Differential effects of cholera toxin on guanine nucleotide regulation of beta-adrenergic agonist high affinity binding and adenylate cyclase activation in frog erythrocyte membranes

The guanine nucleotide regulatory protein(s) regulates both adenylate cyclase activity and the affinity of adenylate cyclase-coupled receptors for hormones or agonist drugs. Cholera toxin catalyzes the covalent modification of the nucleotide regulatory protein of adenylate cyclase systems. Incubation of frog erythrocyte membranes with cholera toxin and NAD+ did not substantially alter the dose dependency for guanine nucleotide activation of adenylate cyclase activity. In contrast, toxin treated membranes demonstrated a 10 fold increase in the concentrations of guanine nucleotide required for a half maximal effect in regulating beta-adrenergic receptor affinity for the agonist (+/-) [3H]hydroxybenzylisoproterenol. The data emphasize the bifunctional nature of the guanine nucleotide regulatory protein and suggest that distinct structural domains of the guanine nucleotide regulatory protein may mediate the distinct regulatory effects on adenylate cyclase and receptor affinity for agonists.     

Functional modification of the guanine nucleotide regulatory protein after desensitization of turkey erythrocytes by catecholamines

Densensitization of turkey erythrocytes by exposure to the beta-adrenergic agonist (-)isoproterenol leads to decreased activation of adenylate cyclase by agonist, NaF, and guanyl-5'-yl imido diphosphate, with no reduction in the number of beta-adrenergic receptors. Interactions between the receptor and the guanine nucleotide regulatory protein (N protein) also seem to be impaired. These observations suggest that a component distal to the beta-adrenergic receptor may be a locus of modification. Accordingly we examined the N protein to determine whether it was altered by desensitization. The rate at which (-)isoproterenol stimulated the release of [3H]GDP from the N protein was substantially lower in membranes prepared from desensitized cells, providing further evidence for uncoupling of the receptor and the N protein. The amount of N protein in membranes from control and desensitized cells was compared by labeling the 42,000 Mr component of the N protein with [32P]NAD+ and cholera toxin; no significant difference was found. However, significantly more N protein (p less than .001) was solubilized by cholate extraction of desensitized membranes, suggesting an altered association of the N protein with the membrane after desensitization. The functional activity of the N protein was measured by reconstitution of cholate extracts of turkey erythrocyte membranes into S49 lymphoma cyc- membranes. Reconstitution of (-)isoproterenol stimulation of adenylate cyclase activity was reduced significantly (p less than .05) after desensitization. These observations suggest that desensitization of the turkey erythrocyte by (-)isoproterenol results in functional modifications of the guanine nucleotide regulatory protein, leading to impaired interactions with the beta-adrenergic receptor and reduced activation of adenylate cyclase.     

Mechanisms of hormone receptor-effector coupling: the beta-adrenergic receptor and adenylate cyclase

The beta-adrenergic receptors that are coupled to adenylate cyclase have provided a model system for studying the mechanisms by which a plasma membrane receptor is coupled to a well-defined biochemical effector. The beta 2-adrenergic receptors from frog erythrocyte membranes have been purified to homogeneity and the ligand-binding subunit has been identified as a glycoprotein with an approximate molecular weight of 58,000. This subunit has also been identified with the use of newly developed photoaffinity reagents. Under the influence of agonist hormones (H), the receptors (R) form transient complexes with another component of this system, termed the nucleotide regulatory protein (N). Formation of this ternary complex, HRN, leads to the dissociation of GDP from N and the interaction of stimulatory GTP with N. N charged with GTP appears to activate the catalytic moiety of the adenylate cyclase enzyme. Although some striking analogies have been found for the mechanisms by which inhibitory receptors interact with adenylate cyclase, much less is known about the molecular properties of the components involved and the ways in which they interact to dampen adenylate cyclase activity in the plasma membrane.     

The molecular size of adenylate cyclase in the absence and presence of nucleotide and hormone effectors

The molecular size of adenylate cyclase solubilized from frog erythrocyte membranes by digitonin extraction has been determined by chromatography on Sepharose 6B. Regardless of whether the membranes are exposed to catecholamines, GPP(NH)P, NaF or no effector prior to solubilization, the apparent molecular size of the adenylate cyclase enzyme is the same. Furthermore, a similar elution profile for the enzyme is observed when the catalytic activity in the eluates is measured in the presence of Mn++, rather than Mg++. Since it is generally assumed that the persistent activation of adenylate cyclase by GPP(NH)P requires interaction of the catalytic moiety with the guanine nucleotide regulatory site, it appears that the adenylate cyclase activity detected in the column eluates represents an intact catalytic-regulatory site complex. The adenylate cyclase activity derived from catecholamine pretreated frog erythrocyte membranes does not co-elute with catecholamine-occupied beta-adrenergic receptors, indicating that the agonist-promoted increase in apparent receptor size observed here and in earlier studies does not represent a physical coupling of the receptor and the adenylate cyclase enzyme.     

Mechanism of guanine nucleotide regulatory protein-mediated inhibition of adenylate cyclase. Studies with isolated subunits of transducin in a reconstituted system

The retinal nucleotide regulatory protein, transducin, can substitute for the inhibitory guanine nucleotide-binding regulatory protein (Ni) in inhibiting adenylate cyclase activity in phospholipid vesicle systems. In the present work we have assessed the roles of the alpha (alpha T) and beta gamma (beta gamma T) subunit components in mediating this inhibition. The inclusion of either a preactivated alpha T . GTP gamma S (where GTP gamma S is guanosine 5'-O-(thiotriphosphate)) complex, or the beta gamma complex, in phospholipid vesicles containing the pure human erythrocyte stimulatory guanine nucleotide-binding regulatory protein (Ns) and the resolved catalytic moiety of bovine caudate adenylate cyclase (C) resulted in inhibition of the GppNHp-stimulated (where GppNHp is guanyl-5'-yl imidodiphosphate) activity (by approximately 30-60 and 90%, respectively, at 2 mM MgCl2). The inhibitions by both of these subunit species are specific for the Ns-stimulated activity with neither alpha T . GTP gamma S nor beta gamma T having any direct effect on the intrinsic activity of the catalytic moiety. Increasing the MgCl2 concentration in the assay incubations significantly decreases the inhibitions by both alpha T . GTP gamma S and beta gamma T. Similarly, when the pure hamster lung beta-adrenergic receptor is included in the lipid vesicles with Ns and C, the levels of inhibition of the GppNHp-stimulated activity by both alpha T . GTP gamma S and beta gamma T are reduced compared to those obtained in vesicles containing just Ns and C (but not stimulatory receptor). These inhibitions are reduced still further under conditions where the agonist stimulation of adenylate cyclase activity is maximal, i.e. when stimulating with isoproterenol plus GTP. In these cases the alpha T . GTP gamma S inhibitory effects are completely eliminated and the inhibitions observed with holotransducin can be fully accounted for by the beta gamma T complex. The ability of the beta-adrenergic receptor to relieve these inhibitions suggests that the receptor may remain coupled to Ns (or alpha s) during the activation of the regulatory protein and the stimulation of adenylate cyclase. These results also suggest that under physiological conditions the beta gamma subunit complex is primarily responsible for mediating the inhibition of adenylate cyclase activity.     

Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding.

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.     

Alteration in the protein components of catecholamine-sensitive adenylate cyclase during maturation of rat reticulocytes

The maturing rat reticulocyte was used as a model system in which to study developmental changes in the protein components of hormone-sensitive adenylate cyclase. Plasma membranes from rat erythrocytes display 10 to 20% of the adenylate cyclase activity and 30 to 50% of the beta-adrenergic receptors which are measured in membranes from rat reticulocytes, as noted by others. Reticulocyte membranes also display equal activities in response to (-)-isoproterenol in the presence of either GTP or GTP gamma S, whereas erythrocyte membrane adenylate cyclase is twice as active in the presence of isoproterenol plus GTP gamma S as in the presence of isoproterenol plus GTP. We have studied this system in greater detail by developing or applying independent assays for the catalytic protein (C) and the guanine nucleotide-binding regulatory protein (G/F) of adenylate cyclase. C was assayed in membranes by its intrinsic Mn2+-stimulated activity. It was also measured by reconstituting membranes with saturating amounts of GTP gamma S-activated G/F, yielding an operationally defined Vmax for the catalyst. By either assay, reticulocytes display about 3-fold greater C activity than do erythrocytes. G/F was assayed by its ability to confer GTP gamma S-stimulated activity upon C (which was supplied by membranes of cyc- S49 lymphoma cells). This assay indicates that reticulocyte membranes contain about 3 times as much G/F as do erythrocyte membranes. Cholera toxin and [32P]NAD were used to [32P]ADP-ribosylate the 45,000- and 52,000-dalton subunits of G/F. Total incorporation of 32P into these subunits decreased 3- to 4-fold with reticulocyte maturation. The ratio of label in the 52,000-dalton peptide to that in the 45,000-dalton peptide decreased from 0.29 in reticulocyte membranes to 0.14 in erythrocyte membranes. The apparently coordinate decrease in the amounts of C, G/F, and beta-adrenergic receptors suggest that the stoichiometry between these components is maintained during maturation, and may account for the decrease in adenylate cyclase in the membranes. However, the qualitative changes in responsiveness to hormones in the presence of GTP or GTP gamma S may be related to loss or proteolysis of the 52,000-dalton subunit of G/F.     

The guanine nucleotide-binding regulatory component of adenylate cyclase in human erythrocytes

The guanine nucleotide-binding regulatory component of adenylate cyclase (G/F) has been purified from human erythrocyte membranes. It is composed of two major polypeptides with molecular weights of 35,000 and 45,000. When cyc- S49 lymphoma cell plasma membranes are reconstituted with purified human erythrocyte G/F, stimulation of adenylate cyclase by beta-adrenergic agonists, guanine nucleotides, and fluoride is restored. Binding of GTP gamma S to human erythrocyte G/F and GTP gamma S-mediated activation of the protein are closely correlated. The agreement between the apparent dissociation constants for these two reactions suggests that the measured binding site is identical to the site responsible for activation. A 41,000-dalton protein has been identified as a contaminant of preparations of G/F that have been purified by four successive chromatographic steps. This protein serves as a specific substrate for ADP-ribosylation and labeling by islet activating protein (IAP) and [32P]NAD, and it appears to contribute an additional high-affinity guanine nucleotide binding site to such preparations.     

Phorbol diester treatment promotes enhanced adenylate cyclase activity in frog erythrocytes

Incubation of intact frog erythrocytes with 12-O-tetradecanoyl phorbol-13-acetate (TPA), a tumor-promoting phorbol diester which activates protein kinase C, results in an approximate two- to threefold increase in subsequently tested beta-adrenergic agonist-stimulated adenylate cyclase activity. This increase is due to an elevation in the Vmax of the enzyme rather than to a change in affinity for the agonist. TPA treatment of frog erythrocytes does not alter the affinity (KD) or the binding capacity (Bmax) for the beta-adrenergic antagonist [125I]cyanopindolol. In addition, agonist/[125I]cyanopindolol competition curves are not affected by TPA pretreatment nor is their sensitivity to guanine nucleotides. Incubation of frog erythrocyte membranes alone with TPA does not promote sensitization or activation of adenylate cyclase activity. Pretreatment of intact frog erythrocytes with TPA also produces approximately two- to threefold increases in basal, guanine nucleotide-, prostaglandin E1-, forskolin-, NaF-, and MnCl2-stimulated adenylate cyclase activities in frog erythrocyte membranes. This enhancement of adenylate cyclase activity by TPA is induced rapidly (t1/2 approximately equal to 5 min) and with an EC50 of about 10(-7) to 10(-6) M. Other tumor-promoting phorbol diesters or phorbol diester-like compounds including 4 beta-phorbol 12,13-dibutyrate, 4 beta-phorbol 12,13-didecanoate, and mezerein are effective in promoting enhanced adenylate cyclase activity. In contrast, phorbols such as 4 beta-phorbol, 4 alpha-phorbol 12,13-didecanoate, and 4-O-methylphorbol 12-myristate 13-acetate, which are inactive in tumor promotion and which do not activate protein kinase C, do not affect frog erythrocyte adenylate cyclase activity. These data are suggestive of a protein kinase C-mediated phosphorylation of one of the adenylate cyclase components that is distal to the receptor, i.e., the nucleotide regulatory and/or catalytic components.     

Rapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N

beta-Adrenergic receptors from turkey erythrocyte membranes have been purified 1000-4000-fold using alprenolol-Sepharose affinity chromatography. Addition of deoxycholate solubilized egg phosphatidylcholine to the beta-adrenergic receptor, that is 5-10% pure and in 0.1% digitonin, followed by Sephadex G-50 gel filtration in buffers containing 30 mM MgCl2 results in 65-70% of the receptor being incorporated into phospholipid vesicles. The beta-adrenergic receptor as detected by photoaffinity labeling using [125I]azidobenzylpindolol in membranes and after alprenolol-Sepharose chromatography is a Mr = 40,000 peptide. Addition of deoxycholate extracts of human erythrocyte membranes, which contain the guanine nucleotide stimulatory regulatory protein of adenylate cyclase (Ns) but not beta-adrenergic receptor, were used to reconstitute a guanine nucleotide-mediated change in agonist affinity for the receptor. These results demonstrate that the alprenolol-Sepharose affinity purified beta-adrenergic receptor is functional in both ligand binding and coupling to Ns. The procedure is rapid, efficient and should be generally applicable to beta-adrenergic receptor and Ns from several different membrane systems.     

Evidence that forskolin activates turkey erythrocyte adenylate cyclase through a noncatalytic site

The diterpene forskolin has been reported to activate adenylate cyclase in a manner consistent with an interaction at the catalytic unit. However, some of its actions are more consistent with an interaction at the coupling unit that links the hormone receptor to the adenylate cyclase activity. This report adds support to the latter possibility. Under conditions that lead to stimulation of adenylate cyclase in turkey erythrocyte membranes by GTP, forskolin also becomes more active. Additional evidence to support an influence of forskolin upon adenylate cyclase via the GTP-coupling protein N includes the following: (i) forskolin, at submaximal concentrations, leads to enhanced sensitivity and responsiveness of isoproterenol-dependent adenylate cyclase activity in turkey erythrocyte membranes; (ii) under specified conditions, the nucleotide GDP, an inhibitor of the stimulating nucleotide GTP and its analog, guanyl imidodiphosphate (Gpp(NH)p), also markedly inhibits the action of forskolin; (iii) both Gpp(NH)p and forskolin are associated with a decrease in agonist affinity for the beta-adrenergic receptor. However, actions of forskolin in the turkey erythrocyte are not identical to those of GTP: (i) forskolin is never as potent as Gpp(NH)p in activating adenylate cyclase; (ii) the magnitude of synergism between isoproterenol and forskolin is not equal to that observed with isoproterenol and Gpp(NH)p; (iii) at high concentrations, forskolin inhibits antagonist binding to the beta-receptor. Forskolin appears to have several sites of action in the turkey erythrocyte membrane, including an influence upon the adenylate cyclase regulatory protein N.     

Hepatic adenylate cyclase. Development-dependent coupling to the beta-adrenergic receptor in the neonate

Guanine nucleotide-dependent modulation of agonist binding to the beta-receptor reflects coupling of the receptor to the nucleotide regulatory protein. Similarly, guanine nucleotide-dependent stimulation of adenylate cyclase can be used as an index of coupling between the regulatory protein and the catalytic unit of the cyclase. Using both approaches we have studied coupling in the beta-adrenergic receptor-adenylate cyclase system in rabbit liver during neonatal development. With [3H]dihydroalprenolol as ligand, the Bmax was relatively unchanged (200-300 fmol/mg of protein) between birth and end of day 1 and was similar to adult values. Guanyl-5'-yl imidodiphosphate-dependent shift in agonist (l-isoproterenol) competition curves was biphasic, decreasing from 10-fold in membranes isolated from animals at term to about 6-fold in membranes from 6-h-old neonates, and increasing progressively in older animals to a maximal measurable value of 42-fold in the adult. The ability of guanyl-5'-yl imidodiphosphate, GTP, GTP plus isoproterenol, NaF, or forskolin to activate adenylate cyclase was also biphasic and age-dependent. With Mn2+ the measured activity was not at any time greater than the activity at term. Pretreatment of membranes with cholera toxin resulted in differential levels of enhancement of adenylate cyclase activity wherein much lower enhancement was observed in membranes from neonatal animals. With [32P]NAD as substrate, cholera toxin-catalyzed ADP-ribosylation of membranes indicated development-dependent accumulation of Ns peptides. From these results we suggest that there is a decreased efficiency in the coupling of the beta-adrenergic receptor to hepatic adenylate cyclase in early neonatal life. The molecular basis for the biphasic nature of the coupling is presently unclear.     

A reconstitution assay for the guanine nucleotide regulatory protein of the adenylate cyclase system using turkey erythrocyte membranes as the acceptor preparation

The turkey erythrocyte beta-adrenergic receptor-adenylate cyclase system has the unusual property that neither GTP nor Gpp(NH)p are effective in activating adenylate cyclase unless a beta-agonist is present simultaneously. This property results in essentially no basal activity and the inability of GTP or Gpp(NH)p alone to activate the catalytic moiety. In this study, we have exploited these characteristics to utilize turkey erythrocyte membranes as the acceptor preparation in a reconstitution assay. Rat reticulocyte or turkey erythrocyte membranes that have been activated with isoproterenol and Gpp(NH)p followed by solubilization with sodium cholate serve as the donor source of the guanine nucleotide regulatory protein (N). By reconstituting this Gpp(NH)p-activated N protein, it has been found that: (1) exogenous Gpp(NH)p-associated N could activate the catalytic unit of adenylate cyclase in turkey erythrocyte membranes; (2) this system can be used to assay N protein activity; (3) the endogenous pathway for activation of turkey erythrocyte membrane adenylate cyclase by hormones and fluoride remains qualitatively functional; and (4) the effects of combined activation via the endogenous and exogenous pathways are additive and saturable.     

Modification of AlF-4- and receptor-stimulated phospholipase C activity by G-protein beta gamma subunits

Turkey erythrocyte membranes possess a phospholipase C that is markedly activated by P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of [3H]inositol-labeled turkey erythrocyte membranes with guanine nucleotide regulatory protein (G-protein) beta gamma subunits resulted in inhibition of both AlF-4-stimulated adenylate cyclase and AlF-4-stimulated phospholipase C activities. The apparent potency (K0.5 approximately 1 microgram or 20 pmol of beta gamma/mg of membrane protein) of beta gamma subunits for inhibition of each enzyme activity was similar and occurred with beta gamma purified by different methodologies from turkey erythrocyte, bovine brain, or human placenta membranes. In contrast to the effect on AlF-4-stimulated activity, the stimulatory effect on phospholipase C of the P2Y-purinergic receptor agonist 2-methylthioadenosine 5'-triphosphate in the presence of guanine nucleotides was potentiated by 50-100% in a concentration-dependent manner by reconstitution of beta gamma subunits. beta gamma subunits did not affect the K0.5 value of 2-methylthioadenosine 5'-triphosphate for the stimulation of phospholipase C activity. These results indicate that beta gamma subunits influence phospholipase C activity in a concentration range similar to that necessary for regulation of adenylate cyclase activity and suggest the involvement of a G-protein possessing an alpha beta gamma heterotrimeric structure in coupling hormone receptors to phospholipase C.     

Desensitization of β-Adrenergic Receptor-Coupled Adenylate Cyclase in Cerebral Cortex After In Vivo Treatment of Rats with Desipramine

Continuous treatment (1-10 days) of rats with desipramine (10 mg/kg, twice per day) caused desensitization of the beta-adrenergic receptor-coupled adenylate cyclase system of cerebral cortical membranes. The decrease in the isoproterenol-stimulated adenylate cyclase activity was more rapid and greater than the decrease in the number of beta-adrenergic receptors in membranes during treatment of the membrane donor rats with desipramine, indicating that the desensitization occurring at an early stage of the treatment was not accounted for solely by the decrease in the receptor number. Neither the guanine nucleotide regulatory protein (N) nor the adenylate cyclase catalyst was impaired by the drug treatment, since there was no decrease in the cyclase activity measured in the presence or absence of GTP, guanyl-5'-yl-beta-gamma-imidodiphosphate [Gpp(NH)p], NaF, or forskolin. Gpp(NH)p-induced activation of membrane adenylate cyclase developed with a lag time of a few minutes in membranes from control or drug-treated rats. The lag was shortened by the addition of isoproterenol, indicating that beta-receptors were coupled to N in such a manner as to facilitate the exchange of added Gpp(NH)p with endogenous GDP on N. This effect of isoproterenol rapidly decreased during the drug treatment of rats. Thus, functional uncoupling of the N protein from receptors was responsible for early development of desensitization of beta-adrenergic receptor-mediated adenylate cyclase in the cerebral cortex during desipramine therapy.     

Influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase in rat striatal membranes

The influence of pertussis toxin on the effects of guanine nucleotide on adenylate cyclase activity were investigated in rat striatal membranes. GTP promoted and inhibited the activity at 1 and 100 microM, respectively. The inhibitory effects of GTP were abolished by pretreatment of the membranes with pertussis toxin. GppNHp (guanyl-5'-y1-beta,gamma-imidodiphosphate) exerted only stimulatory effects and pertussis toxin did not affect the effects of GppNHp. GDP at 10 and 100 microM caused significant inhibition which was completely suppressed by pertussis toxin. It is suggested that guanine nucleotide regulates the affinity of as in stimulatory GTP-binding regulatory protein to either beta gamma or catalytic units of adenylate cyclase in a flip-flop manner. Inhibitory GTP-binding regulatory protein seems to play a regulatory role in inhibiting alpha s activity supplying the beta gamma heterodimer.     

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.     

G regulatory proteins and muscarinic receptor signal transduction in mucous acini of rat submandibular gland

The involvement of G regulatory proteins in muscarinic receptor signal transduction was examined in electrically permeabilized rat submandibular acinar cells. The guanine nucleotide analog, GTP gamma S, caused the dose dependent hydrolysis of membrane phosphatidylinositol 4,5-bisphosphate to release IP3. This response was insensitive to pertussis toxin treatment and was duplicated by NaF but not by GDP beta S. Enhanced IP3 synthesis was observed with a combination of GTP gamma S and carbachol. Exogenous IP3, as well as carbachol and GTP gamma S, provoked the release of sequestered 45Ca2+ from non-mitochondrial stores. In intact cells, carbachol significantly reduced the level of cyclic AMP induced by the beta-adrenergic agonist, isoproterenol, to 69% of its normal value. Pertussis toxin abolished this inhibitory action of carbachol on cyclic nucleotide levels. These results suggest that muscarinic receptors are coupled to two separate G regulatory proteins in submandibular mucous acini-the pertussis toxin-insensitive Gp of the phosphoinositide transduction pathway associated with elevated cytosolic calcium levels, and the pertussis toxin-sensitive Gi inhibitory protein of the adenylate cyclase complex.     

Attenuation of chick heart adenylate cyclase by muscarinic receptors after pertussis toxin treatment

Pertussis toxin selectively modifies the function of Ni, the inhibitory guanine nucleotide binding protein of the adenylate cyclase complex. In chick heart membranes, guanine nucleotide activation of Ni resulted in a decrease in the apparent affinity of the muscarinic receptor for the agonist oxotremorine, inhibition of basal adenylate cyclase activity, and the attenuation of adenylate cyclase by oxotremorine. Treatment of chicks with pertussis toxin caused the covalent modification of 80-85% of cardiac Ni. After this treatment Gpp(NH)p had no effect on muscarinic receptor affinity and GTP stimulated basal adenylate cyclase activity. In contrast, the GTP-dependent attenuation of adenylate cyclase caused by muscarinic receptors was unaffected.     

Heterologous desensitization of the inhibitory A1 adenosine receptor-adenylate cyclase system in rat adipocytes. Regulation of both Ns and Ni

Adenosine, acting via A1 adenosine receptors, can inhibit adenylate cyclase activity in adipocytes. To assess the effects of chronic adenosine agonist exposure on the A1 adenosine receptor system of adipocytes, rats were infused with (-)-phenylisopropyladenosine or vehicle for 6 days and membranes were prepared. Basal as well as isoproterenol-, sodium fluoride-, and forskolin-stimulated adenylate cyclase activities were significantly increased (approximately 2-fold) in membranes from treated animals. (-)-Phenylisopropyladenosine-mediated inhibition of forskolin-stimulated adenylate cyclase activity was significantly (p = 0.0001) attenuated in membranes from treated rats (20.1 +/- 2.1% inhibition) versus controls (31.6 +/- 2.3% inhibition). Prostaglandin E1-induced inhibition of forskolin-stimulated adenylate cyclase activity was also attenuated: 11.7 +/- 3.6 versus 23.2 +/- 4.6% (p = 0.001). Using the A1 adenosine receptor agonist radioligand (-)-N6-(3-[125I]iodo-4-hydroxyphenylisopropyl)adenosine, 32% fewer high affinity binding sites were detected in membranes from treated animals (p less than 0.04). Photoaffinity labeling with N6-2-(3-[125I]iodo-4-azidophenyl)ethyladenosine revealed no gross difference in receptor structure. The number of beta-adrenergic receptors as well as the percentage of receptors in the high affinity state as assessed by (-)-3-[125I]iodocyanopindolol binding were the same in both groups. In membranes from treated rats, the amount of [alpha-32P]NAD incorporated by pertussis toxin into the alpha subunit of the inhibitory guanine nucleotide regulatory protein (Ni) was decreased by 37 +/- 11%. Concurrently, the quantity of label incorporated by cholera toxin into the alpha subunit of the stimulatory guanine nucleotide regulatory protein (Ns) was increased by 44 +/- 14% in treated membranes. Finally, the capacity of Ns solubilized from treated membranes to stimulate adenylate cyclase activity when reconstituted into cyc- S49 lymphoma cell membranes was enhanced by approximately 50% compared to control. Thus, heterologous desensitization, manifested by a diminished capacity to inhibit adenylate cyclase and an enhanced responsiveness to stimulatory effectors, can be induced in the A1 adenosine receptor-adenylate cyclase system of adipocytes. A decrease in Ni alpha subunit concomitant with an increase in Ns alpha subunit quantity and activity may represent the biochemical mechanism of desensitization in this system.