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Gbetagamma binds directly to the third intracellular (i3) loop subdomain of the M(3)-muscarinic receptor (MR). In this report, we identified the Gbetagamma binding motif and G-protein-coupled receptor kinase (GRK2) phosphorylation sites in the M(3)-MR i3 loop via a strategy of deletional and site-directed mutagenesis. The Gbetagamma binding domain was localized to Cys(289)-His(330) within the M(3)-MR-Arg(252)-Gln(490) i3 loop, and the binding properties (affinity, influence of ionic strength) of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain were similar to those observed for the entire i3 loop. Site-directed mutagenesis of the M(3)-MR-Cys(289)-His(330) i3 loop subdomain indicated that Phe(312), Phe(314), and a negatively charged region (Glu(324)-Asp(329)) were required for interaction with Gbetagamma. Generation of the full-length M(3)-MR-Arg(252)-Gln(490) i3 peptides containing the F312A mutation were also deficient in Gbetagamma binding and exhibited a reduced capacity for phosphorylation by GRK2. A similar, parallel strategy resulted in identification of major residues ((331)SSS(333) and (348)SASS(351)) phosphorylated by GRK2, which were just downstream of the Gbetagamma binding motif. Full-length M(3)-MR constructs lacking the 42-amino acid Gbetagamma binding domain (Cys(289)-His(330)) or containing the F312A mutation exhibited ligand recognition properties similar to wild type receptor and also effectively mediated agonist-induced increases in intracellular calcium following receptor expression in Chinese hamster ovary and/or COS 7 cells. However, the M(3)-MRDeltaCys(289)-His(330) and M(3)-MR(F312A) constructs were deficient in agonist-induced sequestration, indicating a key role for the Gbetagamma-M(3)-MR i3 loop interaction in receptor regulation and signal processing.

The distribution and properties in brain of the alpha subunits of the major bovine brain Go isoforms, GoA, GoB and GoC, were characterized. The alpha(o)A and alpha(o)B isoforms arise from alternative splicing of RNAs from a single alpha(o) gene, whereas alpha(o)C is a deamidated form of alpha(o)A. All three Go isoforms purify from brain with different populations of betagamma dimers. This variable subunit composition of Go heterotrimers is likely a consequence of their functional differences. This study examined the biochemical properties of the alpha(o) isoforms to see if these properties explain the variable betagamma composition of their heterotrimers. The brain distribution of alpha(o)B differed substantially from that of alpha(o)A and alpha(o)C, as did its guanine nucleotide binding properties. The unique subunit composition of GoB can be explained by its expression in different brain regions. The alpha(o)A and alpha(o)C showed slight differences in guanine nucleotide binding properties but no preference for particular betagamma dimers when reassociated with a heterogeneous betagamma pool. The alpha(o)C protein occurred in a constant ratio to alpha(o)A throughout the brain, but was a much larger percent of total brain alpha(o) than previously thought, approximately 35%. These results suggest that alpha(o)A is a precursor of alpha(o)C and that the association of G(o)alpha subunits with different betagamma dimers reflects the function of an adaptive, G-protein signaling mechanism in brain.

The phenotype of a Ser to Asn mutation at position 54 of the alpha subunit of G(s)(N54-alpha(s)) was characterized in transient transfection experiments in COS and HEK293 cells. Expression of either wild type or N54-alpha(s) increased basal cAMP levels. In contrast, expression of wild type alpha(s), potentiated agonist-stimulated cAMP levels, while expression of N54-alpha(s)caused a decrease. Thus, the N54-alpha(s) mutant possesses a conditional dominant negative phenotype, suppressing preferentially hormone-stimulated effects.

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.

The structural differences between two major forms of the alpha subunit of the heterotrimeric G protein GO were found to be due to deamidation of either of two Asn residues near the C-terminus of the proteins, in a region involved in receptor recognition. GO is the most abundant heterotrimeric G protein in mammalian brain. Two forms of the protein, GOA and GOB, are known to be generated by alternative splicing of a single GOalpha gene. A third isoform, alphaOC, represents about 1/3 of the alphaO protein in brain and is related to alphaOA, from which it is thought to be generated by protein modification. Mass spectrometry and chemical derivatization of tryptic fragments of the proteins were used to localize the structural difference between alphaOA and alphaOC to a C-terminal peptide. Sequence analysis of a C-terminal chymotryptic fragment both by ion trap mass spectrometry and by Edman degradation identified Asn346 and Asn347 of alphaOA as alternative deamidation sites in alphaOC. These structural differences have immediate implications for G protein function, as they occur in a conformationally sensitive part of the protein involved in receptor recognition and activation. Since Asn347 is a conserved residue present in most G protein alpha subunits outside the alphas family, these observations may have general significance for many G proteins. Deamidation may be a component of a novel process for modifying or adapting cellular responses mediated by G proteins.

The G protein gamma5 subunit is selectively associated with specific G protein alpha subunits [Wilcox, M. D., et al. (1995) J. Biol. Chem. 270, 4189] and is localized preferentially in focal adhesion plaques [Hansen, C. A., et al. (1996) J. Cell Biol. 126, 811]. What determines the differential association of G proteins and their subunits with specific cellular structures or compartments is not clear, but one factor could be variation in the pattern of processing of the proteins. To study gamma5 subunit diversity and modifications, G protein subunits were fractionated on an HPLC phenyl column and analyzed with a gamma5-specific antiserum. The gamma5 eluted from the column as two peaks of immunoreactivity. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry and electrospray ionization tandem mass spectrometry revealed that the first immunoreactive peak corresponded to the predicted gamma5 isoform (N-terminally acetylated after removal of methionine, C-terminally geranylgeranylated and carboxymethylated with removal of the last three amino acids), while the second peak of immunoreactivity contained a gamma5 isoform isoprenylated at the C-terminus but retaining its three terminal amino acids. This alternatively processed protein is the predominant gamma5 subunit isoform associated with Go and Gi proteins purified from bovine brain. These results describe a new C-terminal processing pattern for G protein gamma subunits and establish the principle that G protein gamma subunits can be heterogeneously modified at their C-termini. This is a site on the gamma subunit critical for membrane and protein-protein interactions of G proteins. These results open the possibility that one determinant of the localization of G proteins in cells could be the pattern of processing of their gamma subunit constituents.

We report the direct interaction of Gbetagamma with the third intracellular (i3) loop of the M2- and M3-muscarinic receptors (MR) and the importance of this interaction relative to effective phosphorylation of the receptor subdomain. The i3 loop of the M2- and the M3-MR were expressed in bacteria and purified as glutathione S-transferase fusion proteins for utilization as an affinity matrix and to generate substrate for receptor subdomain phosphorylation. In its inactive heterotrimeric state stabilized by GDP, brain G-protein did not associate with the i3 peptide affinity matrix. However, stimulation of subunit dissociation by GTPgammaS/Mg2+ resulted in the retention of Gbetagamma, but not the Galpha subunit, by the M2- and M3-MR i3 peptide resin. Purified Gbetagamma bound to the M3-MR i3 peptide with an apparent affinity similar to that observed for the Gbetagamma binding domain of the receptor kinase GRK2 and Bruton tyrosine kinase, whereas transducin betagamma was not recognized by the M3-MR i3 peptide. Effective phosphorylation of the M3-MR peptide by GRK2 required both Gbetagamma and lipid as is the case for the intact receptor. Incubation of purified GRK2 with the i3 peptide in the presence of Gbetagamma resulted in the formation of a functional ternary complex in which Gbetagamma served as an adapter protein. Such a complex provides a mechanism for specific spatial translocation of GRK2 within the cell positioning the enzyme on its substrate, the activated receptor. The apparent ability of Gbetagamma to act as a docking protein may also serve to provide an interface for this class of membrane-bound receptors to an expanded array of signaling pathways.

The heterotrimeric G proteins are extensively involved in the regulation of cells by extracellular signals. The receptors that control them are often the targets of drugs. There are many isoforms of each of the three subunits that make up these proteins. Thus far, genes for at least sixteen alpha subunits, five beta subunits, and eleven gamma subunits have been identified. In addition, some of these proteins have splice variants or are differentially modified. Based upon what is already known, there are well over a thousand possible G protein heterotrimer combinations. The role of subunit diversity in heterotrimer formation and its effect on signaling by G proteins are still not well understood. However, many current lines of research are leading toward an understanding of these roles. The functional significance of subunit heterogeneity is related to the mechanisms used by G proteins to transmit and integrate the many signals coming into cells through this system. Described here are the basic mechanisms by which G proteins integrate cellular responses, the possible role of subunit heterogeneity in these mechanisms, and the evidence for and against their physiological significance. Recent studies suggest the likely possibility that subunit heterogeneity plays an important role in signaling by G proteins. This role has the potential to extend substantially the flexibility of G proteins in mediating cellular responses to extracellular signals. However, the details of this are yet to be worked out, and they are the subject of many different avenues of research.

Receptor activation of heterotrimeric G proteins dissociates G alpha from the G betagamma complex, allowing both to regulate effectors. Little is known about the effector-interaction regions of G betagamma. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto Gbeta to identify residues on Gbeta that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (Gbeta 86-105) and 115-135 (Gbeta 115-135) from Gbeta. The Gbeta 86-105 peptide inhibited G betagamma stimulation of AC2 and blocked G betagamma inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the Gbeta 115-135 peptide were similar to those of Gbeta 86-105 but Gbeta 115-135 was less efficacious in blocking G betagamma inhibition of AC1. Substitution of Tyr-124 with Val in the Gbeta 115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of Gbeta as a surface that is involved in transmitting signals to effectors.

Purified bovine brain G-protein was used in a solution phase assay to identify membrane-associated proteins that influenced the activation of heterotrimeric G-proteins. Detergent-solubilized membrane extracts from the neuroblastoma-glioma cell hybrid NG108-15, but not the parent C6B4 glioma cell line, increased [35S]GTPgammaS binding to purified G-protein by approximately 460%. The G-protein activator was heat-sensitive, and the magnitude of its action was related to the amount of extract protein. The biophysical and biochemical properties of the G-protein activator were determined using DEAE ion exchange chromatography, gel filtration, and a lectin affinity matrix. In the presence of added GDP (1 microM), the enriched G-protein activator increased the initial rate of [35S]GTPgammaS binding to brain G-protein by up to 4-fold. In the absence of added GDP, the G-protein activator elicited an initial burst in [35S]GTPgammaS binding to brain G-protein within the first 30 s, after which the rate of nucleotide binding to G-protein was similar in the absence or presence of the G-protein activator. The stimulation of nucleotide binding to brain G-protein by the activator was also observed after resolution of Galpha from Gbetagamma. The G-protein activator was distinct from other proteins (neuromodulin, tubulin, and beta-amyloid precursor protein) that influence nucleotide binding to G-protein, indicating the existence of a novel signal accelerator.

The region encoded by amino acids 956-982 of adenylyl cyclase 2 is important for Gbetagamma stimulation. Interactions of a peptide encoding the 956-982 region of adenylyl cyclase 2 (QEHAQEPERQYMHIGTMVEFAYALVGK (QEHA peptide)) with Gbetagamma subunits were studied. QEHA peptide was covalently attached to beta subunit of free Gbetagamma by the cross-linker N-succinimidyl(4-iodoacetyl)aminobenzoate. Cross-linking was proportional to the amount of QEHA peptide added; other control peptides cross-linked minimally. When Go was used, very little cross-linking was observed with GDP and EDTA, but upon activation by guanosine 5'-3-O-(thio)triphosphate and Mg2+, specific cross-linking of the QEHA peptide to Gbeta was observed. We conclude that beta subunits of G proteins contain effector interaction domains that are occluded by Galpha subunits in the heterotrimer. Molecular modeling studies used to dock the QEHA peptide on to Gbeta indicate that amino acids 75-165 of Gbeta may be involved in effector interactions.

The complete covalent structure of the gamma 2 subunit of bovine brain G proteins was determined by peptide mapping using matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry and by partial sequencing using tandem mass spectrometry. Fragments were identified in proteolytic digests corresponding to all predicted internal sequences of gamma 2, but not for N- or C-terminal sequences. Fragments consistent with modifications of the terminal peptides were observed, however. The presence of these postulated modifications was verified by tandem mass spectrometry. These studies define the structure of the native protein and provide a basis for comparison to expressed proteins.

Among subfamilies of G-protein-coupled receptors, agonists initiate several cell signaling events depending on the receptor subtype (R) and the type of G-protein (G) or effector molecule (E) expressed in a particular cell. Determinants of signaling specificity/efficiency may operate at the R-G interface, where events are influenced by cell architecture or accessory proteins found in the receptor's microenvironment. This issue was addressed by characterizing signal transfer from R to G following stable expression of the alpha 2A/D adrenergic receptor in two different membrane environments (NIH-3T3 fibroblasts and the pheochromocytoma cell line, PC-12). Receptor coupling to endogenous G-proteins in both cell types was eliminated by pertussis toxin pretreatment and R-G signal transfer restored by reconstitution of cell membranes with purified brain G-protein. Thus, the receptor has access to the same population of G-proteins in the two different environments. In this signal restoration assay, agonist-induced activation of G was 3-9-fold greater in PC-12 as compared with NIH-3T3 alpha 2-adrenergic receptor transfectants. The cell-specific differences in signal transfer were observed over a range of receptor densities or G-protein concentration. The augmented signal transfer in PC-12 versus NIH-3T3 transfectants occurred despite a 2-3-fold lower level of receptors existing in the R-G-coupled state (high affinity, guanyl-5'-yl imidodiphosphate-sensitive agonist binding), suggesting the existence of other membrane factors that influence the nucleotide binding behavior of G-protein in the two cell types. Detergent extraction of PC-12 but not NIH-3T3 membranes yielded a heat-sensitive, macromolecular entity that increased 35S-labeled guanosine 5'-O-(thiotriphosphate) binding to brain G-protein in a concentration-dependent manner. These data indicate that the transfer of signal from R to G is regulated by a cell type-specific, membrane-associated protein that enhances the agonist-induced activation of G.

The effects of acute exposure to 25 mM ethanol on high voltage-activated, L-type Ca2+ channels in undifferentiated and nerve growth factor-treated pheochromocytoma (PC-12) cells were examined using conventional, whole-cell, patch-clamp techniques. Acute exposure to 25 mM ethanol inhibited macroscopic L-type Ca2+ currents in undifferentiated PC-12 cells significantly more than in nerve growth factor-treated PC-12 cells. Intracellular infusion with guanosine-5'-O-(2-thio)diphosphate or pretreatment with pertussis toxin reduced ethanol inhibition in undifferentiated cells without altering inhibition in nerve growth factor-treated cells, suggesting the involvement of a G protein in ethanol inhibition of Ca2+ channels in undifferentiated cells. Intracellular infusion with an affinity-purified antibody that recognizes the carboxyl termini of alpha i1 and alpha i2 significantly reduced ethanol inhibition in undifferentiated cells, in contrast to the effects of antibodies that recognize the carboxyl termini of alpha oA and alpha oB. None of these antibodies reduced ethanol inhibition in nerve growth factor-treated cells. These results indicate that Gi1 alpha or Gi2 alpha mediates ethanol inhibition of L-type Ca2+ channel currents in undifferentiated but not in nerve growth factor-treated PC-12 cells.

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.

The diversity of the gamma subunits in bovine brain G protein preparations was investigated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Analysis of these G protein mixtures revealed at least four gamma subunit masses by the following four criteria. 1) The measured masses were in the same mass range as the predicted molecular weights of gamma isoforms. 2) The masses were reproducible between the same or different preparations of G proteins. 3) The masses were independent of the matrix used for MALDI analysis. 4) The masses comigrated with the gamma subunit, as part of the heterotrimer, the beta gamma dimer, or the separated gamma subunit. These measured masses were compared with those calculated from cDNA sequences of known bovine brain gamma isoforms with the addition of plausible post-translational modifications. The mass of each spectral peak was consistent with the calculated mass for only one of four known bovine brain gamma subunit isoforms, but the data suggest modifications of the gamma subunits in addition to those already known or suspected at their carboxyl termini. Besides these four major masses, several additional, less resolved spectral peaks were observed whose measured masses did not correlate with any known gamma subunit or plausible modification. MALDI mass spectrometry promises to be a powerful technique for the analysis of the diversity of the gamma subunit in G proteins and for the characterization of their post-translational modifications.

The properties of a Gs alpha mutant with an Asn substituted for Ser at position 54, designated mutant 54Asn alpha s, were studied after expression in S49 alpha s-deficient (cyc-) cells. Ser-54 in alpha s is comparable to Ser-17 in Ras, which is involved in binding Mg2+ associated with bound nucleotide. 54Asn alpha s did not restore either hormone-induced cyclic AMP production in intact cyc- cells or hormone-induced adenylyl cyclase activation in membranes isolated from these cells. The defect was a failure of ligand-bound receptor to activate 54Asn alpha s, since the mutant protein retained the ability to activate adenylyl cyclase in isolated membranes in the presence of GTP or GTP gamma S. Guanine nucleotide regulation of mutant alpha s suggested that it has increased guanine nucleotide exchange rates and an increased preference for diphosphates over triphosphates. Hormone stimulation magnified the preference of 54Asn alpha s for diphosphates, which could account for its inability to be activated by receptor. The properties of this mutant are discussed in terms of similarities to and differences with the analogous RasH mutant, which has been shown to interfere with endogenous Ras function in cells.

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of alpha- and beta gamma-subunits. The a-subunits of both forms were recognized by an alpha o-specific antiserum, but not by any of the alpha i-specific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant alpha o1. Both the recombinant alpha o1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of alpha i2. H-660 has been shown previously to recognize alpha o and alpha i (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned alpha o1. The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of alpha o1. Hence alpha oA and alpha oB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned alpha o2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTP gamma S) rapidly as well. However Go A binds GTP gamma S about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant alpha o (alpha o1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.

Pertussis toxin ADP-ribosylation and Western blot analysis using G protein-specific antibodies were used to study G protein expression in mouse oocytes, eggs, and early embryos. A pertussis toxin (PT) substrate of about 40 kDa was observed in all stages, but its level was stage dependent. It decreased dramatically between germinal vesicle stage oocytes and unfertilized eggs, remained relatively constant through the early 2-cell stage, and then declined again with each cell division, reaching the lowest level at the 8- to 16-cell stage. Its level, or perhaps that of a different substrate, then increased at the blastocyst stage. Western blot analysis with antisera to the G protein alpha subunit indicated that the decrease between germinal vesicle stage oocytes and unfertilized eggs was less pronounced for the alpha subunit itself than for the PT substrate. Antisera to G protein beta subunit revealed that the difference in the amount of this subunit in germinal vesicle-stage oocytes versus unfertilized eggs was even greater than that of the PT ADP-ribosylation substrate. These results suggest that during oocyte maturation G protein beta gamma levels decline to a greater extent than alpha levels. Additional evidence supporting this hypothesis was obtained by showing that addition of exogenous beta gamma to unfertilized egg preparations increased the amount of PT substrate. These results indicate that G protein subunit expression is differentially regulated during oocyte maturation.

S49 mouse lymphoma cells contain a beta-adrenergic receptor coupled to Gs that stimulates adenylyl cyclase and a somatostatin receptor coupled to Gi that inhibits adenylyl cyclase. Membranes from these cells were used to compare the inhibitory effects of somatostatin and G protein beta gamma complex to determine under what conditions beta gamma is likely to be a mediator of somatostatin action. Somatostatin was equally effective at inhibiting basal adenylyl cyclase activity in the presence of GTP, forskolin-stimulated activity, and hormone-stimulated activity. G protein beta gamma was more effective at inhibiting basal activity than was somatostatin, and these effects were partially additive. In the presence of forskolin, the two inhibitors were equally effective and not additive. In the presence of isoproterenol, beta gamma was much less effective than somatostatin, and the two inhibitors did not have additive or synergistic effects. At very high concentrations beta gamma did inhibit isoproterenol stimulation of adenylyl cyclase, although its effects were not saturating even at 100 micrograms/ml. Under conditions where beta gamma did inhibit hormone stimulation, beta gamma was a mixed inhibitor of isoproterenol stimulation, proportionally decreasing the maximum effect of the hormone and increasing the half-maximally effective concentration. Somatostatin, on the other hand, was a simple noncompetitive inhibitor of isoproterenol stimulation. These results indicate that beta gamma and somatostatin inhibit adenylyl cyclase by different mechanisms, at least in the presence of hormones that stimulate the enzyme. It is proposed that alpha i is the primary mediator of hormone inhibition of adenylyl cyclase when Gs is activated by a hormone, but that beta gamma may have a role as a mediator of inhibition of basal activity.

The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.

Ns and Ni, the regulatory proteins affecting adenylyl cyclase, and transducin, the guanine nucleotide-binding protein from rod outer segments of the eye, are structurally and functionally related proteins. Of these, the alpha subunits are between 39 and 42 kDa in mass, beta subunits are all of 35 kDa in mass, and gamma subunits are much smaller, of approximately 5-8 kDa in mass. We compared, by two-dimensional peptide mapping of iodinated peptides, the beta and gamma subunits of human erythrocyte Ns, human erythrocyte Ni, the beta gamma complex derived from purification of bovine brain N proteins, and frog and bovine eye transducins. We found that gamma subunits in human erythrocyte Ns and Ni and in bovine brain beta gamma complex are indistinguishable by this approach. In contrast, gamma subunits associated with frog and bovine transducin differed markedly between each other and from N protein-associated gamma. beta subunits, on the other hand, yielded essentially indistinguishable peptide maps regardless of whether derived from N proteins or from transducin and regardless also of species of origin: human versus bovine versus frog. These results suggest that the gamma subunit may impart functional heterogeneity of this family of proteins which is evident in the N proteins on the one hand and the transducin proteins on the other.

Pertussis toxin (islet-activating protein) treatment of intact Swiss 3T3 cells causes a time- and dose-dependent loss of availability of a 40 kDa membrane protein for toxin-catalyzed ADP-ribosylation in subsequent incubations with [32P]NAD. In parallel, [3H]thymidine uptake by quiescent cells stimulated with fresh serum, cell doubling time and cell saturation density are all decreased 30-50%. These results cannot be accounted for by the potential effect of the toxin on cell cAMP levels. They suggest that a pertussis toxin substrate, probably Gi, modulates some component of growth regulation in Swiss 3T3 cells.

Modification of bovine brain G proteins by an N-hydroxysuccinimide ester of biotin has been studied. In the presence of GDP, but in the absence of Mg2+, neither guanine nucleotide binding nor GTPase activity of the protein was altered by modification using less than 1.25 mM biotin derivative with 1 mg/ml G protein. Under these conditions the alpha subunit was modified more extensively than the beta and gamma subunits. However, biotinyl-alpha was less readily bound to streptavidin-agarose than was the less modified beta subunit. Biotinyl-beta gamma was isolated from the modified, intact G protein and further characterized to determine if biotinylation alters its functional properties. Isolated biotinyl-beta gamma and unmodified beta gamma were equivalent based upon: 1) inhibition of the S49 cell membrane adenylyl cyclase, 2) changes in hydrodynamic parameters after being recombined with isolated alpha and treated with guanine nucleotides or complexes of fluoride and aluminum, and 3) competition for isolated alpha binding to biotinyl-beta gamma immobilized previously on streptavidin-agarose. Biotinyl-beta gamma prebound to streptavidin-agarose was 70-100% functional, based upon binding of isolated alpha subunits. Estimates of the affinity of alpha binding to biotinyl-beta gamma indicate that bovine brain alpha 41 has a 10-15-fold higher affinity for beta gamma than does alpha 39. Nonhydrolyzable guanine nucleotides and complexes of fluoride and aluminum decreased binding of either alpha 39 or alpha 41 to biotinyl-beta gamma, and these effects were dependent upon the amount of Mg2+ present. GTP decreased binding of alpha 39, but not alpha 41, to biotinyl-beta gamma. These results indicate that GTP can affect G protein subunit interactions and that its effects do not necessarily require an intact membrane environment or the participation of activating receptors or other membrane-associated proteins. They further indicate that biotinylation of beta gamma does not alter its functional properties and that it can be used for studying G protein subunit interactions.

The mechanism by which Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclases, regulate the activity of the catalytic component (C) of adenylyl cyclase was investigated using cyc-S49 cell membranes which contain a functional inhibitory regulatory protein (Ni) but not the active subunit of the stimulatory regulatory protein (Ns). To this end, purified Ns protein was preactivated (Ns) in solution with guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) and Mg2+, and then added to cyc- membranes under conditions where Ni was either unactivated or activated (Ni) by GTP gamma S and Mg2+. Activation of Ni in cyc- membranes resulted in a lowered expression of Ns activity under all conditions tested. Upon dilution of the reactants (Ns and cyc- membranes) the reconstituted activity declined in proportion to the dilution with an approximate t 1/2 of 30-45 min, being unaffected by activation of Ni. Postactivation of Ni after reconstitution of cyc- membranes with Ns resulted in a time-dependent decline in Ns activity to a level that was the same as that obtained when Ns was added to cyc- membranes with preactivated Ni. These data indicated that the effects of Ns on C are of a reversible type. The following indicated that Ns and Ni affect C activity in a noncompetitive manner: (a) the per cent reduction in Ns activity due to activation of Ni was constant and independent of the concentration of Ns, (b) double reciprocal plots of activities reconstituted in control and Ni-containing cyc- membranes versus Ns concentration were linear with an unaltered apparent Km for Ns, and (c) the onset of inhibition of C prereconstituted with Ns was much faster (approximate t 1/2 = 2-5 min) than expected if it were due to occupancy of a common site on C left vacant by Ns.(ABSTRACT TRUNCATED AT 400 WORDS)