The PI3K-mediated activation of CRAC independently regulates adenylyl cyclase activation and chemotaxis

The ability of a cell to detect an external chemical signal and initiate a program of directed migration along a gradient comprises the fundamental process called chemotaxis. Investigations in Dictyostelium discoideum and neutrophils have established that pleckstrin homology (PH) domain-containing proteins that bind to the PI3K products PI(3,4)P2 and PI(3,4,5)P3, such as CRAC (cytosolic regulator of adenylyl cyclase) and Akt/PKB, translocate specifically to the leading edge of chemotaxing cells. CRAC is essential for the chemoattractant-mediated activation of the adenylyl cyclase ACA, which converts ATP into cAMP, the primary chemoattractant for D. discoideum. The mechanisms by which CRAC activates ACA remain to be determined. We now show that in addition to its essential role in the activation of ACA, CRAC is involved in regulating chemotaxis. Through mutagenesis, we show that these two functions are independently regulated downstream of PI3K. A CRAC mutant that has lost the capacity to bind PI3K products does not support chemotaxis and shows minimal ACA activation. Finally, overexpression of CRAC and various CRAC mutants show strong effects on ACA activation with little effect on chemotaxis. These findings establish that chemoattractant-mediated activation of PI3K is important for the CRAC-dependent regulation of both chemotaxis and adenylyl cyclase activation.     

G-protein oncogenes in acromegaly.

G-proteins belong to a family of proteins which share the common properties of GTP binding and hydrolysis. Heterotrimeric G-proteins are composed of alpha-, beta- and gamma-subunits. The alpha-subunit which differs from one G-protein to another contains the GDP/GTP binding site and has intrinsic GTPase activity. The receptor occupancy causes displacement of bound GDP by GTP, dissociation of free beta gamma-dimer and alpha-GTP complex, interaction of the activated alpha-GTP complex with intracellular effectors, such as enzymes and ion channels. The turn off of the reaction is due to the GTPase activity which causes the hydrolysis of GTP to GDP. G-proteins are essential for transferring hormonal signals from cell surface receptors to intracellular effectors. Since G-proteins generate intracellular effectors involved in cell growth, G-protein genes have the propensity to be converted into oncogenes. In fact, mutations in the alpha-subunit of Gs (the G-protein involved in the activation of adenylyl cyclase) have been demonstrated in 40% of human GH secreting pituitary adenomas. Single amino acid substitutions replacing Arg 201 with either Cys or His or Gln 227 with either Arg or Leu cause constitutive activation of adenylyl cyclase by inhibiting GTPase (gsp oncogene). The same mutations were identified in about 10% of thyroid adenomas and in the McCune-Albright syndrome.     

Examining the efficiency of receptor/G-protein coupling with a cleavable beta2-adrenoceptor-gsalpha fusion protein.

Reconstitution of high-affinity agonist binding at the beta2-adrenoceptor (beta2AR) expressed in Sf9 insect cells requires a large excess of the stimulatory G-protein of adenylyl cyclase, Gsalpha, relative to receptor [R. Seifert, T. W. Lee, V. T. Lam & B. K. Kobilka, (1998) Eur. J. Biochem. 255, 369-382]. In a fusion protein of the beta2AR and Gsalpha (beta2AR-Gsalpha), which has only a 1 : 1 stoichiometry of receptor and G-protein, high-affinity agonist binding and agonist-stimulated GTP hydrolysis, guanosine 5'-O-(3-thiotriphosphate) (GTP[S]) binding and adenylyl cyclase (AC) activation are more efficient than in the nonfused coexpression system. In order to analyze the stability of the receptor/G-protein interaction, we constructed a fusion protein with a thrombin-cleavage site between beta2AR and Gsalpha (beta2AR-TS-Gsalpha). beta2AR-TS-Gsalpha efficiently reconstituted high-affinity agonist binding, agonist-stimulated GTP hydrolysis, GTP[S] binding and AC activation. Thrombin cleaves approximately 70% of beta2AR-TS-Gsalpha molecules in Sf9 membranes. Thrombin cleavage did not impair high-affinity agonist binding and GTP[S] binding but strongly reduced ligand-regulated GTPase activity and AC activity. We conclude that fusion of the beta2AR to Gsalpha promotes tight physical association of the two partners and that this association remains stable for a single activation/deactivation cycle even after cleavage of the link between the receptor and G-protein. Dilution of Gsalpha in the membrane and release of activated Gsalpha into the cytosol can both prevent cleaved beta2AR-TS-Gsalpha from undergoing multiple activation/deactivation cycles.     

The GppNHp-activated adenylyl cyclase complex from turkey erythrocyte membranes can be isolated with its beta gamma subunits.

The adenylyl cyclase complex, derived from turkey erythrocyte membranes, was activated using guanosine 5'-[beta, gamma-imido]triphosphate (Gpp[NH]p) and separated under low-detergent and low-salt conditions using conventional molecular-sieve chromatography followed by high-pressure ion-exchange and molecular-sieve chromatography. Although the complex remains activated with Gpp[NH]p throughout the isolation, the beta gamma subunits copurify with the cyclase. The stoichiometry of the cyclase to the alpha subunit of the stimulatory guanosine-nucleotide-binding regulatory protein (alpha s) to the beta subunit is close to unity, demonstrating that the beta gamma subunits do not dissociate from the Gs.cyclase complex (Gs, guanosine-nucleotide-binding regulatory protein) upon activation of the enzyme. If the final purification step was performed at high-salt concentrations, the beta gamma subunits could be separated from the alpha s.cyclase complex. Previously reported results on bovine brain cyclase also showed that the Gs.cyclase complex remains intact subsequent to activation by hormone and Gpp[NH]p [Marbach, I., Bar-Sinai, A., Minich, M. and Levitzki, A. (1990) J. Biol. Chem. 265, 9999-10,004]. These results, using adenylyl cyclase from two different sources, support our previous kinetic experiments which first suggested that beta gamma subunits are not released from Gs upon cyclase activation. We, therefore, argue that the mode of adenylyl cyclase inhibition by the inhibitory guanosine-nucleotide-binding regulatory protein cannot be via shifting the alpha s to beta gamma equilibrium as is commonly believed, and an alternate hypothesis is proposed.     

A MAP kinase necessary for receptor-mediated activation of adenylyl cyclase in Dictyostelium

Analysis of a developmental mutant in Dictyostelium discoideum which is unable to initiate morphogenesis has shown that a protein kinase of the MAP kinase/ERK family affects relay of the cAMP chemotactic signal and cell differentiation. Strains in which the locus encoding ERK2 is disrupted respond to a pulse of cAMP by synthesizing cGMP normally but show little synthesis of cAMP. Since mutant cells lacking ERK2 contain normal levels of both the cytosolic regulator of adenylyl cyclase (CRAC) and manganese-activatable adenylyl cyclase, it appears that this kinase is important for receptor-mediated activation of adenylyl cyclase.     

Diabetes reduces right atrial beta-adrenergic signaling but not agonist stimulation of heart rate in swine

This study assessed the effects of streptozotocin diabetes in swine on the heart rate response to beta-adrenergic stimulation the adenylyl cyclase signal transduction pathway. Diabetic animals (n = 9) were hyperglycemic compared to the control group (n = 10) (12.6 +/- 1.0 vs. 3.53 +/- 0.29 mM). There were no significant differences between the diabetic and nondiabetic groups in the heart rate response to isoproterenol, however, there was a significant reduction (14%) in beta-adrenergic receptor density in the right atrium in the diabetic (61 +/- 3 fmol/mg protein) versus the nondiabetic group (71 +/- 3) (P < 0.05). The content of guanosine triphosphate binding regulatory proteins (Gs and Gi) in the right atrium was not affected by diabetes, nor was adenylyl cyclase activity under unstimulated conditions or with receptor-dependent stimulation with isoproterenol. On the other hand, adenylyl cyclase activity was 34% lower when directly stimulated with forskolin, and it was reduced by 23% when stimulated through Gs with Gpp(NH)p. In conclusion, beta-adrenergic stimulation of heart rate with isoproteronol and the receptor-dependent signal transduction pathway remained intact in the right atrium of diabetic swine despite reduced beta-adrenergic receptor density, G-protein content, and direct stimulation of adenylyl cyclase activity.     

The G protein beta subunit is essential for multiple responses to chemoattractants in Dictyostelium

Increasing evidence suggests that the beta gamma-subunit dimers of heterotrimeric G proteins play a pivotal role in transducing extracellular signals. The recent construction of G beta null mutants (g beta-) in Dictyostelium provides a unique opportunity to study the role of beta gamma dimers in signaling processes mediated by chemoattractant receptors. We have shown previously that g beta- cells fail to aggregate; in this study, we report the detailed characterization of these cells. The g beta- cells display normal motility but do not move towards chemattractants. The typical GTP- regulated high affinity chemoattractant-binding sites are lost in g beta- cells and membranes. The g beta- cells do not display chemoattractant-stimulated adenylyl cyclase or guanylyl cyclase activity. These results show that in vivo G beta links chemoattractant receptors to effectors and is therefore essential in many chemoattractant-mediated processes. In addition, we find that G beta is required for GTP gamma S stimulation of adenylyl cyclase activity, suggesting that the beta gamma-dimer activates the enzyme directly. Interestingly, the g beta- cells grow at the same rate as wild-type cells in axenic medium but grow more slowly on bacterial lawns and, therefore, may be defective in phagocytosis.     

Chemoattractant-mediated Rap1 activation requires GPCR/G proteins

Rap1 is rapidly activated upon chemoattractant stimulation and plays an important role in cell adhesion and cytoskeletal reorganization during chemotaxis. Here, we demonstrate that G-protein coupled receptors and G-proteins are essential for chemoattractant-mediated Rap1 activation in Dictyostelium. The rapid Rap1 activation upon cAMP chemoattractant stimulation was absent in cells lacking chemoattractant cAMP receptors cAR1/cAR3 or a subunit of the heterotrimeric G-protein complex Gα2. Loss of guanylyl cyclases GCA/SGC or a cGMP-binding protein GbpC exhibited no effect on Rap1 activation kinetics. These results suggest that Rap1, a key regulator for the regulation of cytoskeletal reorganization during cell movement, is activated through the G-protein coupled receptors cAR1/cAR3 and Gα2 proteins in a way independent of the cGMP signaling pathway.     

Beta subunit copurifies with GppNHp-activated adenylyl cyclase

Previous kinetic studies (Tolkovsky, A.M., Braun, S., and Levitzki, A. (1982) Proc. Natl. Acad. Sci. U. S.A. 79, 213-222) and biochemical studies (Arad, H., Rosenbusch, J., and Levitzki, A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6579-6583) from our laboratory suggest that Gs or alpha s remain associated with the catalytic subunit of adenylyl cyclase (C) throughout the activation cycle of adenylyl cyclase by hormone receptors. In this study we have purified GppNHp-activated bovine brain adenylyl cyclase over 3000-fold under mild solution conditions. We demonstrate that although the enzyme is permanently activated it retains the beta subunit when bound to a forskolin-agarose affinity column as long as it is not exposed to high salt concentrations. The stoichiometry of alpha s to beta to C is close to unity, suggesting that beta gamma subunits do not dissociate from Gs upon its activation. The complex gamma beta alpha s (GppNHp). C dissociates partially when migrating on a Superose 12 fast protein liquid chromatography molecular-seiving column. This partial dissociation probably results from the relatively diluted state of the enzyme at a high degree of purity. Prolonged ultracentrifugation of the complex also causes partial dissociation of the beta gamma subunits from alpha s (GppNHp). C. The apparent contradiction between the results reported here and the observation that beta gamma subunits inhibit cyclase activity when added to platelet membranes (Katada, T., Bokoch, G. M., Northrup, J. K., Ui, M., and Gilman, A. G. (1984a) J. Biol. Chem. 259, 3568-3577) is discussed. We suggest an alternative model to account for this inhibitory effect of added beta gamma subunits.     

Participation of Tubulin in the Stimulatory Regulation of Adenylyl Cyclase in Rat Cerebral Cortex Membranes

Abstract: This study examined effects of tubulin on the activation of adenylyl cyclase in rat cerebral cortex membranes. Tubulin, prepared from rat brain by polymerization with the hydrolysis-resistant GTP analogue 5'-guanylylimidodiphosphate (GppNHp) caused significant activation of the enzyme by ∼156% under conditions in which stimulation rather than inhibition of the enzyme was favored. Tubulin-GppNHp activated isoproterenol-sensitive adenylyl cyclase, potentiated forskolin-stimulated activity of the enzyme, and reduced agonist binding affinity for β-adrenergic receptors. When tubulin, polymerized with the hydrolysis-resistant photoaffinity GTP analogue [³²P] P ³(4-azidoanilido)- P ¹-5'-GTP ([³²P]AAGTP), was incubated with cerebral cortex membranes, AAGTP was transferred from tubulin to G_sα as well as G_iα. These results suggest that, in rat cerebral cortex membranes, the tubulin dimer participates in the stimulatory regulation of adenylyl cyclase by transferring guanine nucleotide to G_sα, as well as affecting the G_i-mediated inhibitory pathway.     

Spatial expression patterns of genes involved in cyclic AMP responses in Dictyostelium discoideum development

The spatial expression patterns of genes involved in cyclic adenosine monophosphate (cAMP) responses during morphogenesis in Dictyostelium discoideum were analyzed by in situ hybridization. Genes encoding adenylyl cyclase A (ACA), cAMP receptor 1, G-protein alpha2 and beta subunits, cytosolic activator of ACA (CRAC and Aimless), catalytic subunit of protein kinase A (PKA-C) and cAMP phosphodiesterases (PDE and REG-A) were preferentially expressed in the anterior prestalk (tip) region of slugs, which acts as an organizing center. MAP kinase ERK2 (extracellular signal-regulated kinase-2) mRNA, however, was enriched in the posterior prespore region. At the culmination stage, the expression of ACA, CRAC and PKA-C mRNA increased in prespore cells in contrast with the previous stage. However, no alteration in the site of expression was observed for the other mRNA analyzed. Based on these findings, two and four classes of expression patterns were catalogued for these genes during the slug and culmination stages, respectively. Promoter analyses of genes in particular classes should enhance understanding of the regulation of dynamic and coordinated gene expression during morphogenesis.     

RACK1 binds to a signal transfer region of G betagamma and inhibits phospholipase C beta2 activation

Receptor for Activated C Kinase 1 (RACK1), a novel G betagamma-interacting protein, selectively inhibits the activation of a subclass of G betagamma effectors such as phospholipase C beta2 (PLCbeta2) and adenylyl cyclase II by direct binding to G betagamma (Chen, S., Dell, E. J., Lin, F., Sai, J., and Hamm, H. E. (2004) J. Biol. Chem. 279, 17861-17868). Here we have mapped the RACK1 binding sites on G betagamma. We found that RACK1 interacts with several different G betagamma isoforms, including G beta1gamma1, Gbeta1gamma2, and Gbeta5gamma2, with similar affinities, suggesting that the conserved residues between G beta1 and G beta5 may be involved in their binding to RACK1. We have confirmed this hypothesis and shown that several synthetic peptides corresponding to the conserved residues can inhibit the RACK1/G betagamma interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of G beta1 and have little overlap with the G alpha subunit binding interface. Additional experiments indicate that the G betagamma contact residues for RACK1, in particular the positively charged amino acids within residues 44-54 of G beta1, are also involved in the interaction with PLCbeta2 and play a critical role in G betagamma-mediated PLCbeta2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G betagamma effector by competing for its binding to the signal transfer region of G betagamma.     

Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters

Human platelets, prelabeled with [32P]phosphate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37 degrees C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G-proteins and the catalytic moiety. Less than 0.01 mol of [32P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the alpha-subunit of the GTP binding protein Gi. TPA, however, caused the incorporation of 0.67-1.1 mol of [32P]phosphate per mol of catalyst while 0.13-0.2 mol were found in the absence of TPA. Lack of modification of the alpha-subunit of Gi was also indicated by the results of reconstitution experiments with purified Gi alpha from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added G1 alpha, than that from TPA treated cells. While beta, gamma-subunits were like-wise inhibitory no difference dependent on platelet-pretreatment could be observed.     

Characterization of the extra-large G protein alpha-subunit XLalphas. II. Signal transduction properties

In the preceding paper (Pasolli, H. A., Klemke, M., Kehlenbach, R. H. , Wang, Y., and Huttner, W. B. (2000) J. Biol. Chem. 275, 33622-33632), we report on the tissue distribution and subcellular localization of XLalphas (extra large alphas), a neuroendocrine-specific, plasma membrane-associated protein consisting of a novel 37-kDa XL domain followed by a 41-kDa alphas domain encoded by exons 2-13 of the Galphas gene. Here, we have studied the signal transduction properties of XLalphas. Like Galphas, XLalphas undergoes a conformational change upon binding of GTPgammaS (guanosine 5'-O-(thio)triphosphate), as revealed by its partial resistance to tryptic digestion, which generated the same fragments as in the case of Galphas. Two approaches were used to analyze XLalphas-betagamma interactions: (i) ADP-ribosylation by cholera toxin to detect even weak or transient XLalphas-betagamma interactions and (ii) sucrose density gradient centrifugation to reveal stable heterotrimer formation. The addition of betagamma subunits resulted in an increased ADP-ribosylation of XLalphas as well as an increased sedimentation rate of XLalphas in sucrose density gradients, indicating that XLalphas interacts with the betagamma dimer. Surprisingly, however, XLalphas, in contrast to Galphas, was not activated by the beta2-adrenergic receptor upon reconstitution of S49cyc(-) membranes. Similarly, using photoaffinity labeling of pituitary membranes with azidoanilide-GTP, XLalphas was not activated upon stimulation of pituitary adenylyl cyclase-activating polypeptide (PACAP) receptors or other Galphas-coupled receptors known to be present in these membranes, whereas Galphas was. Despite the apparent inability of XLalphas to undergo receptor-mediated activation, XLalphas-GTPgammaS markedly stimulated adenylyl cyclase in S49cyc(-) membranes. Moreover, transfection of PC12 cells with a GTPase-deficient mutant of XLalphas, XLalphas-Q548L, resulted in a massive increase in adenylyl cyclase activity. Our results suggest that in neuroendocrine cells, the two related G proteins, Galphas and XLalphas, exhibit distinct properties with regard to receptor-mediated activation but converge onto the same effector system, adenylyl cyclase.     

Modulation of ion channel function by P2Y receptors

P2Y receptors are classified as P2 purinergic receptors that belong to the superfamily of G-protein coupled receptors. They are distinguishable from P1 (adenosine) receptors in that they bind adenine and/or uracil nucleotide triphosphates or diphosphates depending on the subtype. Over the past decade, P2Y receptors have been cloned from a variety of tissues and species. Eight functional subtypes have been characterized. Nucleotide binding produces activation of specific G-proteins that in turn regulate the function of membrane bound enzymes including phospholipase C and adenylyl cyclase. Certain P2Y receptor subtypes possess a PDZ domain located at the end of the C-terminal region of the receptor. PDZ domains have been established as sites for protein-protein interaction, thus providing a possible mechanism for receptor modulation of membrane protein function independent of G-protein activation. In this review we discuss recent findings that suggest that P2Y receptors can modulate the function of ion channels through multiple protein-protein interactions at the plasma membrane that do not directly involve G-protein activation.     

The regulation of adenylate cyclase by guanine nucleotides in Dictyostelium discoideum membranes

Extracellular cAMP induces the activation of adenylate cyclase in Dictyostelium discoideum cells. Conditions for both stimulation and inhibition of adenylate cyclase by guanine nucleotides in membranes are reported. Stimulation and inhibition were induced by GTP and non-hydrolysable guanosine triphosphates. GDP and non-hydrolysable guanosine diphosphates were antagonists. Stimulation was maximally twofold, required a cytosolic factor and was observed only at temperatures below 10 degrees C. An agonist of the cAMP-receptor-activated basal and GTP-stimulated adenylate cyclase 1.3-fold. Adenylate cyclase in mutant N7 could not be activated by cAMP in vivo; in vitro adenylate cyclase was activated by guanine nucleotides in the presence of the cytosolic factor of wild-type but of not mutant cells. Preincubation of membranes under phosphorylation conditions has been shown to alter the interaction between cAMP receptor and G protein [Van Haastert (1986) J. Biol. Chem. in the press]. These phosphorylation conditions converted stimulation to inhibition of adenylate cyclase by guanine nucleotides. Inhibition was maximally 30% and was not affected by the cytosolic factor involved in stimulation. In membranes obtained from cells that were treated with pertussis toxin, adenylate cyclase stimulation by guanine nucleotides was as in control cells, whereas inhibition by guanine nucleotides was lost. When cells were desensitized by exposure to cAMP agonists for 15 min, and adenylate cyclase was measured in isolated membranes, stimulation by guanine nucleotides was lost while inhibition was retained. These results suggest that Dictyostelium discoideum adenylate cyclase may be regulated by Gs-like and Gi-like activities, and that the action of Gs but not Gi is lost during desensitization in vivo and by phosphorylation conditions in vitro.     

GTPgammaS regulation of a 12-transmembrane guanylyl cyclase is retained after mutation to an adenylyl cyclase

DdGCA is a Dictyostelium guanylyl cyclase with a topology typical for mammalian adenylyl cyclases containing 12 transmembrane-spanning regions and two cyclase domain. In Dictyostelium cells heterotrimeric G-proteins are essential for guanylyl cyclase activation by extracellular cAMP. In lysates, guanylyl cyclase activity is strongly stimulated by guanosine 5'-3-O-(thio) triphosphate (GTPgammaS), which is also a substrate of the enzyme. DdGCA was converted to an adenylyl cyclase by introducing three point mutations. Expression of the obtained DdGCA(kqd) in adenylyl cyclase-defective cells restored the phenotype of the mutant. GTPgammaS stimulated the adenylyl cyclase activity of DdGCA(kqd) with properties similar to those of the wild-type enzyme (decrease of K(m) and increase of V(max)), demonstrating that GTPgammaS stimulation is independent of substrate specificity. Furthermore, GTPgammaS activation of DdGCA(kqd) is retained in several null mutants of Galpha and Gbeta proteins, indicating that GTPgammaS activation is not mediated by a heterotrimeric G-protein but possibly by a monomeric G-protein.     

Down-regulation of cell surface cyclic AMP receptors and desensitization of cyclic AMP-stimulated adenylate cyclase by cyclic AMP in Dictyostelium discoideum. Kinetics and concentration dependence

cAMP binds to Dictyostelium discoideum surface receptors and induces a transient activation of adenylatecyclase, which is followed by desensitization. cAMP also induces a loss of detectable surface receptors (down-regulation). Cells were incubated with constant cAMP concentrations, washed free of cAMP, and cAMP binding to surface receptors and cAMP-induced activation of adenylate cyclase were measured. cAMP could induce maximally 65% loss of binding activity and complete desensitization of cAMP-stimulated adenylate cyclase activity. Half-maximal effects for down-regulation were observed at 50 nM cAMP and for desensitization at 5 nM cAMP. Down-regulation was rapid with half-times of 4, 2.5, and 1 min at 0.1, 1, and 10 microM cAMP, respectively. Similar kinetic data have been reported for desensitization (Dinauer, M.C., Steck, T.L., and Devreotes, P.N. (1980) J. Cell Biol. 86, 554-561). Down-regulation and desensitization were not reversible at 0 degrees C. Down-regulation reversed slowly at 20 degrees C with a half-time of about 1 h. Resensitization of adenylate cyclase was biphasic showing half-times of 4 min and about 1 h, respectively; the contribution of the rapidly resensitizing component was diminished when down-regulation of receptors was enhanced. These results suggest that cAMP-induced down-regulation of receptors and desensitization of adenylate cyclase stimulation proceed by at least two steps. One step is rapidly reversible, occurs at low cAMP concentrations, and induces desensitization without down-regulation, while the second step is slowly reversible, requires higher cAMP concentrations, and also induces down-regulation.     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.