Changes in the phosphorylation state of the inhibitory guanine-nucleotide-binding protein Gi-2 in hepatocytes from lean (Fa/Fa) and obese (fa/fa) Zucker rats

Treatment of intact, 32Pi-labelled hepatocytes from lean Zucker rats with a range of agents including 12-O-tetradecanoyl-phorbol 13-acetate (TPA), vasopressin, and angiotensin II elicited substantial increases in the phosphorylation of the alpha-subunit of the inhibitory G protein of adenylate cyclase (alpha Gi-2). These agonist-induced phosphorylations of alpha Gi-2 were associated with loss of Gi function as assessed by the ability of low concentrations of guanylyl 5'-[beta,gamma imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity. Hepatocytes from obese Zucker rats displayed a resistance to both agonist-induced phosphorylation of alpha Gi-2 and to p[NH]ppG-mediated inhibition of adenylate cyclase. The basal level of alpha Gi-2 phosphorylation in hepatocytes from obese Zucker rats was considerably greater at 1.06 +/- 0.09 mol phosphate/mol alpha Gi-2 than in hepatocytes from lean animals which gave 0.54 +/- 0.09 mol phosphate/mol alpha Gi-2. Incubation with TPA (10 ng/ml, 15 min) approximately doubled the level of phosphorylation of alpha Gi-2 in the hepatocytes from lean animals but had little effect on the phosphorylation of alpha Gi-2 in hepatocytes from obese animals. Incubation of hepatocytes from lean animals with ligands which lead to the phosphorylation of alpha Gi-2 abolished the ability of low concentrations of p[NH]ppG to inhibit adenylate cyclase expressed in isolated membranes. Treatment of hepatocyte plasma membranes from lean but not obese Zucker rats with pure protein kinase C led to the phosphorylation of alpha Gi-2. The resistance to protein-kinase-C-mediated phosphorylation in hepatocyte membranes from obese animals could be overcome by treatment of the membranes with alkaline phosphatase. These results indicate that the defect in guanine-nucleotide-mediated 'Gi function' seen in obese Zucker rats may be due to an inactivating phosphorylation of alpha Gi-2.     

Interactions in platelets between G proteins and the agonists that stimulate phospholipase C and inhibit adenylyl cyclase

Platelet responses to agonists are believed to be mediated by at least two pertussis toxin-sensitive guanine nucleotide-binding (G) proteins: Gi which inhibits adenylyl cyclase and Gp, which stimulates phospholipase C. The present studies compare the properties of Gi and Gp and examine their interactions with the receptors for various platelet agonists. In permeabilized platelets and platelet membranes, pertussis toxin [32P]ADP-ribosylated a protein(s) (alpha 41) which migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis fractionally below rabbit and bovine alpha i (Mr = 41,000). Prior exposure of the platelets to an agonist inhibited the [32P]ADP-ribosylation of alpha 41 to an extent which correlated with the pattern of responses to that agonist. Thrombin, which elicited responses that were mediated by both Gi and Gp, decreased radiolabeling by greater than 90%. Epinephrine, which was functionally coupled only to Gi, decreased radiolabeling by 50%, as did vasopressin and platelet-activating factor (PAF), which were coupled only to Gp. U46619, a thromboxane analog which neither inhibited cAMP formation nor caused pertussis toxin-sensitive phosphoinositide hydrolysis, had no effect on 32P-ADP-ribosylation. These results suggest that either G alpha 41 regulates more than one enzyme or that alpha subunits from more than one G protein comigrate within alpha 41. Two-dimensional electrophoresis was used to test the latter possibility. Upon isoelectric focusing, alpha 41 resolved into two distinct subspecies. However, these appear to be minor variants rather than functionally distinct alpha subunits since: 1) both proteins produced the same proteolytic fragments after digestion with chymotrypsin or Staphylococcus aureus V8 protease and 2) preincubation of the platelets with agonists, including those which appear to interact in intact platelets solely with Gp (PAF and vasopressin) or solely with Gi (epinephrine), inhibited the [32P]ADP-ribosylation of both proteins to the same extent. The pattern of functional responses produced by some of the agonists was found to depend upon the conditions used for the assay. Although unable to inhibit cAMP formation in intact platelets, both PAF and vasopressin caused pertussis toxin-sensitive inhibition of adenylyl cyclase in isolated membranes. Collectively, these observations suggest that 1) in platelets a single pertussis toxin-sensitive, alpha 41-containing G protein may be involved in the regulation of both adenylyl cyclase and phospholipase C and 2) additional constraints which are altered during membrane isolation may help to determine which enzyme is coupled to which agonist.     

Multiple defects occur in the guanine nucleotide regulatory protein system in liver plasma membranes of obese (fa/fa) but not lean (Fa/Fa) Zucker rats: loss of functional Gi and abnormal Gs function

Hepatocyte membranes from both lean and obese Zucker rats exhibited adenylate cyclase activity that could be stimulated by glucagon, forskolin, NaF and elevated concentrations of p[NH]ppG. In membranes from lean animals, functional Gi was detected by the ability of low concentrations of p[NH]ppG to inhibit forskolin-activated adenylate cyclase. This activity was abolished by treatment of hepatocytes with either pertussis toxin or the phorbol ester TPA, prior to making membranes for assay of adenylate cyclase activity. In hepatocyte membranes from obese animals no functional Gi activity was detected. Quantitative immunoblotting, using an antibody able to detect the alpha subunit of Gi, showed that hepatocyte plasma membranes from both lean and obese Zucker rats had similar amounts of Gi-alpha subunit. This was 6.2 pmol/mg plasma membrane for lean and 6.5 pmol/mg plasma membrane for obese animals. Using thiol pre-activated pertussis toxin and [32P]-NAD+, similar degrees of labelling of the 40 kDa alpha subunit of Gi were found using plasma membranes of both lean and obese Zucker rats. We suggest that liver plasma membranes from obese Zucker rats express an inactive Gi alpha subunit. Thus lesions in liver Gi functioning are seen in insulin-resistant obese rats and in alloxan- and streptozotocin-induced diabetic rats which also show resistance as regards the acute actions of insulin. Liver plasma membranes of obese animals also showed an impairment in the coupling of glucagon receptors to Gs-controlled adenylate cyclase, with the Kd values for activation by glucagon being 17.3 and 126 nM for lean and obese animals respectively. Membranes from obese animals also showed a reduced ability for high concentration of p[NH]ppG to activate adenylate cyclase. The use of [32P]-NAD+ and thiol-preactivated cholera toxin to label the 43 kDa and 52 kDa forms of the alpha-subunit of Gs showed that a reduced labelling occurred using liver plasma membranes from obese animals. It is suggested that abnormalities in the levels of expression of primarily the 52 kDa form of alpha-Gs may give rise to the abnormal coupling between glucagon receptors and adenylate cyclase in liver membranes from obese (fa/fa) Zucker rats.     

Chronic Electroconvulsive Treatment Augments Coupling of the GTP-Binding Protein Gs to the Catalytic Moiety of Adenylyl Cyclase in a Manner Similar to That Seen with Chronic Antidepressant Drugs

A significant increase of guanylylimidodiphosphate (GppNHp)-, fluoride-, and forskolin-stimulated adenylyl cyclase was observed in synaptic membrane preparations from rat cerebral cortex subsequent to chronic electroconvulsive shock (ECS) treatment. This effect required at least five treatments over a course of 10 days. The inhibition of adenylyl cyclase induced by GppNHp was not affected by these treatments. The dissociation constant (KD) and maximal binding for the photoaffinity GTP analog, [32P]P3-(4-azidoanilido)-P1-5'-GTP [( 32P]AAGTP), to each of the synaptic membrane G proteins also were unchanged after ECS treatment. Nonetheless, the transfer of [32P]AAGTP from Gi to Gs, which we suggest is indicative of the coupling between Gs and the adenylyl cyclase catalytic moiety, was accelerated by chronic ECS treatment but not by acute or sham treatment. Furthermore, chemical uncoupling of Gs from adenylyl cyclase rendered membranes from treated animals indistinguishable from controls. Finally, in all cases tested, membranes prepared from animals subjected to chronic treatment with amitriptyline or iprindole showed similar changes in the Gs-mediated activation of adenylyl cyclase. Acute treatments produced effects similar to controls, and liver and kidney membranes from animals receiving chronic treatment showed no changes in adenylyl cyclase despite the marked changes seen in brain. These results suggest that chronic administration of ECS enhances coupling between Gs and adenylyl cyclase enzyme and modifies interactions between Gs and Gi.     

Pertussis toxin-mediated ADP-ribosylation of rabbit luteal Gi uncouples enkephalin inhibition of adenylyl cyclase

1. Some of the actions of pertussis toxin on the rabbit luteal adenylyl cyclase system were analyzed. 2. Incubation of luteal membranes with pertussis toxin and [32P]NAD resulted in the [32P]ADP-ribosylation of a 40,000 Da protein that is distinct from the proteins ADP-ribosylated by cholera toxin. 3. Pertussis toxin specific [32P]ADP-ribosylation was time-dependent and dependent upon the concentration of pertussis toxin present during the incubation. 4. Pertussis toxin mediated [32P]ADP-ribosylation was enhanced by ATP, ADP, adenylyl imidodiphosphate, GTP, guanosine-5'-O-(2-thiodiphosphate), guanosine-5'-O-(3-thiotriphosphate), and NaF but not AMP or guanylyl imidodiphosphate [GMP-P(NH)P]. 5. Treatment of luteal membranes with NAD and pertussis toxin prevents GTP and enkephalin but not GMP-P(NH)P mediated inhibition of forskolin stimulated adenylyl cyclase, demonstrating the existence of a functional Gi in the rabbit corpus luteum.     

The alpha subunit of the stimulatory guanine-nucleotide-binding regulatory protein forms high-molecular-mass aggregates, concomitant with iloprost-induced desensitization of human platelet adenylyl cyclase.

Prolonged treatment of human platelets with the prostacyclin analog iloprost led to desensitization of the response to various prostaglandin derivatives. However, basal adenylyl cyclase activity and stimulation by agents acting directly via Gs, the stimulatory guanine-nucleotide-binding regulatory protein of adenylyl cyclase, were likewise decreased. Reconstitution of desensitized membranes with purified Gs from turkey erythrocytes indicated no alteration in the catalyst itself. However, the function of Gs (in cholate extracts) appeared to be severely impaired when reconstituted with adenylyl cyclase catalyst. Modification of Gs was also indicated by its altered sedimentation in sucrose density gradients. From Western blots, the alpha subunit of Gs, alpha s, from control platelets sedimented as a 5.6S species, while that from desensitized cells appeared at higher S values (in a polydisperse distribution). Activation by guanosine 5'-[gamma-thio]triphosphate of Gs from control platelets shifted alpha s to 3.5-3.7S, while activation of Gs from desensitized platelets induced such shift only for a minor portion of alpha s. This small fraction alone appeared to be susceptible to ADP-ribosylation by cholera toxin/[32P]NAD. Furthermore, an antibody directed against the C-terminal hexadecapeptide of alpha s precipitated much less alpha s from cholate extracts derived from desensitized platelets. Modification of alpha s during desensitization was also suggested from cross-linking experiments using the homobifunctional agent bismaleimidohexane: alpha s from desensitized platelets formed a single product of 80 kDa, while that from untreated platelets yielded a doublet (100 kDa and 110 kDa).     

Two distinct Gi-proteins mediate formyl peptide receptor signal transduction in human leukemia (HL-60) cells

In membranes of myeloid differentiated HL-60 cells, the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine stimulates phospholipase C via a pertussis toxin-sensitive G-protein but does not inhibit adenylyl cyclase. In these membranes, the chemotactic peptide markedly stimulates the cholera toxin-dependent [32P]ADP-ribosylation of two proteins with approximate molecular masses of 40 and 41 kDa, respectively. The radiolabeled proteins comigrate on sodium dodecyl sulfate-polyacrylamide gels with the two pertussis toxin substrates present in HL-60 membranes, alpha i2 and alpha i3. The effect of the chemotactic peptide is blocked by treatment of intact HL-60 cells with pertussis toxin. Peptide mapping studies using Staphylococcus aureus protease V8 reveal that the two radiolabeled proteins are structurally distinct. Thus, the agonist-activated formyl peptide receptor functionally interacts with two distinct pertussis toxin substrates, most likely with Gi2 and Gi3. As the third Gi protein, Gi1, appears to be absent from both HL-60 cells and from systems that clearly reveal hormonal inhibition of adenylyl cyclase, the results strongly suggest that primary structure alone does not suffice to determine which effector mechanism is regulated by a given Gi-protein.     

G-proteins of fat-cells. Role in hormonal regulation of intracellular inositol 1,4,5-trisphosphate.

Pertussis toxin abolishes hormonal inhibition of adenylate cyclase, hormonal stimulation of inositol 1,4,5-trisphosphate accumulation in rat fat-cells, and catalyses the ADP-ribosylation of two peptides, of Mr 39,000 and 41,000 [Malbon, Rapiejko & Mangano (1985) J. Biol. Chem. 260, 2558-2564]. The 41,000-Mr peptide is the alpha-subunit of the G-protein, referred to as Gi, that is believed to mediate inhibitory control of adenylate cyclase by hormones. The nature of the 39,000-Mr substrate for pertussis toxin was investigated. The fat-cell 39,000-Mr peptide was compared structurally and immunologically with the alpha-subunits of two other G-proteins, Gt isolated from the rod outer segments of bovine retina and Go isolated from bovine brain. After radiolabelling in the presence of pertussis toxin and [32P]NAD+, the electrophoretic mobilities of the fat-cell 39,000-Mr peptide and the alpha-subunits of Go and Gt were nearly identical. Partial proteolysis of these ADP-ribosylated proteins generates peptide patterns that suggest the existence of a high degree of homology between the fat-cell 39,000-Mr peptide and the alpha-subunit of Go. Antisera raised against purified G-proteins and their subunits were used to probe immunoblots of purified Gt, Gi, Go, and fat-cell membrane proteins. Although recognizing the 36,000-Mr beta-subunit band of Gt, Gi, Go and a 36,000-Mr fat-cell peptide, antisera raised against Gt failed to recognize either the 39,000- or the 41,000-Mr peptides of fat-cells or the alpha-subunits of Go and Gi. Antisera raised against the alpha-subunit of Go, in contrast, recognized the 39,000-Mr peptide of rat fat-cells, but not the alpha-subunit of either Gi or Gt. These data establish the identity of Go, in addition to Gi, in fat-cell membranes and suggest the possibility that either Go or Gi alone, or both, may mediate hormonal regulation of adenylate cyclase and phospholipase C.     

Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.     

Cholera toxin action on rabbit corpus luteum membranes: effects on adenylyl cyclase activity and adenosine diphospho-ribosylation of the stimulatory guanine nucleotide-binding regulatory component

Cholera toxin elicited 5- to 7-fold stimulation of adenylyl cyclase activity. Half-maximal activation was at 4.42 micrograms/ml cholera toxin. Cholera toxin-mediated activation was time dependent. At 0.1 mM ATP, both guanosine triphosphate (GTP) and nicotinamide adenine dinucleotide (NAD+) were required for cholera toxin activation of luteal adenylyl cyclase. The concentrations of GTP and NAD+ required for half-maximal activation were 1 and 200 microM, respectively. The GTP requirement could be eliminated by increasing the ATP concentration to 1.0 mM. Guanosine-5'-O-(2-thiodiphosphate) [GDP beta S] did not support cholera toxin activation of the luteal enzyme. Cholera toxin treatment increased GTP-stimulated activity, did not significantly alter guanyl-5'-yl imidodiphosphate [GMP-P(NH)P]-stimulated activity, and depressed NaF-stimulated activity. Furthermore, toxin treatment resulted in a 3.4-fold reduction in the Kact values for ovine luteinizing hormone (oLH) to activate adenylyl cyclase. A similar reduction in Kact values for oLH was obtained when concentration-effect curves performed in the presence of GMP-P(NH)P were compared to those performed in the presence of GTP. In addition, luteal membranes treated with cholera toxin and [32P]NAD+ were subjected to autoradiographic analysis following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This treatment resulted in the [32P] adenosine diphospho (ADP)-ribosylation of a 45,000-dalton protein doublet, corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (Ns). As with activation of adenylyl cyclase activity, cholera toxin-specific [32P] ADP-ribosylation was time dependent and increased with increasing concentrations of cholera toxin. GTP, GMP-P(NH)P, and NaF, but not GDP beta S, were capable of supporting [32P] ADP-ribosylation of the protein doublet. oLH did not alter the ability of cholera toxin to ADP-ribosylate the protein activation of luteal adenylyl cyclase activity is due to the ADP-ribosylation of the alpha subunit of Ns and the concomitant inhibition of a GTPase associated with adenylyl cyclase.     

Hormonal regulation of Gi2 alpha-subunit phosphorylation in intact hepatocytes.

Hepatocytes contain the Gi2 and Gi3 forms of the 'Gi-family' of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity ('Gi'-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a 'Gi-like' G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.     

Evidence for a rabbit luteal ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase.

1. An ADP-ribosyltransferase activity which appears to be capable of activating adenylyl cyclase was identified in a plasma membrane fraction from rabbit corpora lutea and partially characterized by comparing the properties of the luteal transferase with those of cholera toxin. 2. Incubation of luteal membranes in the presence of GTP and varying concentrations of NAD resulted in concentration-dependent increases in adenylyl cyclase activity. 3. Stimulation of adenylyl cyclase by NAD and cholera toxin plus NAD was observed in the presence of GTP but not in the presence of guanosine-5'-O-(2-thiodiphosphate) or guanyl-5'-yl imidodiphosphate. 4. NAD or cholera toxin plus NAD reduced the Kact values for luteinizing hormone to activate adenylyl cyclase 3- to 3.5-fold. 5. NAD or cholera toxin plus NAD increased the extent to which cholate extracts from luteal membranes were able to reconstitute adenylyl cyclase activity in S49 cyc- mouse lymphoma membranes. 6. It was necessary to add ADP-ribose and arginine to the incubation mixture in order to demonstrate cholera toxin-specific ADP-ribosylation of a protein corresponding to the alpha subunit of the stimulatory guanine nucleotide-binding regulatory component (alpha Gs). 7. Treatment of luteal membranes with NAD prior to incubation in the presence of [32P]NAD plus cholera toxin resulted in reduced labeling of alpha Gs. 8. Endogenous ADP-ribosylation of alpha Gs was enhanced by Mg but was not altered by guanine nucleotide, NaF or luteinizing hormone and was inhibited by cAMP. 9. Incubation of luteal membranes in the presence of [32P]ADP-ribose in the absence and presence of cholera toxin did not result in the labeling of any membrane proteins.     

Determination of G-protein levels, ADP-ribosylation by cholera and pertussis toxins and the regulation of adenylyl cyclase activity in liver plasma membranes from lean and genetically diabetic (db/db) mice

Liver plasma membranes prepared from genetically diabetic (db/db) mice expressed levels of Gi alpha-2, Gi alpha-3 and G-protein beta-subunits that were reduced by some 75, 63 and 73% compared with levels seen in membranes from lean animals. In contrast, there were no significant differences in the expression of the 42 and 45 kDa forms of Gs alpha-subunits. Pertussis toxin-catalysed ADP-ribosylation of membranes from lean animals identified a single 41 kDa band whose labelling was reduced by some 86% in membranes from diabetic animals. Cholera toxin-catalysed ADP-ribosylation identified two forms of Gs alpha-subunits whose labelling was about 4-fold greater in membranes from diabetic animals compared with those from lean animals. Maximal stimulations of adenylyl cyclase activity by forskolin (100 microM), GTP (100 microM), p[NH]ppG (100 microM), NaF (10 mM) and glucagon (10 microM) were similar in membranes from lean and diabetic animals, whereas stimulation by isoprenaline (100 microM) was lower by about 22%. Lower concentrations (EC50-60 nM) of p[NH]ppG were needed to activate adenylyl cyclase in membranes from diabetic animals compared to those from lean animals (EC50-158 nM). As well as causing activation, p[NH]ppG was capable of eliciting a pertussis toxin-sensitive inhibitory effect upon forskolin-stimulated adenylyl cyclase activity in membranes from both lean and diabetic animals. However, maximal inhibition of adenylyl cyclase activity in membranes from diabetic animals was reduced to around 60% of that found using membranes from lean animals. Pertussis toxin-treatment in vivo enhanced maximal stimulation of adenylyl cyclase by glucagon, isoprenaline and p[NH]ppG through a process suggested to be mediated by the abolition of functional Gi activity. The lower levels of expression of G-protein beta-subunits, in membranes from diabetic compared with lean animals, is suggested to perturb the equilibria between holomeric and dissociated G-protein subunits. We suggest that this may explain both the enhanced sensitivity of adenylyl cyclase to stimulation by p[NH]ppG in membranes from diabetic animals and the altered ability of pertussis and cholera toxins to catalyse the ADP-ribosylation of G-proteins in membranes from these two animals.     

Simultaneous coupling of alpha 2-adrenergic receptors to two G-proteins with opposing effects. Subtype-selective coupling of alpha 2C10, alpha 2C4, and alpha 2C2 adrenergic receptors to Gi and Gs.

Coupling of the three alpha 2-adrenergic receptor (alpha 2AR) subtypes to Gi and Gs was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the alpha 2AR agonist UK-14304, alpha 2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, alpha 2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of alpha 2C10 to both Gi and Gs. To isolate functional Gs and Gi coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only alpha 2C10-mediated inhibition (approximately 50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only alpha 2C10-mediated stimulation (approximately 60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl-terminal portion of Gs alpha blocked this functional alpha 2AR.Gs coupling to the same extent as that found for beta 2AR.Gs coupling. In addition to functional Gs coupling, we also verified direct, agonist-dependent, physical coupling of alpha 2AR to Gs alpha. In agonist-treated membranes, an agonist-receptor-Gs alpha complex was immunoprecipitated with a specific alpha 2C10 antibody, and the Gs component identified by both western blots using Gs alpha antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the alpha 2AR subtypes, we investigated whether G-protein coupling was subtype-selective, using UK-14304 and cells with the same alpha 2AR expression levels (approximately 5 pmol/mg). Coupling to Gi was equivalent for alpha 2C10, alpha 2C4, and alpha 2C2: 53.4 +/- 8.8% versus 54.9 +/- 1.0% versus 47.6 +/- 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to Gs were found between the three alpha 2AR subtypes: stimulation of adenylyl cyclase was 57.9 +/- 6.3% versus 30.7 +/- 1.1% versus 21.8 +/- 1.7% for alpha 2C10, alpha 2C4, and alpha 2C2, respectively. Thus, alpha 2AR have the potential to couple physically and functionally to both Gi and Gs; for Gi coupling we found a rank order of alpha 2C10 = alpha 2C4 = alpha 2C2, while for Gs coupling, alpha 2C10 greater than alpha 2C4 greater than alpha 2C2.     

Site of pertussis toxin-induced ADP-ribosylation on Gi is critical for receptor modulation of GDP interaction with Gi

In this study, the influence of the inhibitory mu-opioid receptor on the potencies of 5'-guanosine alpha-thiotriphosphate (GTP gamma S) and GDP at the inhibitory GTP-binding protein (Gi) were investigated in an adenylyl cyclase system. It was hoped that a receptor-mediated change in the potency of either GTP gamma S or GDP in affecting adenylyl cyclase activity may elucidate how a receptor alters cyclase activity via its G-protein. In an adenylyl cyclase system employing 5'-adenylyl imidodiphosphate as substrate, GTP gamma S, a nonhydrolyzable analog of GTP, inhibited forskolin-stimulated adenylyl cyclase activity in the absence of morphine; morphine failed to significantly affect the apparent potency of GTP gamma S. GDP blocked the GTP gamma S-induced inhibition of adenylyl cyclase; morphine profoundly diminished the ability of GDP to block the inhibitory effect of GTP gamma S. The IC50 values of GTP gamma S were 0.02 +/- 0.01, 0.18 +/- 0.04, and 2.2 +/- 0.5 microM in the absence of other drugs, in the presence of a combination of 100 microM GDP and morphine, and in the presence of 100 microM GDP, respectively. GDP blocked the inhibitory effect of GTP gamma S (0.3 microM) in a concentration-dependent manner; the EC50 for GDP was 16 +/- 2.6 microM in the absence of morphine and 170 +/- 32 microM in the presence of morphine. Exposure of 7315c cells to pertussis toxin for 3 h resulted in a small decrease in the potency of GTP gamma S in inhibiting cyclase. However, the relative potency of GDP in blocking the GTP gamma S-mediated inhibition of cyclase was increased: the EC50 values of GDP were 11 +/- 4 and 0.81 +/- 0.2 microM in untreated and pertussis toxin-treated membranes, respectively. In untreated membranes, there was a brief lag in the GTP gamma S-induced inhibition of adenylyl cyclase; morphine diminished this lag. In membranes treated with pertussis toxin, there was an exaggerated lag in the onset of GTP gamma S inhibition of adenylyl cyclase activity; morphine could no longer affect this lag. Thus, uncoupling the mu-opioid receptor from Gi appeared to increase the affinity of Gi for GDP. These data suggest that the effect of an inhibitory receptor is to decrease the affinity of Gi for GDP by virtue of its interaction with the carboxy-terminal region of Gi alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Major pertussis-toxin-sensitive GTP-binding protein of bovine lung. Purification, characterization and production of specific antibodies

Two GTP-binding proteins which can be ADP-ribosylated by islet-activating protein, pertussis toxin, were purified from the cholate extract of bovine lung membranes. Both proteins had the same heterotrimeric structure (alpha beta gamma), but the alpha subunits were dissociated from the beta gamma when they were purified in the presence of AlCl3, MgCl2 and NaF. The molecular mass of the alpha subunit of the major protein (designated GLu, with beta gamma) was 40 kDa and that of the minor one was 41 kDa. The results of peptide mapping analysis of alpha subunits with a limited proteolysis indicated that GLu alpha was entirely different from the alpha of brain Gi or Go, while the 41-kDa polypeptide was identical with the alpha of bovine brain Gi. The kinetics of guanosine 5'-[3-O-thio]triphosphate (GTP[gamma S]) binding to GLu was similar to that to lung Gi but quite different from that to brain Go. On the other hand, incubation of GLu alpha at 30 degrees C caused a rapid decrease of GTP[gamma S] binding, the inactivation curve being similar to that of Go alpha but different from that of Gi alpha. The alpha subunits of lung Gi and GLu did not react with the antibodies against the alpha subunit of bovine brain Go. The antibodies were raised in rabbits against GLu alpha and were purified with a GLu alpha-Sepharose column. The purified antibodies reacted not only with GLu alpha but also with the 41-kDa protein and purified brain Gi alpha. However, the antibodies adsorbed with brain Gi alpha reacted only with GLu alpha, indicating antisera raised with GLu alpha contained antibodies that recognize both Gi alpha and GLu alpha, and those specific to GLu alpha. These results further indicate that GLu is different from Gi or Go. Anti-GLu alpha antibodies reacted with the 40-kDa proteins in the membranes of bovine brain and human leukemic (HL-60) cells. The beta gamma subunits were also purified from bovine lung. The beta subunit was the doublet of 36-kDa and 35-kDa polypeptides. The lung beta gamma could elicit the ADP-ribosylation of GLu alpha by islet-activating protein, increase the GTP[gamma S] binding to GLu and protect the thermal denaturation of GLu alpha. The antibodies raised against brain beta gamma cross-reacted with lung beta but not with lung gamma.     

Phosphorylation of Gz in human platelets. Selectivity and site of modification

We have demonstrated previously that the G protein alpha subunit Gz alpha (or Gx alpha) in human platelets is subject to phosphorylation by agents that activate protein kinase C, including phorbol 12-myristate 13-acetate, thrombin, and the thromboxane A2 analog U46619. We examine here the site and selectivity of phosphorylation both in vitro using recombinant G protein alpha subunits and in situ using permeabilized and intact platelets. Protein kinase C catalyzes the rapid and nearly stoichiometric phosphorylation of recombinant Gz alpha, with the modification occurring preferentially for the GDP-bound form of the subunit. Under the same conditions, phosphorylation of recombinant Gi alpha 1, Gi alpha 2, Gi alpha 3, Gs alpha-S, Gs alpha-L, and Go alpha 1 was minimal. Phosphorylation of both rGz alpha and platelet Gz alpha occurs at a serine residue near the amino terminus. This conclusion is supported by phosphoamino acid analysis and the incorporation of radiolabel from [gamma-32P]ATP into the amino-terminal CNBr peptide (residues 2-53 of the encoded protein). One of the antisera used in this study (6354, directed toward residues 24-33) recognizes only the nonphosphorylated form of Gz alpha, providing strong evidence that Ser25 or Ser27 is the site of phosphorylation. Results obtained with 6354 also suggest that phorbol ester-promoted phosphorylation of Gz alpha approaches 1 mol of phosphate per mol of subunit in permeabilized platelets.     

The alpha 2B adrenergic receptor of undifferentiated neuroblastoma x glioma hybrid NG108-15 cells, interacts directly with the guanine nucleotide binding protein, Gi2

In membranes of undifferentiated neuroblastoma x glioma hybrid cell line NG108-15, the apparent specific binding of [3H]yohimbine measured in the presence of 1 microM noradrenaline, was increased substantially by the presence of the poorly hydrolysed analogue of GTP, guanylyl-imidodiphosphate (Gpp[NH]p) or by preincubation of membranes with antibodies against the C-terminal decapeptide of the alpha subunit of the G-protein Gi2. Such an effect was not produced by antibodies against the equivalent region of Go alpha Gi3 alpha or Gs alpha or from non-immune serum. By contrast, total specific binding of [3H]yohimbine was not modified by co-incubation with Gpp[NH]p or by preincubation with the antibodies from any of the anti-G protein antisera. These results demonstrate a direct interaction of the alpha 2B adrenergic receptor of NG108-15 cells with Gi2.     

Adenylyl cyclase and G-proteins in Phytomonas

Phytomonas sp. membranes have an adenylyl cyclase activity which is greater in the presence of Mn2+ than with Mg2+. The Mg2+ and Mn2+ activity ratio varies from one membrane preparation to another, suggesting that the adenylyl cyclase has a variable activation state. A[35S]GTP-gamma-S-binding activity with a Kd of 171 nM was detected in Phytomonas membranes. Incubation of these membranes with activated cholera or pertussis toxin and [adenylate 23P]NAD+ led to incorporation of radioactivity into bands of about 40-44 kDa. Crude membranes were electrophoresed on SDS-polyacrylamide gels and analyzed, by Western blotting, with the 9188 anti-alpha[s] antibody and the AS/7 antibody (anti-alpha[i], anti-alpha[i1], and anti-alpha[i2]. These procedures resulted in the identification of polypeptides of approximately 40-44 kDa. Phytomonas adenylyl cyclase could be activated by treatment of membrane preparations with cholera toxin, in the presence of NAD+, while similar treatment with pertussis toxin did not affect this enzyme activity. These studies indicate that in Phytomonas, adenylyl cyclase activity is coupled to an unknown receptor entity through G alpha[s] proteins.     

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain.

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.