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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.

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 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.

Ns and Ni have been purified without using NaF and Mg as stabilizing agents (Codina, J., Hildebrandt, J.D., Sekura, R.D., Birnbaumer, M., Bryan, J., Manclark, C.R. and Birnbaumer, L. [1984] J. Biol. Chem. 259, in press). Since the submission of that report, several modifications have been introduced to the purification procedure and additional fractions have been processed from which N proteins are obtained. This article describes the updated protocols and presents methodological details not included in the previous publication. The final products are Ns, the stimulatory N, Ni the inhibitory N, both of subunit structure alpha beta gamma, and a Mr = 40,000 protein of beta gamma composition. They are obtained from human erythrocytes.

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 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)

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)

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.

Membrane-bound adenylyl cyclases from ram, dog, and human sperm are unresponsive to fluoride and guanylylimidodiphosphate [GMP-P(NH)P], two agents that stimulate the adenylyl cyclases of somatic cells by an action on the stimulatory guanine nucleotide-binding regulatory (Ns) component of adenylyl cyclase. We have investigated whether this is because the sperm cell catalytic unit is functionally uncoupled from Ns but, nevertheless, capable of interacting with it, or because the sperm cell adenylyl cyclase system is unique and regulated differently from that of somatic cells. Sperm cells were found to be deficient in Ns, as evidenced by the inability of detergent extracts from sperm cell membranes and fractions to reconstitute Ns-mediated regulation of the adenylyl cyclase of cyc- S49 cells. In addition, attempts to label Ns in sperm cell membranes by [32P]ADP ribosylation with cholera toxin revealed that, if present, Ns is less than 1% of that found in human erythrocyte membranes. This, however, was not the only reason for the unresponsiveness of sperm cell adenylyl cyclase, since fluoride stimulation of the sperm cell enzyme could not be induced by reconstituting it with Ns purified from human erythrocytes (hRBC). When intact hRBC membranes were added to sperm cell fractions in the presence of fluoride, the activities that resulted were greater than the sum of the individual activities. This apparent reconstitution of fluoride regulation of sperm cell adenylyl cyclase could be blocked by lima bean trypsin inhibitor and appears to have resulted from proteolytic activation of the hRBC adenylyl cyclase by sperm proteases. Sperm cell membranes also appear to lack a functional inhibitory regulatory protein of the adenylyl cyclase system (Ni), since they did not contain an ADP-ribosylatable substrate for pertussis toxin action. These results suggest that the sperm cell adenylyl cyclase system is unique and different from that of somatic cells. Sperm cells appear to neither contain Ns or Ni nor possess the ability of their adenylyl cyclase system to interact with Ns from an exogenous source.

The final step in a scheme for the purification of the guanine nucleotide- and Mg2+-binding stimulatory regulatory component (Ns) of adenylyl cyclase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from human erythrocyte membranes involves chromatography over hydroxylapatite (HAP) which yields two fractions. The first fraction (HAP I) contains predominantly two peptides that, upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, migrate with Mr values of 39,000 and 35,000. The second fraction (HAP II) contains predominantly Ns formed of two peptides of Mr 42,000 and 35,000. The HAP I, Mr 39,000 peptide is shown to be a substrate for the ADP-ribosylating toxin of Bordetella pertussis (pertussis toxin). Upon sucrose density gradient centrifugation, both the Mr 39,000 and the Mr 35,000 peptides of HAP I migrate at about 4 S. Treatment of HAP I with guanine nucleotide and Mg2+ prior to centrifugation results in a coordinated change in the migration of both peptides to 2 S. It is postulated that HAP I contains an alpha beta heterodimeric protein composed of an alpha subunit of Mr 39,000 and a beta subunit of Mr 35,000. Further, this protein dissociates under the influence of guanine nucleotides and Mg2+ into its individual alpha and beta subunits. Because previous studies have shown that treatment of cells and cell membranes with pertussis toxin results in attenuation of the effects of hormones that inhibit adenylyl cyclase activity, and because this effect correlates with the ADP-ribosylation of a Mr approximately equal to 40,000 peptide, we believe that we have purified a guanine nucleotide- and Mg2+-binding inhibitory regulatory component of adenylyl cyclases--i.e., the Ni.

Adenylyl cyclases are under positive and negative control by guanine nucleotides and hormones. Stimulatory responses are mediated by a guanine nucleotide- and Mg-binding regulatory component (Ns), a protein that has been purified to homogeneity. Inhibitory responses have been hypothesized to be mediated by an analogous regulatory component (Ni) distinct from Ns, but definitive proof for this is lacking and these effects may result from modulation of Ns activity. Recently, Bordetella pertussis toxin has been shown to ADP-ribosylate a peptide that is not part of Ns, and this coincides with attenuation of hormonal inhibition of adenylyl cyclase. We show here that cyc- S49 cells contain a substrate for ADP-ribosylation by pertussis toxin and that the toxin alters GTP dependent inhibition of cyc- adenyl cyclase activity. As cyc- S49 cells do not contain Ns by several criteria, we conclude that Ni is a distinct and separate regulatory component of adenylyl cyclase.