhibits G beta 1 gamma 2-induced GIRK activity in excised membrane patches of Xenopus oocytes expressing GIRK1. High-affinity but partial inhibition is produced by G alpha s-GTP gamma S. G alpha i 1-GTP gamma S also inhibits G beta 1 gamma 2-activated GIRK in atrial myocytes. Antagonistic interactions between G alpha and G beta gamma may be among the mechanisms determining specificity of G protein coupling to GIRKs.

Phosphotransfer reactions as a means of G protein activation.

Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins) serve to transduce information from agonist-bound receptors to effector enzymes or ion channels. Current models of G protein activation-deactivation indicate that the oligomeric GDP-bound form must undergo release of GDP, bind GTP and undergo subunit dissociation, in order to be in active form (GTP bound alpha subunits and free beta gamma dimers) and to regulate effectors. The effect of receptor occupation by an agonist is generally accepted to be promotion of guanine nucleotide exchange thus allowing activation of the G protein. Recent studies indicate that transphosphorylation leading to the formation of GTP from GDP and ATP in the close vicinity, or even at the G protein, catalysed by membrane-associated nucleoside diphosphate kinase, may further activate G proteins. This activation is demonstrated by a decreased affinity of G protein-coupled receptors for agonists and an increased response of G protein coupled effectors. In addition, a phosphorylation of G protein beta subunits and consequent phosphate transfer reaction resulting in G protein activation has also been demonstrated. Finally, endogenously formed GTP was preferentially effective in activating some G proteins compared to exogenous GTP. The aim of this report is to present an overview of the evidence to date for a transphosphorylation as a means of G protein activation (see also refs [1 and 2] for reviews).

The PH domain of Ras-GAP is sufficient for in vitro binding to beta gamma subunits of heterotrimeric G proteins.

1. The noncatalytic domain of Ras-GAP can affect signaling through G protein-coupled receptors by a poorly understood mechanism. 2. In this study, fusion proteins containing elements of the noncatalytic domain of ras-GAP were examined for their ability to bind beta gamma subunits of heterotrimeric G proteins and phosphotyrosine-containing polypeptides. 3. Our results demonstrate that purified beta gamma dimers associated with bacterially expressed GAP proteins and that this association does not require SH2 or SH3 domains but is dependent on the presence of the GAP pleckstrin-homology (PH) domain. In contrast, only the SH2 domains are necessary for binding to tyrosine phosphorylated proteins. 4. These findings raise the possibility that heterotrimeric G proteins might affect functioning of ras-like proteins through beta gamma subunits acting on their regulatory molecules.

Crystal structure of a GA protein βγdimer at 2.1 Å resolution

Many signalling cascades use seven-helical transmembrane receptors coupled to heterotrimeric G proteins (G alpha beta gamma) to convert extracellular signals into intracellular responses. Upon nucleotide exchange catalysed by activated receptors, heterotrimers dissociate into GTP-bound G alpha subunits and G beta gamma dimers, either of which can modulate many downstream effectors. Here we use multiwavelength anomalous diffraction data to solve the crystal structure of the beta gamma dimer of the G protein transducin. The beta-subunit is primarily a seven-bladed beta-propeller that is partially encircled by an extended gamma-subunit. The beta-propeller, which contains seven structurally similar WD repeats, defines the stereochemistry of the WD repeat and the probable architecture of all WD-repeat-containing domains. The structure details interactions between G protein beta- and gamma-subunits and highlights regions implicated in effector modulation for the conserved family of G protein beta gamma dimers.

Intersubunit Surfaces in G Protein αβγ Heterotrimers: ANALYSIS BY CROSS-LINKING AND MUTAGENESIS OF βγ

Heterotrimeric guanine nucleotide binding proteins (G proteins) are made up of alpha, beta, and gamma subunits, the last two forming a very tight complex. Stimulation of cell surface receptors promotes dissociation of alpha from the beta gamma dimer, which, in turn, allows both components to interact with intracellular enzymes or ion channels and modulate their activity. At present, little is known about the conformation of the beta gamma dimer or about the areas of beta gamma that interact with alpha. Direct information on the orientation of protein surfaces can be obtained from the analysis of chemically cross-linked products. Previous work in this laboratory showed that 1,6-bismaleimidohexane, which reacts with cysteine residues, specifically cross-links alpha to beta and beta to gamma (Yi, F., Denker, B. M., and Neer, E. J. (1991) J. Biol. Chem. 266, 3900-3906). To identify the residues in beta and gamma involved in cross-linking to each other or to alpha, we have mutated the cysteines in beta 1, gamma 2, and gamma 3 and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were able to form beta gamma dimers that could interact with the alpha subunit. We found that 1,6-bismaleimidohexane can cross-link beta 1 to gamma 3 but not to gamma 2. The cross-link goes from Cys25 in beta 1 to Cys30 in gamma 3. This cysteine is absent from any of the other known gamma isoforms and therefore confers a distinctive property to gamma 3. The beta subunit in the beta 1 gamma 2 dimer can be cross-linked to an unidentified protein in the rabbit reticulocyte lysate, generating a product slightly larger than cross-linked beta 1 gamma 3. The beta subunit can also be cross-linked to alpha, giving rise to two products on SDS-polyacrylamide gel electrophoresis, both of which were previously shown to be formed by cross-linking beta to Cys215 in alpha o (Thomas, T. C., Schmidt, C. J., and Neer, E. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10295-10299). Mutation of Cys204 in beta 1 abolished one of these two products, whereas mutation of Cys271 abolished the other. Because both alpha-beta cross-linked products are formed in approximately equal amounts, Cys204 and Cys271 in beta are equally accessible from Cys215 in alpha o. Our findings begin to define intersubunit surfaces, and they pose structural constraints upon any model of the beta gamma dimer.

Identification of a three-amino-acid region in G protein gamma 1 as a determinant of selective beta gamma heterodimerization.

Guanine-nucleotide-binding protein beta and gamma subunits belong to large protein families encompassing at least five and ten members, respectively, from mammalian cells. The formation of stable beta gamma heterodimers is a selective process determined by the primary sequences of both the beta and gamma subunit. For example, gamma 2 dimerizes with both beta 1 and beta 2, gamma 1 with beta 1, but not with beta 2. To identify the structural elements of gamma subunits relevant to the selectivity of beta gamma dimerization, we have used the baculovirus-insect cell-expression system to produce chimeric beta and gamma subunits and have studied their dimerization using an assay based on the ability of isoprenylation-resistant gamma subunit mutants to draw beta subunits into the cytosol and including sucrose density gradient analysis of soluble recombinant beta gamma dimers. The results show that replacement of three consecutive residues of gamma 1, Cys36-Cys37-Glu38, by the corresponding residues of gamma 2, Ala33-Ala34-Ala35, suffices to render the mutant gamma 1 subunit capable of forming heterodimers with beta 2. The ability of mutant gamma 1 subunits to dimerize with beta 2 does not correlate with the probability of the mutated region to participate in coiled-coil structures. The tripeptide region identified here as a critical determinant of the selectivity of beta gamma dimer formation is distinct from, but partially overlaps with, the region reported by Lee et al. [Lee, C., Murakami, T. & Simonds, W. F. (1995) J. Biol. Chem. 270, 8779-8784]. The results of this study, therefore, not only extend the region of gamma 1 selecting between beta 1 and beta 2 to the five-residue sequence between Cys36 and Phe40, but also argue against the notion that the hydrophobic terminal residue of this motif represents the key determinant of selective beta gamma interaction.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, but Not N-Methyl-D-aspartate, Activates Mitogen-activated Protein Kinase through G-protein βγ Subunits in Rat Cortical Neurons

alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor channels play important roles in plasticity, neurotransmission, and neurotoxicity in the central nervous system. AMPA, but not N-methyl-D-aspartate (NMDA), receptor signaling in rat cortical neurons was found to involve a G-protein coupled to a protein kinase cascade. While both NMDA and AMPA activated p42 mitogen-activated protein kinase (MAPK) in neurons, only AMPA-induced MAPK was inhibited by pertussis toxin. AMPA, but not NMDA, caused an association of a G-protein beta subunit with a Ras, Raf kinase, and MAP/ERK kinase (MEK)-1 complex. The evidence indicates that AMPA triggers MAPK activation via a novel mechanism in which G-protein beta gamma dimers released from G alpha bind to a Ras protein complex causing the activation of Ras, Raf kinase, MEK-1, and finally MAPK.

Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway.

In the budding yeast Saccharomyces cerevisiae, the process of conjugation of haploid cells of genotype MATa and MAT alpha to form MATa/alpha diploids is triggered by pheromones produced by each mating type. These pheromones stimulate a cellular response by interaction with receptors linked to a heterotrimeric G protein. Although genetic analysis indicates that the pheromone signal is transmitted through the G beta gamma dimer, the initial target(s) of G protein activation remain to be determined. Temperature-sensitive cells with mutations of the CDC24 and CDC42 genes, which are incapable of budding and of generating cell polarity at the restrictive temperature, are also unable to mate. Cdc24 acts as a guanylyl-nucleotide-exchange factor for the Rho-type GTPase Cdc42, which has been shown to be a fundamental component of the molecular machinery controlling morphogenesis in eukaryotic cells. Therefore, the inability of cdc24 and cdc42 mutants to mate has been presumed to be due to a requirement for generation of cell polarity and related morphogenetic events during conjugation. But here we show that Cdc42 has a direct signalling role in the mating-pheromone response between the G protein and the downstream protein kinase cascade.

The G protein beta subunit is essential for multiple responses to chemoattractants in Dictyostelium.

Increasing evidence suggests that the beta gamma-subunit dimers of heterotrimeric G proteins play a pivotal role in transducing extracellular signals. The recent construction of G beta null mutants (g beta-) in Dictyostelium provides a unique opportunity to study the role of beta gamma dimers in signaling processes mediated by chemoattractant receptors. We have shown previously that g beta- cells fail to aggregate; in this study, we report the detailed characterization of these cells. The g beta- cells display normal motility but do not move towards chemattractants. The typical GTP-regulated high affinity chemoattractant-binding sites are lost in g beta- cells and membranes. The g beta- cells do not display chemoattractant-stimulated adenylyl cyclase or guanylyl cyclase activity. These results show that in vivo G beta links chemoattractant receptors to effectors and is therefore essential in many chemoattractant-mediated processes. In addition, we find that G beta is required for GTP gamma S stimulation of adenylyl cyclase activity, suggesting that the beta gamma-dimer activates the enzyme directly. Interestingly, the g beta- cells grow at the same rate as wild-type cells in axenic medium but grow more slowly on bacterial lawns and, therefore, may be defective in phagocytosis.

S-Prenylated Cysteine Analogs Inhibit Receptor-Mediated G Protein Activation in Native Human Granulocyte and Reconstituted Bovine Retinal Rod Outer Segment Membranes

We have previously shown that the S-prenylated cysteine analogue N-acetyl-S-trans,trans-farnesyl-L-cysteine (L-AFC) inhibits basal and formyl peptide receptor-stimulated binding of guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) to and hydrolysis of GTP by membranes of HL-60 granulocytes and have presented evidence suggesting that this inhibition was not caused by reduced protein carboxyl methylation [Scheer, A., & Gierschik, P. (1993) FEBS Lett. 319, 110-114]. We now report a detailed analysis of the structural properties of S-prenylated cysteine analogues required for this inhibition and demonstrate that S-prenylcysteines also suppress basal and receptor-stimulated GTP[S] binding to human peripheral neutrophil and HL-60 granulocyte membranes when stimulated by formyl peptide and complement C5a, respectively. S-Prenylcysteines did not affect pertussis toxin-mediated [32P]ADP-ribosylation of Gi proteins. The inhibitory effect of L-AFC was reversible and was not mimicked by farnesylic acid. L-AFC also interfered with GTP[S] binding to retinal transducin when stimulated by light-activated rhodopsin in a reconstituted system. This inhibitory effect was fully reversed upon increasing the concentration of either the G protein beta gamma dimer or the activated receptor. On the basis of these results, we suggest that S-prenylated cysteine analogues like L-AFC inhibit receptor-mediated G protein activation by specifically and reversibly interfering with the interaction of activated receptors with G proteins, most likely with their beta gamma dimers, rather than by inhibiting alpha.beta gamma heterotrimer formation.

Bovine Brain GO Isoforms Have Distinct γ Subunit Compositions

The gamma subunit composition of the major bovine brain Go and Gi proteins (GOA, GOB, GOC, Gi1, and Gi2) was characterized using antibodies against specific gamma isoforms. Each of the purified G protein heterotrimers contained a heterogeneous population of gamma subunits, and the profiles of the gamma subunits found with Gi1, Gi2, and GOA were similar. In contrast, each GO isoform had a distinct pattern of associated gamma subunits. These differences were surprising given that all three alpha O isoforms are thought to share a common amino-terminal sequence important for the binding of beta gamma dimers and that the alpha OA and alpha OC proteins may come from the same alpha O1 mRNA. The free alpha OA and alpha OC subunits had unique elution behaviors during MonoQ chromatography, compatible with differences in their post-translational processing. These results indicate that both the alpha and gamma subunit compositions of heterotrimers define the structure of an intact G protein. Furthermore, the exact subunit composition of G protein heterotrimers may depend upon regulated expression of different subunit isoforms or upon cellular processing of alpha subunits.