Ring-deleted analogs of atrial natriuretic factor inhibit adenylate cyclase/cAMP system. Possible coupling of clearance atrial natriuretic factor receptors to adenylate cyclase/cAMP signal transduction system

We have recently shown that atrial natriuretic factor (ANF) inhibits adenylate cyclase activity in rat platelets where only one population of ANF receptors (ANF-R2) is present, indicating that ANF-R2 receptors may be coupled to the adenylate cyclase/cAMP system. In the present studies, we have used ring-deleted peptides which have been reported to interact with ANF-R2 receptors also called clearance receptors (C-ANF) without affecting the guanylate cyclase/cGMP system, to examine if these peptides can also inhibit the adenylate cyclase/cAMP system. Ring-deleted analog C-ANF4-23 like ANF99-126 inhibited the adenylate cyclase activity in a concentration-dependent manner in rat aorta, brain striatum, anterior pituitary, and adrenal cortical membranes. The maximal inhibition was about 50-60% with an apparent Ki between 0.1 and 1 nM. In addition, C-ANF4-23 also decreased the cAMP levels in vascular smooth muscle cells in a concentration-dependent manner without affecting the cGMP levels. The maximal decrease observed was about 60% with an apparent Ki of about 1 nM. Furthermore, C-ANF4-23 was also able to inhibit cAMP levels and progesterone secretion stimulated by luteinizing hormone in MA-10 cell line. Other smaller fragments of ANF with ring deletions were also able to inhibit the adenylate cyclase activity as well as cAMP levels. Furthermore, the stimulatory effects of various agonists such as 5'-(N-ethyl)carboxamidoadenosine, dopamine, and forskolin on adenylate cyclase activity and cAMP levels were also significantly inhibited by C-ANF4-23. The inhibitory effect of C-ANF4-23 on adenylate cyclase was dependent on the presence of GTP and was attenuated by pertussis toxin treatment. These results indicate that ANF-R2 receptors or so-called C-ANF receptors are coupled to the adenylate cyclase/cAMP signal transduction system through inhibitory guanine nucleotide regulatory protein.     

Adenylate Cyclase of Torpedo Synaptosomes Is Inhibited by Calcium and Not Affected by Muscarinic Ligands

Abstract: Cholinergic synaptosomes isolated from the electric organ of Torpedo contain membrane-bound adenylate cyclase activity (∼6 pmol/mg proteidmin), which is dependent on the presence of guanine nucleotides. The activity is strongly dependent on temperature and only slightly affected by NaCl. The Torpedo adenylate cyclase is completely inhibited by low levels of free Ca²⁺ (K₀∼ 0.5 μ M ). This effect is not altered by either trifluoperazine or addition of exogenous calmodulin. Ca³⁺ has no effect on the activation step of the adenylate cyclase by guanyl-5'-yl imidodiphosphate (GppNHp), and Mn²⁺ abolishes the Ca²⁺-dependent inhibition of cyclic AMP synthesis. These findings suggest that Ca²⁺ exerts its effect by direct interaction with a site located on the catalytic subunit. Torpedo synaptosomes contain presynaptic inhibitory muscarinic receptors. The binding of muscarinic agonists to the receptors is modulated (to lower affinity) by GTP. However, muscarinic ligands, examined under a variety of assay conditions, have no effect on adenylate cyclase activity. These results suggest that although both the muscarinic receptor and the adenylate cyclase are coupled to G proteins, they either interact with different G proteins or are situated in different regions of the presynaptic membrane.     

Multistep regulation of Leydig cell function

LH controls Leydig cell steroidogenesis by interaction with specific membrane receptors initiating membrane coupling events. Stimulation of the androgen pathways occurs mainly through cAMP mediated mechanism including LH induced guanyl nucleotide binding, membrane phosphorylation and adenylate cyclase activation. cAMP dependent kinase activation presumably causes phosphorylation of key proteins of the steroidogenic pathway and consequent increase in testosterone production. The hormone also appears to facilitate the androgen stimulus by a cyclic AMP independent mechanism located at the plasma membrane or intracellular sites. The stimulatory event can be negatively influenced by the action of certain peptide hormones (i.e. angiotensin II) through the guanyl nucleotide inhibitory subunit of adenylate cyclase (Gi). In recent studies we have presented evidence for a Ca2+ sensitive kinase system present in purified cell membranes. Gpp(NH)p, GTP, and phospholipid in presence of nanomolar Ca2+ induce phosphate incorporation into Mr 44,500 substrate with marked inhibition at microM Ca2+. Similarly a biphasic pattern of activation was observed with adenylate cyclase activity. Membrane phosphorylation may be a modifier of LH-stimulated adenylate cyclase activity and possibly other LH induced actions in the activated Leydig cell membrane. Furthermore we have defined the stimulatory effects of forskolin on all Leydig cell cyclic AMP pools and have provided additional evidence of functional compartmentalization and/or cAMP independent facilitory stimulus of steroidogenesis by the trophic hormone. The demonstration of a novel high affinity inhibitory action of forskolin upon adenylate cyclase activity and cyclic AMP generation mediated by the Gi subunit of adenylate cyclase has provided a new approach for direct evaluation of functional inhibitory influence of Gi subunit in the Leydig cell. The cultured fetal Leydig cell system has provided a useful model to elucidate mechanisms involved in the development of gonadotropin induced estradiol mediated desensitization of steroidogenesis. We have isolated from the fetal testis a small population (2-5% of total) of transitional cells with morphological characteristics of cells found in 15 day postnatal testis but functional capabilities of the adult cell. We have also demonstrated after appropriate treatment (i.e. estrogen, and frequent or a high gonadotropin dose) the emergence of a functional adult-like cell type from the fetal Leydig cell population.     

Regulation of adenylate cyclase in electropermeabilized Dictyostelium discoideum cells.

In Dictyostelium discoideum cells the enzyme adenylate cyclase is functionally coupled to cell surface receptors for cAMP. Coupling is known to involve one or more G-proteins. Receptor-mediated activation of adenylate cyclase is subject to adaptation. In this study we employ an electropermeabilized cell system to investigate regulation of D. discoideum adenylate cyclase. Conditions for selective permeabilization of the plasma membrane have been described by C.D. Schoen, J. C. Arents, T. Bruin, and R. Van Driel (1989, Exp. Cell Res. 181, 51-62). Only small pores are created in the membrane, allowing exchange of exclusively low molecular weight substances like nucleotides, and preventing the loss of macromolecules. Under these conditions functional protein-protein interactions are likely to remain intact. Adenylate cyclase in permeabilized cells was activated by the cAMP receptor agonist 2'-deoxy cAMP and by the nonhydrolyzable GTP-analogue GTP gamma S, which activates G-proteins. The time course of the adenylate cyclase reaction in permeabilized cells was similar to that of intact cells. Maximal adenylate cyclase activity was observed if cAMP receptor agonist or GTP-analogue was added just before cell permeabilization. If these activators were added after permeabilization adenylate cyclase was stimulated in a suboptimal way. The sensitivity of adenylate cyclase activity for receptor occupation was found to decay more rapidly than that for G-protein activation. Importantly, the adenylate cyclase reaction in permeabilized cells was subject to an adaptation-like process that was characterized by a time course similar to adaptation in vivo. In vitro adaptation was not affected by cAMP receptor agonists or by G-protein activation. Evidently electropermeabilized cells constitute an excellent system for investigating the positive and negative regulation of D. discoideum adenylate cyclase.     

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.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and guanine nucleotide-dependent hormonal inhibition

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta (35,000 Da) subunits are functionally indistinguishable. Gi and Gs both dissociate in the presence of guanine nucleotide analogs or Al3+, Mg2+, and F- in detergent-containing solutions. Several characteristics of Gi- and Gs-mediated regulation of adenylate cyclase activity have been studied in human platelet membranes. The nonhydrolyzable analog of GTP, guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) mimics GTP-dependent hormonal inhibition or stimulation of adenylate cyclase under appropriate conditions. This inhibition or stimulation follows a lag period. The combined addition of epinephrine or prostaglandin E1 with GTP gamma S results in the immediate onset of steady state inhibition or activation. The effects of the GTP analog are essentially irreversible. Fluoride is also an effective inhibitor of prostaglandin E1-stimulated adenylate cyclase, while it markedly stimulates the basal activity of the enzyme. The addition of the resolved 35,000-Da subunit of Gi to membranes results in inhibition of adenylate cyclase, and the resolved 41,000-Da subunit has a stimulatory effect on enzymatic activity. The inhibitory action of the 35,000-Da subunit is almost completely abolished in membranes that have been irreversibly inhibited by GTP gamma S plus epinephrine; this irreversible inhibition is almost completely relieved by the 41,000-Da subunit. Detergent extracts of membranes that have been treated with GTP gamma S plus epinephrine contain free 35,000-Da subunit. The 41,000-Da subunit of Gi contained in such extracts has a reduced ability to be ADP-ribosylated by islet-activating protein (IAP), which implies that this subunit is in the GTP gamma S-bound form. The irreversible inhibition of adenylate cyclase caused by GTP gamma S (plus epinephrine) in membranes is highly correlated with the liberation of free 35,000-Da subunit activity and is inversely related to the 41,000-Da IAP substrate activity in detergent extracts prepared therefrom. The increase in free 35,000-Da subunit activity in extracts and the inhibition of adenylate cyclase activity in GTP gamma S (plus epinephrine)-treated membranes are both markedly inhibited by treatment with IAP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of hormonally regulated adenylate cyclase by the beta gamma subunit of transducin.

Transducin (T), the GTP-binding protein of the retina activates the cGMP phosphodiesterase system, and presents analogies with the proteins GS and Gi which respectively mediate adenylate cyclase activation and inhibition by hormone receptors. These proteins are all comprised of an alpha subunit carrying the GTP-binding site and a beta gamma subunit made of two peptides. The beta peptide (35 kd) appears similar in the three proteins. We demonstrate here that purified T beta gamma inhibits adenylate cyclase from human platelet membranes. This inhibition was observed when adenylate cyclase was stimulated by GTP, prostaglandin E1 (PGE1), NaF and forskolin, but not when stimulated by GTP(gamma)S. In the presence of GTP and forskolin, the T beta gamma-induced maximal inhibition was not additive with the alpha 2-receptor-induced adenylate cyclase inhibition mediated by Gi. Both inhibitions were suppressed at high Mg2+ concentrations, which as also known to dissociate T beta gamma from T alpha-GDP. This suggests that these adenylate cyclase inhibitions are due to the formation of inactive complexes of GS alpha-GDP with T beta gamma or Gi beta gamma. T beta gamma-induced inhibition did not require detergent and could be suppressed by simple washing. T beta gamma effects are dependent on its concentration rather than on its total amount. This suggests that T beta gamma can operate in solution with no integration into the membrane. Similar inhibitory effects of T beta gamma are observed on adenylate cyclase from anterior pituitary and lymphoma S49 cell lines.     

A possible new class of octopamine receptors coupled to adenylate cyclase in the brain of the dipterous Ceratitis capitata. Pharmacological characterization and regulation of 3H-octopamine binding

Octopamine exerts its effects in insects through interaction with at least two classes of receptors, designated octopamine-1 and octopamine-2. Octopamine-2 receptors are positively coupled to adenylate cyclase, while octopamine-1 receptors are not coupled to this enzyme system. Ceratitis capitata brain appears to have octopamine receptors as unique aminergic receptors coupled to adenylate cyclase. These receptors show some pharmacological analogies with respect to octopamine-2 receptors, however they should constitute a new class of octopamine receptors. C. capitata brain octopamine receptors have also been characterized by [3H]octopamine-binding studies, exhibiting similar regulatory mechanisms to other receptors coupled to adenylate cyclase activation.     

3H]GDP release from rat and hamster adipocyte membranes independently linked to receptors involved in activation or inhibition of adenylate cyclase. Differential susceptibility to two bacterial toxins

When rat adipocyte membranes had been labeled with [3H]GTP in the presence of a beta-adrenergic agonist, the subsequent [3H]GDP release was stimulated by beta-agonists or agonists (e.g. glucagon and secretin) of other "activatory" receptors involved in activation of adenylate cyclase, but was not stimulated by agonists (e.g. prostaglandin E1 and adenosine) of "inhibitory" receptors involved in cyclase inhibition. On the contrary, agonists of inhibitory receptors were effective in stimulating GDP release from hamster adipocyte membranes that had been labeled via inhibitory alpha 2-adrenergic receptors, but an activatory receptor agonist such as isoproterenol was not. Thus, the guanine nucleotide regulatory protein (Ni) involved in adenylate cyclase inhibition is an entity distinct from the regulatory protein (Ns) involved in cyclase activation, and multiple activatory or inhibitory receptors are coupled to a respective common pool of Ns or Ni. Preactivated cholera toxin added together with NAD enhanced GDP release from rat adipocyte membranes prelabeled with isoproterenol but was without effect on the release from hamster adipocyte membranes that had been labeled with an alpha-agonist. In sharp contrast, the active subunit of islet-activating protein, pertussis toxin, failed to alter GDP release from the former membrane but completely abolished inhibitory agonist-induced stimulation of GDP release from the latter membrane preparation in the presence of NAD. Thus, the site of action of cholera toxin is Ns, while that of islet-activating protein is Ni. The function of Ni to communicate between inhibitory receptors and adenylate cyclase was lost when it was ADP-ribosylated by islet-activating protein.     

Evidence that human platelet alpha-adrenergic receptors coupled to inhibition of adenylate cyclase are not associated with the subunit of adenylate cyclase ADP-ribosylated by cholera toxin

Exposure of the alpha-adrenergic receptor of the human platelet to agonist prior to solubilization stabilizes a receptor complex of the alpha-adrenergic receptor with the GTP-binding protein(s) which modulates receptor affinity for agonists (Smith, S. K., and Limbird, L. E. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4026-4030). The soluble alpha-adrenergic receptor is characterized by retention of sensitivity to GTP and a faster rate of sedimentation in sucrose gradients than antagonist-occupied or unoccupied receptors. The present studies were undertaken to determine whether the alpha-adrenergic receptor, which is coupled to inhibition of adenylate cyclase, contains the same GTP-binding protein that is involved in activation of adenylate cyclase. The GTP-binding protein that is coupled to activation of adenylate cyclase was labeled with [32P]ADP-ribose using cholera toxin. Incorporation of [32]ADP-ribose into a Mr = 42,000 peptide in human platelet membranes was paralleled by an enhancement of GTP-sensitive catalytic activity in the membranes. However, cholera toxin treatment did not modify alpha-receptor-mediated inhibition of adenylate cyclase or interaction of the alpha-receptor with agonist agents. Moreover, sucrose gradient centrifugation revealed that the [32P]ADP-ribosylated Mr = 42,000 subunit of the stimulatory GTP-binding protein did not appear to associate with the agonist-alpha-receptor complex. These data suggest that the GTP-binding protein that mediates GTP activation of adenylate cyclase in the human platelet membrane is distinct from the GTP-binding protein that modulates alpha-adrenergic receptor affinity for agonist agents and which associates with the receptor in the presence of agonists.     

Photoaffinity Identification of Colchicine-Solubilized Regulatory Subunit from Rat Brain Adenylate Cyclase

Five GTP binding proteins in rat cerebral cortex synaptic membranes were identified by photoaffinity labelling with [3H] or [32P](P3-azido-anilido)-P1-5' GTP (AAGTP). When AAGTP-treated membranes were incubated with colchicine or vinblastine and subsequently washed, a single AAGTP-labelled protein of 42 kD was released into the supernatant. About 30% of the total labelled 42-kD protein was released into supernatants from membranes pretreated with colchicine or vinblastine compared with 15% released from control membranes. The amount of adenylate cyclase regulatory subunit (G unit) remaining in these membranes was assessed with reconstitution studies after inactivating the adenylate cyclase catalytic moiety with N-ethylmaleimide (NEM). Forty to fifty percent of functional G units were lost from membranes treated with colchicine prior to washing. This 40-50% loss of functional G unit after colchicine treatment corresponds to the previously observed 42% loss of NaF and guanylyl-5'-imidodiphosphate [Gpp(NH)p]-activated adenylate cyclase. Release of the AAGTP-labelled 42-kD protein from colchicine-treated synaptic membranes is double that from lumicolchicine-treated membranes. This colchicine-mediated release of 42-kD protein correlates with a doubling of functional G unit released from synaptic membranes after colchicine treatment. These findings suggest multiple populations of the G unit within the synaptic plasma membrane, some of which may interact with cytoskeletal components.     

The inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase. Subunit dissociation and the inhibition of adenylate cyclase in S49 lymphoma cyc- and wild type membranes

The inhibitory and stimulatory guanine nucleotide-binding regulatory components (Gi and Gs) of adenylate cyclase both have an alpha X beta subunit structure, and the beta subunits are functionally indistinguishable. GTP-dependent hormonal inhibition of adenylate cyclase and that caused by guanine nucleotide analogs seem to result from dissociation of the subunits of Gi. Such inhibition can be explained by reduction of the concentration of the free alpha subunit of Gs as a result of its interaction with the beta subunit of Gi in normal Gs-containing membranes. However, inhibition in S49 lymphoma cyc- cell membranes presumably cannot be explained by the Gi-Gs interaction, since the activity of the alpha subunit of Gs is not detectable in this variant. Several characteristics of Gi-mediated inhibition of adenylate cyclase have been studied in both S49 cyc- and wild type membranes. There are several similarities between inhibition of forskolin-stimulated adenylate cyclase by guanine nucleotides and somatostatin in cyc- and wild type membranes. 1) Somatostatin-induced inhibition of the enzyme is dependent on GTP; nonhydrolyzable GTP analogs are also effective inhibitors. 2) The effect of guanosine-5'-(3-O-thio)triphosphate (GTP gamma S) is essentially irreversible, and somatostatin accelerates GTP gamma S-induced inhibition. 3) Inhibition of adenylate cyclase by somatostatin or Gpp(NH)p is attenuated by treatment of cells with islet-activating protein (IAP). 4) Both cyc- and wild type membranes contain the substrate for IAP-catalyzed ADP-ribosylation (the alpha subunit of Gi). 5) beta Subunit activity in detergent extracts of membranes is liberated by exposure of the membranes to GTP gamma S. The alpha subunit of Gi in such extracts has a reduced ability to be ADP-ribosylated by IAP, which implies that this subunit is in the GTP gamma S-bound form. The resolved subunits of Gi have been tested as regulators of cyc- and wild type adenylate cyclase under a variety of conditions. The alpha subunit of Gi inhibits forskolin-stimulated adenylate cyclase activity in cyc-, while the beta subunit stimulates; these actions are opposite to those seen with wild type membranes. The inhibitory effects of GTP plus somatostatin (or GTP gamma S) and the alpha subunit of Gi are not additive in cyc- membranes. In wild type, the inhibitory effects of the hormone and GTP gamma S are not additive with those of the beta subunit.(ABSTRACT TRUNCATED AT 400 WORDS)     

Biochemical characterization of the BETA-adrenergic receptor of the frog erythrocyte

Summary The beta-adrenergic receptor which is coupled to adenylate cyclase in the frog erythrocycte plasma membrane provides a convenient model system for probing the molecular characteristics of an adenylate cyclase coupled hormone receptor. Direct radioligand binding studies with beta-adrenergic agonists and antagonists such as [³H]hydroxybenzylisoproterenol and [³H]dihydroalprenolol have shed new light on the biochemical properties of the receptor as well as on its mode of interaction with other components of the adenylate cyclase system. Agonist binding to the receptor induces a high affinity state of the receptor which can be selectively reverted to a low agonist affinity state by guanyl nucleotides. This agonist-induced high affinity state of the receptor appears to correspond to a receptor moiety which has larger apparent molecular weight and which is probably a complex of the beta-adrenergic receptor and nucleotide regulatory binding protein. Antagonists do not appear capable of inducing or stabilizing the formation of this high affinity receptor-nucleotide site complex.The beta-adrenergic receptors have been solubilized using the plant glycoside digitonin as the detergent and have been highly purified by biospecific affinity chromatography on an alprenolol-agarose affinity support. These highly purified receptor preparations retain all of the binding characteristics observed in the unpurified soluble receptor preparations.Remarkably, antibodies raised in rabbits against affinity chromatography purified preparations of the receptor, themselves bind beta-adrenergic ligands with typical beta-adrenergic specificity. Such antibodies which possess binding sites similar to those of physiological receptors provide useful model systems for further probing the molecular characteristics of beta-adrenergic binding sites.     

Reduction of GTP activation of adenylate cyclase system by its coupling to hormone receptor.

We have examined the characteristics of the adenylate cyclase system from control and butyrate-treated cells. Butyrate treatment results in both an increased number of catecholamine receptors and an induction of a response to the hormone, as reported previously (Tallman, J.F., Smith, C.C., and Henneberry, R.C. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 873-877); in addition, we found that the same treatment reduces the degree of activation of adenylate cyclase by GTP. We have demonstrated in two cell types that this decrease in GTP activation is inversely related to the degree of induction of the hormone response. Furthermore, in plasma membranes isolated from butyrate-treated cells, the hormone receptor is sensitive to GTP; i.e. GTP reduces the affinity of isoproterenol for the receptor. We propose that these changes reflect an interaction between the beta-adrenergic receptor and the nucleotide regulatory component and that this interaction represents, at least in part, the process of coupling. Several possible mechanisms which can account for the change in GTP activation are discussed in terms of our current understanding of the regulation of the adenylate cyclase system.     

Endogenous GTP and the regulation of epinephrine stimulation of adenylate cyclase.

Epinephrine increased adenylate cyclase activity 10 to 15 fold in lysates of the cultured human astrocytoma cell line 132-1N1. GTP had little effect on adenylate cyclase activity of lysed cell preparations either with or without added epinephrine. However, the epinephrine stimulation of adenylate cyclase was essentially lost (less than 90%) when a washed nuclei-free membrane preparation of the cyclase was assayed. A 10 to 15 fold epinephrine stimulation of the membrane adenylate cyclase could be demonstrated if cytosol of GTP were added to the assay with the hormone. The criteria of anion exchange, cation exchange, gel exclusion and paper chromatography indicated that the cytosolic agents which acted synergistically with hormones were GTP and GDP. The apparent Kact's for the synergistic action of GDP and GTP were essentially identical (1.0 muM) and of all the other nucleotides examined only GDP had a potency similar to GTP. However, the effect of GDP was apparently due to its rapid conversion to GTP even in the absence of a regenerating system. With epinephrine pretreatment of the intact 132-1N1 cells there was a specific loss of epinephrine stimulation of adenylate cyclase activity. The hormone pretreatment did not alter the capacity of the cytosol from these desensitized cells to potentiate epinephrine stimulation of the cyclase. Rather, the alteration was in the particulate fraction of the lysate. The desensitization of the membranous cyclase was stable and not reversed by GTP.     

Carbamylcholine inhibits beta-adrenergic receptor-coupled Gs protein function proximal to adenylate cyclase

The specific mechanism by which the inhibitory guanine nucleotide binding protein (Gi) mediates the inhibition of adenylate cyclase activity is still unclear. The subunit dissociation model, based on studies in purified or reconstituted systems, suggests that the beta gamma subunit, which is dissociated with activation of Gi, inhibits the function of the stimulatory guanine nucleotide binding protein (Gs) by reducing the concentration of the free alpha s subunit. In the present study, Gs protein function is determined by measuring cholera toxin-blockable, isoproterenol-induced increases in guanosine triphosphate (GTP) binding capacity to rat cardiac ventricle membrane preparations. Carbamylcholine totally inhibited this beta-adrenergic receptor-coupled Gs protein function. Pretreatment of the cardiac ventricle membrane with pertussis toxin prevented this muscarinic agonist effect. These results confirm the possibility of an inhibitory agonist-receptor coupled effect through Gi on Gs protein function proximal to the catalytic unit of adenylate cyclase in an intact membrane preparation.     

Induction of catecholamine-responsive adenylate cyclase in HeLa cells by sodium butyrate. Evidence for a more efficient stimulatory regulatory component

HeLa cells, when exposed to 5 mM sodium butyrate, increased their responsiveness to isoproterenol and their number of beta-receptors. As untreated HeLa cells have a substantial number of receptors but respond poorly to isoproterenol, the effect of butyrate could be due to quantitative or qualitative changes in beta-receptors or other components of the adenylate cyclase system. Receptors were analyzed by membrane/membrane and membrane/cell fusion techniques. HeLa donor membranes, treated to inactivate regulatory and catalytic components of adenylate cyclase, were fused with Fc cells, which lack beta-receptors. Isoproterenol-stimulated adenylate cyclase activity in the fusates was proportional to the number of receptors present. There appeared to be only quantitative but not qualitative differences in beta-receptors from control and butyrate-treated HeLa. Prostaglandin E1 receptors from neuroblastoma cell membranes were similarly coupled to HeLa adenylate cyclase. The hybrid prostaglandin E1-stimulated activity was lower when acceptor membranes were from control HeLa than when they were from butyrate-treated HeLa cells. These results suggested that butyrate was altering the ability of the regulatory component to interact with receptors. HeLa membranes were extracted with sodium cholate and the extracts used to reconstitute effector-stimulated adenylate cyclase activity in S49 cyc- membranes, which lack a functional regulatory component. Whereas extracts from control and butyrate-treated HeLa were equally effective in restoring NaF-stimulated activity in cyc- membranes, extracts from control HeLa were less efficient in reconstituting isoproterenol- and prostaglandin E1-stimulated activities. We conclude that the poor response of control HeLa to beta-agonists is due to a limited activity of the regulatory component but not the receptor. Butyrate induces quantitative changes in the receptor and qualitative changes in the regulatory component that facilitate its ability to couple to receptors but do not alter its ability to interact with the catalytic component of adenylate cyclase.     

Adenylate cyclase and membrane fluidity

Summary The relationships between membrane fluidity as induced by drug addition and the stimulation of adenylate cyclase by hormones (mainly catecholamines), GTP, Gpp(NH)p and NaF are reviewed. In particular, the data corresponding to pigeon erythrocyte membranes are reviewed and compared with other data published in the literature. A brief summary of the theories involved in fluidity measurements and their significance at the molecular level is also given for anisotropy of fluorescence and electron spin resonance.One of the conclusions is that the cationic drugs and neutral alcohols by perturbing preferentially the inner half-layer of the bilayer induced in pigeon erythrocyte membrane correlated multiphasic changes on fluidity and adenylate cyclase activity.This and other experimental data concerning the regulation of the adenylate cyclase are discussed in regard to a new interpretation of cyclase stimulation: the repressor hypothesis. In cell membrane the catalytic unit C is repressed by its association with a repressor complex made of the hormone receptor R and the regulatory protein N. The activation of cyclase activity is the dissociation of the catalytic unit C from the repressor complex R.N according to the equilibrium: R.N.C (inactive) R.N + C (active). Hormones, metal ions (magnesium), and nucleotides (GTP) are the allosteric ligands which shift this equilibrium towards the dissociation. state with the liberation of the active form, membrane-bound, C unit. Gpp(NH)p, fluoride and forskolin will also shift the equilibrium toward the right. GDP and free receptors favour the associated repressed state of the system.     

Synthesis of 2',3'-O-(2,4,6-trinitrocyclohexadienylidine)guanosine 5'-triphosphate and a study of its inhibitory properties with adenylate cyclase

A fluorescent GTP analog 2',3'-O-(2,4,6-trinitrocyclohexadienylidine) guanosine 5'-triphosphate (TNP-GTP) has been prepared and some of its physical properties characterized. TNP-GTP was found to be a potent inhibitor of chick embryo heart adenylate cyclase as activated by guanyl 5'-(beta,gamma-imido)triphosphate (GppNHp), F-, and forskolin with Ki values in the 8-15 microM range. It also appeared to inhibit substantially basal adenylate cyclase in this system. TNP-GTP demonstrated an effective competition with [3H]GppNHp, binding to membranes equivalently to GppNHp and about three times better than GTP. 8-Azidoguanosine 5'-triphosphate (8N3GTP) mimics GTP activation of chick embryo heart adenylate cyclase and [gamma-32P]8N3GTP is effectively photoincorporated into a 42,000- to 44,000-Mr doublet when proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TNP-GTP effectively prevents this photoincorporation, as does GTP, at concentrations that agree with their respective apparent inhibition and activation binding constants. The data suggest that TNP-GTP could prove to be a valuable tool for studying the mechanisms of GTP regulation of adenylate cyclase and other GTP-regulated systems.     

Evidence that a beta-adrenergic receptor-associated guanine nucleotide regulatory protein conveys guanosine 5'-O-(3-thiotriphosphate)- dependent adenylate cyclase activity

The guanine nucleotide regulatory protein component (N) of the frog erythrocyte membrane adenylate cyclase system appears to form a stable complex with the beta-adrenergic receptor (R) in the presence of agonist (H). This agonist-promoted ternary complex HRN can be solubilized with Lubrol. The guanine nucleotide regulatory protein associated with the solubilized complex can be adsorbed either to GTP-Sepharose directly or to wheat germ lectin-Sepharose via its interaction with the receptor which is a glycoprotein. Guanosine 5'-O-(3-thiotriphosphate)(GTP gamma S) can be used to elute the guanine nucleotide regulatory protein from either Sepharose derivative. The resulting N.GTP gamma S complex conveys nucleotide-dependent adenylate cyclase activity when combined with a Lubrol-solubilized extract of turkey erythrocyte membranes. The ability to observe GTP gamma S-dependent reconstitution of adenylate cyclase activity in the eluate from either resin required the formation of the HRN complex prior to solubilization. The N protein can be identified by its specific [32P]ADP ribosylation catalyzed by cholera toxin in the presence of [32P]NAD+. The existence of a stable HRN intermediate complex is supported by the observation that agonist pretreatment of frog erythrocyte membranes results in a 100% increase in the amount of 32P-labeled N protein eluted from the lectin-Sepharose in the presence of GTP gamma S compared to membranes pretreated with either antagonist or agonist plus GTP. Our results therefore provide evidence that the same guanine nucleotide-binding protein that associates with the beta-adrenergic receptor in the presence of agonist mediates adenylate cyclase activation.