Multiple Interactions of Unsaturated Fatty Acids with Opiate and Ouabain Binding Sites and β-Adrenergic Sensitive Adenylate Cyclase System

Abstract

The unsaturated fatty acids oleic, linoleic and arachidonic inhibited binding of ligands to the ouabain, opiate, and β-adrenergic plasma membrane receptors. Low concentrations of fatty acids slightly increased the binding of ouabain to its binding sites. The effect of these fatty acids on β-adrenergic sensitive adenylate cyclase was more complex. 0.2–0.3 mM fatty acids increased adenylate cyclase activity, while higher concentrations of arachidonic and linoleic acids, but not oleic acid     

Modulation of the β-Adrenergic Receptor-Coupled Adenylate Cyclase by Chemical Inducers of Differentiation: Effects on β Receptors and the Inhibitory Regulatory Protein Gi

Abstract

Several drugs known to induce differentiation in tumor cells were analyzed for their effects on the β-adrenergic receptor-coupled adenylate cyclase system in two human carcinoma cell lines, HeLa and A431. Each of the drugs was tested alone or in combination with sodium butyrate (NaBu), a known inducer of this signal transduction system. Puromycine amino nucleoside (PMAN) caused the largest increase in β-adrenergic receptors in HeLa cells followed by hexamethylenebisacetamide (HMBA) whereas 5′-azacytidine (5AZC) was ineffective. In addition, PMAN but not the others acted together with NaBu to elevate receptor levels 12-fold over control values. In contrast, HMBA and 5AZC were much more effective on A431 cells, PMAN caused only a slight increase in β receptors and none of the drugs acted in concert with NaBu. The increase in β receptors was usually accompanied by a corresponding increase in isoproterenol-stimulated adenylate cyclase activity. These effects of the drugs appeared to require protein synthesis as they were blocked by cycloheximide. In addition, some of the drugs caused a substantial decrease in basal adenylate cyclase activity. This effect on basal activity was abolished in cells treated with pertussis toxin, which ADP-ribosylates the inhibitory GTP-binding protein, G_i. Both HeLa and A431 cells contained a 41 kDalton substrate for the toxin which corresponds to the α; subunit of G_i. The G_i subunit was ADP-ribosylated by the toxin to a similar extent in membranes from control and drug-treated cells. Thus, the drugs appear to induce quantitative changes in β-adrenergic receptors and qualitative changes in G_i which results in a highly responsive β-adrenergic-stimulated adenylate cyclase.     

LOW CONCENTRATIONS OF LITHIUM INHIBIT THE SYNTHESIS OF CYCLIC AMP AND CYCLIC GMP IN THE RAT PINEAL GLAND

Abstract— Lithium chloride (2 m m ) significantly inhibited the increases in cyclic AMP and in cyclic GMP caused by norepinephrine or high concentrations of potassium in intact rat pineal glands. Adenylyl cyclase activity in homogenates and its stimulation by isoproterenol, a β-adrenergic agonist, were also inhibited. Lithium reduced the apparent V _max of isoproterenol-stimulated adenylyl cyclase activity without significantly affecting the apparent affinity for isoproterenol. There was no effect on the binding of the antagonist [³H]dihydroalprenolol to the β-adrenergic receptors, nor on the competition for binding sites by isoproterenol. Inhibition of adenylyl cyclase activity by lithium was inversely related to the magnesium concentration in the reaction mixture. There was no differential effect of lithium on adenylyl cyclase activity from supersensitive vs subsensitive glands. Lithium may inhibit cyclic nucleotide synthesis by interfering with the role of divalent cations.     

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase

The binding of [³H](?)-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay. The specificity of the binding sites is strikingly similar to what might be expected of the β-adrenergic receptor binding sites which mediate stimulation of adenylate cyclase by catecholamines in these membranes. The sites bind β-adrenergic antagonists and agonists with affinities which are directly related to their antagonist or agonist potency on the frog erythrocyte membrane adenylate cyclase. Binding shows strict stereospecificity with (?)-isomers exhibiting two orders of magnitude higher affinities than (+)-isomers. Dissociation constants for potent β-adrenergic antagonists are in the range of 10^?9 – 10^?8M whereas those for β-adrenergic agonists are about two orders of magnitude higher (≥ 10^?6M).     

beta-adrenergic supersensitivity in rat brainstem during clonidine withdrawal

The clonidine withdrawal syndrome was studied in the rat by measuring β-adrenergic responses as isoproterenol stimulated cyclic 3′, 5′-Adenosine monophosphate accumulation in brain slices and β-adrenergic membrane receptors as [³H] dihydroalprenolol binding. Supersensitivity of cyclic AMP accumulation was evident in brain-stems of clonidine-treated animals 18 and 24 hours after the last dose, but not in cerebral cortex. In addition there was no indication of changes in either number or affinity of beta-receptors in brainstem. The similarity of these findings to changes in adenylate cyclase activity seen during opiate withdrawal is intriguing.     

Independent variation of β-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes

Isoproterenol-stimulated adenylate cyclase activity in membrane preparations of reticulocyte-rich (90%) erythrocytes from phenylhydrazine-treated rats is 9 times greater than in untreated animals (1% reticulocytes); basal and fluoride-stimulated activities are also enhanced 2 and 4-fold respectively. In contrast, the number of β-adrenergic receptor sites detected by the binding of ¹²⁵I-hydroxybenzylpindolol (¹²⁵I-HYP) is increased only 40% in these same preparations. The dissociation constant (K_D) of ¹²⁵I-HYP and the IC₅₀ of (-)-isoproterenol for receptor binding sites are unchanged, as is the EC₅₀ of (-)-isoproterenol for activation of adenylate cyclase. The disproportionately large increase in the activity of the isoproterenol-sensitive adenylate cyclase, compared with the small increase in the number of ¹²⁵I-HYP binding sites indicates that the functions of catecholamine recognition and consequent adenylate cyclase response can vary independently and suggest that the receptor and the cyclase may be autonomous molecular entities.     

Evidence for a defect in the number of β-adrenergic receptors and in the adenylate cyclase responsiveness to guanine nucleotides in fat cells after adrenalectomy

Receptor binding studies (?)-[³H]dihydroalprenolol as the ligand revealed, in adrenalectomized rat fat cells, a 50% decrease in the number of β-adrenergic receptors. er cell with no change in the receptor affinity for this ligand. Adrenalectomy caused no change in the binding affinity for isoproterenol of both high affinity and low affinity populations of the β-adrenergic receptors. Guanine nucleotide sensitivity of the agonist binding to β-receptors was also unaltered by adrenalectomy. Adrenalectomy caused a 30–40% decrease in the maximal response of adenylate cyclase to (?)-isoproterenol only when guanine nucleotides were present in the assay, without altering the (?)-isoproterenol concentration giving half-maximal adenylate cyclase stimulation (K_act values). The maximal response of adenylate cyclase to Gpp(NH)p also was lower in adrenalectomized membranes, indicating a defect at the guanine nucleotide regulatory site. Removal of adenosine by addition of adenosine deaminase failed to reverse the decreased adenylate cyclase response to isoproterenol in adrenalectomized rats. However, in intact fat cells, in which cyclic AMP accumulation in response to isoproterenol was decreased by adrenalectomy, removal of adenosine almost completely corrected this defect. These results indicate that the observed changes in the number of β-adrenergic receptors and in the ability of guanine nucleotides to stimulate adenylate cyclase, though explaining the decreased adenylate cyclase responsiveness to catecholamines, do probably not contribute significantly to the mechanism by which adrenalectomy decreases the lipolytic responsiveness of adipocyte to catecholamines. In addition, this study also suggests that the increased sensitivity to adenosine of lipolysis reported in adipocytes from adrenalectomized rats may result from an action of adenosine at a post-adenylate cyclase step, possibly on the cyclic AMP phosphodiesterase.     

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes.

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.     

Properties of β-adrenergic receptors in untreated and butyrate-threated HeLa cells

HeLa cells contain receptors on their surface which are β-adrenergic in nature. The binding of (?)-[³H]dihydroalprenolol is rapid, reversible, stereo-specific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (?)-isoproterenol > (?)-epinephrine > (?)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5′-yl-imidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (?)-isoproterenol and Gpp(NH)p leads to approximately additive rathen than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5 mM sodium butyrate there is an increase in the number of β-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (?)-isoproterenol to activate adenylate cyclase. Other properties of the β-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

Cholesterol modulation of β-adrenergic receptor characteristics

Cholesterol, a major structural component of plasma membranes, has a profound influence on cell surface receptor characteristics and on adenylate cyclase activity. β-Adrenergic receptor number, adenylate cyclase activity, and receptor-cyclase coupling were assessed in rat lung membranes following preincubation with cholesteryl hemisuccinate. β-Adrenergic receptor number increased by 50% without a change in antagonist affinity. However, β-adrenergic receptor affinity for isoproterenol increased 2-fold as a result of an increase in the affinity of the isoproterenol high-affinity binding site. This increase in agonist affinity did not potentiate hormone-stimulated adenylate cyclase activity, which decreased 3-fold following cholesterol incorporation. However, the ratio of isoproterenol to GTP-stimulated activity was unchanged with cholesterol. Stimulation distal to the receptor by GTP, NaF, GppNHp, Mn²⁺ and forskolin also demonstrated 50–80% reduced enzyme activity following cholesterol incorporation. These data suggest that membrane cholesterol incorporation decreases catalytic unit activity without affecting transduction of the hormone signal.     

Elevated level of beta-adrenergic receptors in hepatocytes from regenerating rat liver. Time study of [125I]iodocyanopindolol binding following partial hepatectomy and its relationship to catecholamine-sensitive adenylate cyclase

Hepatocytes from regenerating rat liver show an enhanced epinephrine-sensitive adenylate cyclase activity and cAMP response, which may be involved in triggering of the cell proliferation. We have determined adrenergic receptors and adenylate cyclase activity in hepatocytes isolated at various time points after partial hepatectomy. The number of beta-adrenergic receptors, measured by binding of [125I]iodocyanopindolol ([125I]CYP) to a particulate fraction prepared from isolated hepatocytes, increased rapidly after partial hepatectomy as compared with sham-operated or untreated controls. The maximal increase, which was observed at 48 h, was between 5- and 6-fold (from approximately 1 800 to approximately 10 500 sites per cell). Thereafter, the number of beta-adrenergic receptors decreased gradually. Competition experiments indicated beta 2-type receptors. Parallelism was found between the change in the number of beta 2-adrenergic receptors and the isoproterenol-responsive adenylate cyclase activity. The number of alpha 1-adrenergic receptors, determined by binding of [3H]prazosin, was transiently lowered by about 35% at 18-24 h, with no significant change in Kd. Although the results of this study do not exclude the possibility of post-receptor events, they suggest that the increased number of beta 2-adrenergic receptors is a major factor responsible for the enhanced catecholamine-responsive adenylate cyclase activity in regenerating liver.     

Beta-Adrenergic receptors: solubilization of (-)(3H)alprenolol binding sites from frog erythrocyte membranes.

The β-adrenergic receptors ((?)[³H]alprenolol binding sites) present in a purified preparation of frog erythrocyte membranes have been solubilized with digitonin and assayed by equilibrium dialysis with (?)[³H]alprenolol. At a concentration of 0.5–1% the detergent solubilizes about 80% of the receptor binding activity. The soluble receptor sites are not sedimented at centrifugal forces up to 105,000 xg for two hours, pass freely through Millipore filters of 0.22 μ pore size and fractionate on Sepharose 6B gel with an apparent molecular weight of 130–150,000 in the presence of digitonin. The soluble receptor sites retain all of the binding characteristics of the membrane-bound receptors. β-adrenergic agonists and antagonists compete with (?)[³H]alprenolol for occupancy of the soluble sites with affinities which are directly related to their β-adrenergic potency on membrane-bound adenylate cyclase.     

Alterations in cerebral β-adrenergic receptor-adenylate cyclase system induced by halothane,ketamine and ethanol

The effect of halothane, ketamine and ethanol on β-adrenergic receptor adenylate cyclase system was studied in the brain of rats. An anesthetic concentration of halothane and ketamine added in vitro decreased the stimulatory effect of norepinephrine on cyclic AMP formation in slices from the cerebral cortex. On the other hand, ethanol increased the basal activity of cerebral adenylate cyclase without affecting on the norepinephrine-stimulated activity. The increase of the basal activity induced by ethanol was not antagonized by propranolol, a β-adrenergic antagonist. In the crude synaptosomal (P₂) fraction, these drugs had no significant effect on the basal adenylate cyclase activity, binding of [³H]dihydroalprenolol to β-receptor, and binding of [³H]guanylylimido diphosphate ([³H]Gpp(NH)p) to guanyl nucleotide binding site. In contrast, the adenylate cyclase activity stimulated by Gpp(NH)p or NaF was significantly inhibited by an anesthetic concentration of these drugs. An anesthetic concentration of these drugs increased the membrane fluidity of P₂ fraction monitored by the fluorescence polarization technique. The addition of linoleic acid (more than 500 μM) also induced not only the increase of fluidity, but also the decrease of Gpp(NH)p- or NaF-stimulated adenylate cyclase activity in the cerebral P₂ fraction. The present results suggest that general anesthetics may interfere with the guanyl nucleotide binding regulatory protein-mediated activation of cerebral adenylate cyclase by disturbing the lipid region of synaptic membrane.     

Effects of drugs on pigeon erythrocyte membrane and asymmetric control of adenylate cyclase by the lipid bilayer

In pigeon erythrocyte membrane, the β-adrenergic receptor and the enzyme adenylate cyclase can be uncoupled in two different ways depending on the type of drug used.Cationic drugs: chlorpromazine, methochlorpromazine, tetracaine, $\text{n-}\text{octylamine}$ and a neutral alcohol, octanol, abolished alprenolol receptor binding ability and in the same range of concentration of the drug, sensitized adenylate cyclase to fluoride or Gpp(NH)p stimulation. Anionic drugs: di- and trinitrophenols, indomethacin and octanoic acid did not affect the total number of β-adrenergic receptor sites and, with the exception of trinitrophenol, did not change the association constant for alprenolol but they abolished the stimulation of adenylate cyclase by isoproterenol, fluoride or Gpp(NH)p. These modifications of the adenylate cyclase system occurred in a range of drug concentration where cell shape and protection against hemolysis were also affected.As chemical composition varies widely from one drug to another, it is suggested that these effects are largely nonspecific and mediated by the lipid bilayer. They are probably related to a preferential sidedness of action of the drugs in the lipid bilayer, displaying the role of an asymmetric control of the adenylate cyclase system in the membrane by the two halves of this bilayer.     

Stimulation of mammalian adenylate cyclase activity with guanosine 5′-tetraphosphate and other guanine nucleotides

Guanosine 5′-tetraphosphate (GTP₄) stimulated mammalian adenylate cyclase activity at concentrations down to 1 μM. Greater stimulatory activity was apparent with lung than with heart, brain or liver from the rat. At a concentration of 0.1 mM, GTP₄ stimulated lung adenylate cyclase activity from rat, guinea pig and mouse about four-fold. Other guanine nucleotides such as GTP, GDP, GMP, guanosine 3′, 5′-monophosphate and 5′-guanylylimidodiphosphate (GMP · PNP) also stimulated mammalian adenylate cyclase activity. GMP · PNP irreversibly activated, whereas GTP₄ and GTP reversibly activated adenylate cyclase. Adenosine 5′-tetraphosphate (ATP₄) stimulated rat lung and liver but inhibited rat heart and brain adenylate cyclase activities. Lung from guinea pig and mouse were not affected by ATP₄. The formation of cyclic AMP by GTP₄-stimulated rat lung adenylate cyclase was verified by Dowex-50 (H⁺), Dowex 1-formate and polyethyleneimine cellulose column chromatography. GTP₄ was at least three times more potent than 1-isoproterenol in stimulating rat lung adenylate cyclase activity. The β-adrenergic receptor antagonist propranolol blocked the effect of 1-isoproterenol but not that of GTP₄, thus, suggesting that GTP₄ and β-adrenergic agonists interact with different receptor sites on membrane-bound adenylate cyclase. Stimulation of rat lung and liver adenylate cyclase activities with 1-isoproterenol was potentiated by either GTP₄ or GMP. PNP, thus indicating that GTP₄ resembles other guanine nucleotides in their capacity to increase the sensitivity of adenylate cyclase to β-adrenergic agonists. Stimulation of adenylate cyclase activity by guanine derivatives requires one or more free phosphate moieties on the 5 position of ribose, as no effect was elicited with guanine, guanosine, guanosine 2′-monophosphate, guanosine 3′-monophosphate or guanosine 2′,5′-monophosphate. Ribose, ribose 5-phosphate, phosphate and pyrophosphate were inactive. Pyrimidine nucleoside mono-, di-, tri- and tetraphosphates elicited negligible effects on mammalian adenylate cyclase activity.     

"Antibodies raised against beta-adrenergic receptors stimulate adenylate cyclase"

Antibodies raised in mice against β-adrenergic receptors purified from turkey erythrocyte membranes, specifically bind to cells which possess a β-adrenergic receptor and immunoprecipitate radiolabelled purified receptor. These antibodies stimulate the adenylate cyclase activity of the turkey erythrocytes, although they do not compete with the catecholamine hormones for binding to the β-adrenergic receptor. Thus the receptor-antibody interaction, although occuring at another site than the receptor-hormone interaction, may still trigger the enzymatic activity.     

Uncoupling of alpha-adrenoceptor-mediated inhibition of human platelet adenylate cyclase by N-ethylmaleimide

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and is inhibited by epinephrine via alpha-adrenoceptors. Both agonists, epinephrine more than PGE1, increase the activity of a low Km GTPase in platelet membranes. Pretreatment of intact platelets or platelet membranes with the sulfhydryl reagent, N-ethylmaleimide (NEM), abolished the inhibition of the adenylate cyclase and the concomitant stimulation of the GTPase by epinephrine. In contrast, stimulation of the adenylate cyclase by PGE1 was not affected or even increased by NEM pretreatment; only at high NEM concentrations were both basal and PGE1-stimulated activities decreased. Similarly, the PGE1-induced activation of the low Km GTPase was not or was only partially reduced by NEM. Adenylate cyclase activation by stable GTP analogs, NaF, and cholera toxin was also not decreased by NEM pretreatment. Exposure of intact platelets to NEM did not reduce alpha-adrenoceptor number and affinities for agonists and antagonists, as determined by [3H]yohimbine binding in platelet particles. The data indicate that NEM uncouples alpha-adrenoceptor-mediated inhibition of platelet adenylate cyclase, leaving the receptor recognition site and the adenylate cyclase itself relatively intact. Although the effect of NEM may be based on a reaction with the alpha-adrenoceptor site interacting with a coupling component, the selective loss of the adenylate cyclase inhibition together with an even increased stimulation of the enzyme by PGE1 suggests that there are two at least partially distinct regulatory sites involved in opposing hormonal regulations of adenylate cyclase activity, with that involved in hormonal inhibition being highly susceptible to inactivation by NEM.     

Structure and function of cholera toxin and hormone receptors

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active ¹²⁵I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside G_MI. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyme to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the “active” subunit of the toxin can specifically adsorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergentsolubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct pertubation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.     

In Vivo Partial Inactivation of Dopamine D1 Receptors Induces Hypersensitivity of Cortical Dopamine-Sensitive Adenylate Cyclase: Permissive Role of α1-Adrenergic Receptors

As shown by autoradiography, peripheral injections of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a dose-dependent decrease of [3H]SCH 23390 and [3H]prazosin high-affinity binding sites in the rat prefrontal cortex. EEDQ showed similar efficacy in inactivating cortical and striatal dopamine (DA) D1 receptors, whereas prazosin-sensitive alpha 1-adrenergic receptors were more sensitive to the action of the alkylating agent, as for all doses of EEDQ tested (from 0.8 to 3 mg/kg, i.p.), the decrease in cortical [3H]SCH 23390 binding was less pronounced than that of [3H]prazosin. The effects of EEDQ on [3H]SCH 23390 binding and DA-sensitive adenylate cyclase activity were then simultaneously compared in individual rats. In the striatum, whatever the dose of EEDQ used, the decrease of DA-sensitive adenylate cyclase activity was always lower than that of D1 binding sites, suggesting the occurrence of a large proportion of spare D1 receptors. In the prefrontal cortex, a significant increase in DA-sensitive adenylate cyclase activity was observed in rats treated with a low dose of EEDQ (0.8 mg/kg), this effect being associated with a slight reduction in [3H]SCH 23390 binding sites (-20%). Parallel decreases in the enzyme activity and D1 binding sites were observed with higher doses. The EEDQ-induced supersensitivity of DA-sensitive adenylate cyclase did not occur in rats in which the decrease in [3H]prazosin binding sites was higher than 35%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exfoliation of the β-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed ‘exosomes’ into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, G_s, and the inhibitory, G_i, regulatory components) and β-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of G_s or G_i in the exosomes was detected by ADP ribosylation using [α-³²P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal G_s was almost as active as the membrane-derived G_s in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc⁻ cells, which lack a functional G_s. The ability of exosomal G_s to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane G_s. The density of β-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated β-adrenergic receptors seem to be impaired in their ability to couple to and activate G_s. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the β-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.