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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.

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 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 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.

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.

cyc- S49 cell membranes contain an adenylyl cyclase activity which is stimulated by forskolin and inhibited by guanine nucleotides and NaF. These inhibitory effects are mediated by an inhibitory guanine nucleotide-binding regulatory component (Ni) affecting the adenylyl cyclase catalytic unit (Hildebrandt, J. D., Sekura, R. D., Codina, J., Iyengar, R., Manclark, C. R., and Birnbaumer, L. (1983) Nature (Lond.) 302, 706-709). Since cyc- S49 cells do not contain a stimulatory guanine nucleotide-binding regulatory component (Ns), these membranes were used to study the requirements and kinetics of activation of Ni in the absence of Ns. Activation of Ni by guanyl-5'-yl imidodiphosphate was time-dependent (i.e. hysteretic) and pseudo-irreversible. Although GTP and guanosine 5'-(beta-thio)diphosphate could prevent the inhibition caused by guanyl-5'-yl imidodiphosphate if added simultaneously with it, they could not reverse the inhibited state induced by previous exposure to guanyl-5'-yl imidodiphosphate. Activation of Ni had an absolute requirement for Mg2+. Unlike the activation of Ns, however, which requires millimolar concentrations of Mg2+ in the absence of hormonal stimulation, activation of Ni requires only micromolar concentrations of the divalent cation. These results support the contention that hormones which activate Ni or Ns do so by altering different parameters of a similar activation mechanism.

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)

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.

The subunit composition of the Ns and Ni, the human erythrocyte stimulatory and inhibitory regulatory proteins of adenylyl cyclase, respectively, were analyzed by a sodium dodecyl sulfate-containing discontinuous urea and polyacrylamide gradient gel electrophoresis system designed for the study of low molecular weight polypeptides. This system disclosed that these proteins, in addition to their known alpha and beta subunits, contain an additional small peptide of apparent molecular weight of 5,000 (5K). This “5K peptide” is also present in preparations of another protein which we termed “40K protein” on the basis of its hydrodynamic behavior and whose primary protein constituent is the Mr 35,000 beta subunit of the above regulatory proteins. Analyzing Ni, the 5K peptide was functionally related to the protein by showing that its apparent Stokes radius changes from 5.9 to 5.1 nm after treatment with guanyl-5'-yl imidodiphosphate and magnesium in parallel with the alpha and beta subunits. These data are interpreted as evidence for the existence of a third subunit associated with the regulatory proteins of adenylyl cyclase. We call this subunit gamma and propose a minimum subunit structure for these proteins of the alpha beta gamma type.

Treatment of MDCK cells with glucagon results in decreases in glucagon, NaF and prostaglandin E1-stimulated adenylyl cyclase activities, indicating the occurrence of a heterologous desensitization process. The extent of desensitization was time and glucagon concentration dependent. Maximal desensitization (30-50% decrease in stimulation by various effectors) was obtained by 4 h at 100 nM glucagon. Glucagon also induced homologous desensitization since after treatment, the Kact of glucagon was specifically increased. Treatment of cells with 10 microM 8-bromoadenosine 3':5'-monophosphate or 10 microM forskolin resulted in decreased hormonal (glucagon and prostaglandin E1) stimulation without any decrease in the stimulation by nonhormonal effectors (NaF, forskolin, and guanyl-5'-yl imidodiphosphate). The stimulatory regulator (Ns) of the adenylyl cyclase system was analyzed after desensitization with glucagon and no measurable changes in the apparent levels of the alpha s subunits of Ns or the activity of Ns as assessed by reconstitution of the cyc- S49 cell membrane adenylyl cyclase were detected. Levels of the alpha i subunit of the inhibitory regulator (Ni) were monitored by labeling with [32P]NAD and pertussis toxin. Membranes of glucagon-treated cells showed a 2-fold increase in the amount of alpha i labeled. Addition of pure Ns to glucagon-treated MDCK cell membranes restored full stimulation by NaF but did not restore stimulation by prostaglandin E1 or glucagon. It is concluded that glucagon induces heterologous and homologous desensitization of the MDCK cell adenylyl cyclase. The locus of the heterologous desensitization is at the level of the regulatory components. Decreased stimulation is thought to occur due to either an increase in the levels of Ni or due to altered interactions between the subunits of Ni.

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 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.

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 effect of GTP analogs and Mg on the structure of Ns and Ni, the stimulatory and inhibitory regulatory components of adenylyl cyclase, were studied in a comparative manner. Both N proteins, which are alpha beta gamma heterotrimers that differ in their alpha subunits, when exposed to GTP analogs underwent a Mg-dependent conformational change that was not dependent on subunit dissociation. This was seen both as change in sedimentation behavior at 4 degrees C from 4 S to about 3 S and by a property of the new conformation to retain guanine nucleotide tightly bound to it. Warming to 32 degrees C promoted subunit dissociation, each protein giving a mixture of 2 S alpha G and 2 S beta gamma complexes. For both Ns and Ni, these reactions were reversible: 2 S complexes of Ns and Ni associated to 3 S forms on cooling to 4 degrees C, provided the Mg concentration was at or below 10 mM and detergent concentration was below 1%, and the 3 S complexes of these proteins reverted to 4 S forms and released the nucleotide in the cold on chelation of free Mg with EDTA. Reconstitution assays with Ns-deficient membranes from cyc- S49 lymphoma cells revealed that the 3 S form of Ns is a "pre-active" form of the protein. The scheme below summarizes these findings, where G represents a guanine nucleotide. (Formula: see text) Ns and Ni differ in that more Mg is necessary to promote the 4 S to 3 S conversion of Ns than of Ni, and in that both the 2 S to 3 S to 4 S conversions proceeded more readily with Ni than with of Ns. Mg could not be shown to promote subunit dissociation. The above scheme is suggested as a plausible description of the reaction sequence leading from an unactivated to an activated N protein.

Methods were developed to adequately extract, separate and, without the use of NaF as stabilizing agent, purify to better than 90% purity human erythrocyte Ns and Ni, the stimulatory and inhibitory guanine nucleotide- and Mg-binding regulatory components of adenylyl cyclases, as well as a protein containing Mr = 35,000 subunits. On the basis of a functional assay for Ns, it was purified about 5,000-fold from starting washed erythrocyte membranes with a yield of about 10%. A typical purification yields from 60 units of outdated human blood, between 500 and 1,000 micrograms of pure Ns, and a similar amount of Ni. Pure Ns and Ni contain each at least one alpha and one beta subunit (Northup, J.K., Sternweis, P.C., Smigel, M.D., Schleifer, L.S., Ross, E.M., and Gilman, A.G. (1980) Proc. Natl. Acad. Sci. U.S.A. 74, 6516-6520; Codina, J., Hildebrandt, J.D., Iyengar, R., Birnbaumer, L., Sekura, R.D., and Manclark, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 77, 4276-4280). Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate at varying acrylamide concentrations yielded Mr values of 42,000 and 40,000 for the alpha subunits of Ns and Ni, and of 35,000 for the beta subunits of Ns and Ni. Two-dimensional thin layer analysis of tryptic peptides obtained from digesting 125I-labeled subunits of Ns and Ni confirmed the finding of Manning, D., and Gilman, A.G. (1983) J. Biol. Chem. 258, 7059-7063) that while their alpha subunits are clearly different, their beta subunits are the same. Hydrodynamic analysis of the molecular weights of the nondenatured proteins showed behavior consistent with Mr = 95,500 for Ns, the same for Ni, and Mr = 40,000 for the protein containing the Mr = 35,000 beta subunit. Sedimentation coefficients and Stokes radii of the purified Ns were indistinguishable from those of Ns activity present in initial cholate extracts from human erythrocyte membranes. Further, the overall kinetics with which Ns activity in cholate extracts and Ns activity in the purified protein reconstituted the Ns-deficient adenylyl cyclase system of cyc- S49 cells was also indistinguishable. We conclude that we have purified the native unactivated form of Ns, and by serendipity the Ni, as well as a protein containing the 35 kDa beta subunit of Ns and Ni.(ABSTRACT TRUNCATED AT 400 WORDS)