Induction of adenylate cyclase in a mammary carcinoma cell line by human corticosteroid-binding globulin

Corticosteroid-Binding globulin (CBG) is a plasma protein that binds certain steroid hormones, mainly cortisol and progesterone. It has been demonstrated recently that specific binding sites for this protein exist on cell membranes. In this communication we establish that binding to these sites results in the induction of adenylate cyclase activity and the accumulation of cAMP in MCF-7 cells. These events are critically dependent upon a steroid being bound to CBG. These data are consistent with the hypothesis that CBG is a prohormone which is activated when cortisol is bound to it.     

Histidine regulation of cyclic AMP metabolism in cultured renal epithelial LLC-PK1 cells.

L-Histidine and imidazole (the histidine side chain) significantly increase cAMP accumulation in intact LLC-PK1 cells. This effect is completely inhibited by isobutylmethylxanthine (IBMX). Histidine and imidazole stimulate cAMP phosphodiesterase activity in soluble and membrane fractions of LLC-PK1 cells suggesting that the IBMX-sensitive effect of these agents to stimulate cAMP formation is not due to inhibition of cAMP phosphodiesterase. Histidine and imidazole but not alanine (the histidine core structure) increase basal, GTP-, forskolin-, and AVP-stimulated adenylate cyclase activity in LLC-PK1 membranes. Two other amino acids with charged side chains (aspartic and glutamic acids) increase AVP-stimulated but neither basal- nor forskolin-stimulated adenylate cyclase activity. This suggests that multiple amino acids with charged side chains can regulate selected aspects of adenylate cyclase activity. To better define the mechanism of histidine regulation of adenylate cyclase, membranes were detergent-solubilized which prevents histidine and imidazole potentiation of forskolin-stimulated adenylate cyclase activity and suggests that an intact plasma membrane environment is required for potentiation. Neither pertussis toxin nor indomethacin pretreatment alter imidazole potentiation of adenylate cyclase. IBMX pretreatment of LLC-PK1 membranes also prevents imidazole to potentiate adenylate cyclase activity. Since IBMX inhibits adenylate cyclase coupled adenosine receptors, LLC-PK1 cells were incubated in vitro with 5'-N-ethylcarboxyamideadenosine (NECA) which produced a homologous pattern of desensitization of NECA to stimulate adenylate cyclase activity. Despite homologous desensitization, histidine and imidazole potentiation of adenylate cyclase was unaltered. These data suggest that histidine, acting via an imidazole ring, potentiates adenylate cyclase activity and thereby increases cAMP formation in cultured LLC-PK1 epithelial cells. This potentiation requires an intact plasma membrane environment, occurs independent of a pertussis toxin-sensitive substrate and of products of cyclooxygenase, and is inhibited by IBMX. This IBMX-sensitive pathway does not involve either inhibition of cAMP phosphodiesterase activity or a stimulatory adenosine receptor coupled to adenylate cyclase.     

Adenylate cyclase in Saccharomyces cerevisiae is a peripheral membrane protein.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains the CYR1 polypeptide, responsible for catalyzing formation of cyclic AMP (cAMP) from ATP, and two RAS polypeptides, which mediate stimulation of cAMP synthesis of guanine nucleotides. By analogy to the mammalian enzyme, models of yeast adenylate cyclase have depicted the enzyme as a membrane protein. We have concluded that adenylate cyclase is only peripherally bound to the yeast membrane, based on the following criteria: (i) substantial activity was found in cytoplasmic fractions; (ii) activity was released from membranes by the addition of 0.5 M NaCl; (iii) in the presence of 0.5 M NaCl, activity in detergent extracts had hydrodynamic properties identical to those of cytosolic or NaCl-extracted enzyme; (iv) antibodies to yeast adenylate cyclase identified a full-length adenylate cyclase in both membrane and cytosol fractions; and (v) activity from both cytosolic fractions and NaCl extracts could be functionally reconstituted into membranes lacking adenylate cyclase activity. The binding of adenylate cyclase to the membrane may have regulatory significance; the fraction of activity associated with the membrane increased as cultures approached stationary phase. In addition, binding of adenylate cyclase to membranes appeared to be inhibited by cAMP. These results indicate the existence of a protein anchoring adenylate cyclase to the membrane. The identity of this protein remains unknown.     

Sex hormone-binding globulin: Anatomy and physiology of a new regulatory system

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds a number of circulating steroid hormones (testosterone, dihydrotestosterone and estradiol) with high affinity, thus regulating their free concentration in plasma. In addition to binding steroids, SHBG itself binds to receptor sites on plasma membranes with somewhat unusual kinetics. Both the off and on rates are quite slow. The steroid-binding and membrane-binding functions are interwined in what is clearly an allosteric relationship. Occupation of SHBG's steroid-binding site by a steroid inhibits its ability to bind to its membrane receptor-binding site. This inhibition is not related to a steroid's biological activity. Metabolites of steroids without biological activity, e.g. 2-methoxyestradiol, actively inhibit SHBG's interaction with its membrane receptor. However, if unliganded SHBG is allowed to bind to its receptor on intact cells, and an appropriate steroid hormone then is introduced, adenylate cyclase is activated and intracellular cAMP increases. This function is specific for steroids with biological activity, 2-methoxyestradiol has no activity in this arena. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.     

The hepatic angiotensin II receptor. II. Effect of guanine nucleotides and interaction with cyclic AMP production

Guanine nucleotides were observed to modify the binding of 125I-angiotensin II to rat hepatic plasma membrane receptors. GTP and its nonhydrolyzable analogues greatly increased the dissociation rate of bound 125I-angiotensin II and altered hormone binding to the receptor under equilibrium conditions. In the absence of GTP, 125I-angiotensin II labeled both high affinity sites (Kd1 = 0.46 nM, N1 = 650 fmol/mg) and low affinity sites (Kd2 = 4.1 nM, N2 = 1740 fmol/mg). In the presence of guanine nucleotides, the affinities of the two sites were unchanged, but the number of high affinity sites decreased markedly to 52 fmol/mg. In analogous experiments using the angiotensin II antagonist, 125I-sarcosine1,Ala8-angiotensin II (125I-saralasin), guanine nucleotides minimally affected the interaction of 125I-saralasin with its receptor, increasing the dissociation rate 1.9-fold and the Kd 1.4-fold. The guanine nucleotide inhibition of agonist binding required a cation such as Na+ or Mg2+, with a maximal effect occurring at about 1 mM Mg2+. In liver plasma membranes prepared in EDTA, angiotensin II inhibited basal and glucagon-stimulated adenylate cyclase activities by 30% and 10%, respectively. Angiotensin II also caused a 40% inhibition of glucagon-stimulated cyclic AMP accumulation in intact hepatocytes, with a half-maximal effect occurring at 1 nM. The inhibition by angiotensin II of adenylate cyclase in membranes and of cAMP levels in intact cells could be reversed by the antagonist sarcosine1,Ile8-angiotensin II. Vasopressin caused a smaller 26% inhibition of glucagon-stimulated cyclic AMP accumulation. The ability of angiotensin II to inhibit cyclic AMP synthesis may provide an explanation for the observed effects of guanine nucleotides on 125I-angiotensin II binding to plasma membranes.     

Specific uncoupling by islet-activating protein, pertussis toxin, of negative signal transduction via alpha-adrenergic, cholinergic, and opiate receptors in neuroblastoma x glioma hybrid cells

Exposure of NG108-15 hybrid cells to islet-activating protein (IAP), pertussis toxin, caused strong ADP-ribosylation of one of the membrane proteins with a molecular weight of 41,000. This ADP-ribosylation was paralleled by decreases in the inhibition of cAMP accumulation in intact cells or associated with reversal of the inhibition of GTP-dependent membrane adenylate cyclase, via alpha-adrenergic, cholinergic muscarinic, or opiate receptors. The affinity of these receptors for agonists was lowered by guanyl-5'-yl beta-gamma-imidodiphosphate (Gpp(NH)p) reflecting their coupling to the guanine nucleotide regulatory protein in this cell line. This effect of Gpp(NH)p was lost in membranes of IAP-treated cells; in the absence of Gpp(NH)p, the affinity for agonist was lower in treated than in nontreated cells. In contrast, the function of these receptors to bind antagonists remained unaltered in IAP-treated cells. Thus, IAP treatment of NG108-15 cells caused specific uncoupling of negative signal transduction from inhibitory receptors to the adenylate cyclase catalytic unit via the guanine nucleotide regulatory protein, as a result of ADP-ribosylation of one of the subunits of the regulatory protein.     

Prostaglandin E receptors in bovine adrenal medulla are coupled to adenylate cyclase via Gi and to phosphoinositide metabolism in a pertussis toxin-insensitive manner

Prostaglandin E (PGE) receptor is coupled to a pertussis toxin-insensitive GTP-binding protein in bovine adrenal medulla, but PGE receptor partially purified from bovine adrenal medulla was functionally reconstituted with Gi into phospholipid vesicles (Negishi, M., Ito, S., Yokohama, H., Hayashi, H., Katada, T., Ui, M., and Hayaishi, O. (1988) J. Biol. Chem. 263, 6893-6900). We demonstrate here that PGE2 inhibited forskolin-induced accumulation of cAMP in cultured bovine chromaffin cells. In plasma membranes prepared from bovine adrenal medulla, PGE2 inhibited forskolin-stimulated adenylate cyclase activity in a GTP-dependent manner. This inhibitory action of PGE2 was abolished by treatment of the membrane with pertussis toxin. Reconstitution of the membranes ADP-ribosylated by pertussis toxin with Gi purified from bovine brain restored the potency of PGE2 to inhibit the adenylate cyclase activity. Inhibition of forskolin-induced cAMP accumulation by PGE2 was also abolished by exposure to the toxin in the cells, indicating that PGE receptors are coupled to Gi. In contrast, PGE2 stimulated the formation of inositol phosphates in chromaffin cells, but this effect was not affected by treatment of the cells with pertussis toxin, suggesting that the PGE receptors are coupled to phosphoinositide metabolism via a pertussis toxin-insensitive G-protein. Both the inhibitory action of cAMP accumulation and stimulation of phosphoinositide metabolism were specific for PGE1 and PGE2, and the Scatchard plot analysis of PGE2 binding to the membrane showed a single high-affinity binding site (Kd = 2 nM). In bovine adrenal chromaffin cells PGE2 enhanced catecholamine release in the presence of ouabain by stimulation of phosphoinositide metabolism (Yokohama, H., Tanaka, T., Ito, S., Negishi, M., Hayashi, H., and Hayaishi, O. (1988) J. Biol. Chem. 263, 1119-1122). We further examined the modulation of catecholamine release by PGE2 through its inhibitory coupling to the adenylate cyclase system. Prior exposure of chromaffin cells to forskolin or dibutyryl-cAMP reduced nicotine-stimulated catecholamine release, and PGE2 attenuated forskolin-induced inhibition of catecholamine release stimulated by nicotine, but not dibutyryl-cAMP-induced inhibition. In the absence of evidence that PGE receptor subtypes exist, these results suggest that the PGE receptor is coupled to two signal transduction systems leading to inhibition of cAMP accumulation via Gi and to production of inositol phosphates via a pertussis toxin-insensitive G-protein, both of which may modulate catecholamine release from bovine chromaffin cells.     

Activation, desensitization, and recycling of frog erythrocyte beta-adrenergic receptors. Differential perturbation by in situ trypsinization

We have utilized limited in situ trypsinization of the adenylate cyclase-coupled beta-adrenergic receptor of frog erythrocytes to probe the processes of receptor activation, desensitization, and recycling. Treatment of intact erythrocytes with trypsin (1 mg/ml) for 1 h at 20 degrees C converts all the receptor peptides (identified by photoaffinity labeling with p-azido-125I-benzylcarazolol) from a Mr approximately 58,000 to a Mr approximately 40,000 species. Nonetheless, the trypsinized beta-adrenergic receptors bind agonists and antagonists with unaltered affinity and with no change in the number of binding sites. Moreover, the ability of the proteolyzed receptors to interact with the nucleotide regulatory protein to form a high affinity guanine nucleotide-sensitive state and to activate adenylate cyclase were also unaltered. However, upon exposure of intact cells to the agonist isoproterenol, trypsinized beta-adrenergic receptors were more rapidly and more completely cleared from the plasma membranes ("down-regulated") than untrypsinized receptors. Whereas down-regulated receptors from nontrypsinized cells appear to recycle to the cell surface after removal of the agonist, internalized trypsinized beta-adrenergic receptors do not recycle to the plasma membrane and appear to be degraded within the cell. Moreover, when internalized receptors, recovered in a light vesicle fraction, were fused with a heterologous adenylate cyclase system, untreated but not trypsinized receptors reconstituted catecholamine stimulation of the enzyme. These data suggest that the beta-adrenergic receptor contains a trypsin-sensitive site which is exposed on the outer surface of the plasma membrane. Proteolysis at this site releases a fragment which though not critically involved in either ligand binding or "effector coupling" might be important for anchoring the receptors in the plasma membrane. These data also suggest that in situ proteolysis of the receptors might serve as a physiological trigger for their internalization and degradation.     

Heterologous desensitization by 1,25-dihydroxyvitamin D-3 of cyclic AMP response to parathyroid hormone in osteoblast-like cells and the role of the stimulatory guanine nucleotide regulatory protein

The influence of 1,25-dihydroxyvitamin D-3 on the cAMP response to parathyroid hormone was studied in the osteoblast-like rat osteosarcoma cells ROS 17/2.8. The stimulation by parathyroid hormone of cAMP production in intact cells and of adenylate cyclase activity in isolated plasma membranes was attenuated by 1,25-dihydroxyvitamin D-3 treatment. This was associated with a reduction of the stimulatory guanine nucleotide regulatory protein, as demonstrated by a lower response to NaF and guanosine 5'-[beta, gamma-imido]triphosphate, and by a lower activity of solubilized plasma membrane extracts in the reconstitution assay. 1,25-dihydroxyvitamin D-3 blunted also the cAMP response to parathyroid hormone in cells incubated with the glucocorticoid dexamethasone, where a higher activity of the adenylate cyclase catalytic unit was observed. Thus, the two steroids appear to affect distinct levels of the adenylate cyclase system. Furthermore, the two hormones also showed an antagonistic effect upon the production of osteocalcin, an osteoblast-specific extracellular matrix protein. The release of this non-collagenous matrix protein by ROS 17/2.8 cells was increased by 1,25-dihydroxyvitamin D-3 and decreased by dexamethasone.     

Role of Ni in coupling angiotensin receptors to inhibition of adenylate cyclase in hepatocytes

Angiotensin II can inhibit glucagon-stimulated cyclic AMP production in hepatocytes and adenylate cyclase activity in hepatic membranes. Pertussis toxin, an exotoxin produced by Bordetella pertussis, was used to investigate the role of the inhibitory guanine nucleotide-binding regulatory protein of adenylate cyclase (Ni) in coupling angiotensin receptors to the adenylate cyclase system. An assay was developed using [32P] NAD+ to quantitate the amount of Ni protein in the membrane and the extent of its ADP-ribosylation catalyzed by toxin. The ability of angiotensin to inhibit adenylate cyclase and interact with its receptor was compared with the degree of modification of Ni in membranes prepared from isolated hepatocytes. In control membranes angiotensin II inhibited basal adenylate cyclase by 35%. When all of the Ni molecules in the membrane were ADP-ribosylated, angiotensin did not inhibit adenylate cyclase. However, the attenuation of angiotensin's effect on cyclase was not linearly correlated with the degree of modification of Ni; ADP-ribosylation of greater than 80% of the Ni was required before a reduction of the angiotensin effect was observed. A possible explanation for this finding is an excess of Ni molecules in the membrane (approximately 3.4 pmol/mg of membrane protein) over angiotensin II receptors (approximately 1.2 pmol/mg of membrane protein). 125I-angiotensin bound to sites in the membrane with two affinities. Computer fitting of the binding isotherms yielded parameters of N1 = 279 fmol/mg protein, Kd1 = 0.2 nM; N2 = 904 fmol/mg protein, Kd2 = 1.4 nM. When all of the Ni molecules in the membrane were ADP-ribosylated, angiotensin bound to only one site with binding parameters of N = 349 fmol/mg protein, Kd = 0.4 nM. GTP-gamma-S caused a 7-fold increase in the Kd of this site to 2.7 nM. Overall, the data indicate that the Ni protein mediates the effect of angiotensin on adenylate cyclase. The observation that GTP-gamma-S can markedly decrease the affinity of angiotensin receptors when all Ni molecules are ADP-ribosylated suggests that angiotensin receptors may couple to other GTP-binding proteins which may mediate the effects of angiotensin in other signal transduction systems.     

Isolation of adenylate cyclase-enriched membranes from mammalian cells using concanavalin A.

Plasma membrane vesicles containing adenylate cyclase and beta-adrenergic receptors were prepared from 1321N1 human astrocytoma cells by a procedure involving the use of concanavalin A to stabilize the plasma membrane to fragmentation and vesiculation upon cell lysis. Treatment of cells with concanavalin A causes these plasma membrane markers to sediment to a higher density of sucrose and in a narrower band than observed with untreated cells. Upon treatment of the heavy membrane fragments with alpha-methylmannoside to remove bound concanavalin A, the enzyme markers again sediment a lower densities of sucrose. This reversible change in sedimentation behavior has been used to obtain preparations of plasma membranes enriched 14- to 21-fold (recovery 25%) in adenylate cyclase activity and about 12-fold (recovery 16%) in beta-adrenergic receptor density, as compared to lysates. The adenylate cyclase of purified membranes responded normally to isoproterenol and prostaglandin E1. Experiments with S49 and YAC mouse lymphoma cells and human skin fibroblasts indicate that this procedure may be adaptable to the isolation of plasma membranes from a variety of cultured cell lines.     

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

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

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.     

Establishment of gonadotropin-responsive murine leydig tumor cell line

Several clonal Leydig tumor cell lines have been established by adapting the transplantable Leydig tumor, M548OP, to culture. One of these cell line, MLTC-1, has been characterized with regard to the gonadotropin-responsive adenylate cyclase system. The binding of 125I-labeled human chorionic gonadotropin (hCG) was blocked by excess unlabeled hCG and lutropin (LH) but not by follitropin, thyrotropin, or insulin, indicating the presence of specific receptors for hCG and LH. Based on the specific binding of hCG to isolated MLTC-1 membranes, the calculated dissociation constant was 1.0 +/- 0.2 X 10(-10) M. The receptors appeared identical to those from normal murine Leydig cells when analyzed by SDS PAGE and sucrose density gradient centrifugation. The molecular weight and sedimentation coefficient were 95,000 daltons and 8.5 S, respectively. MLTC-1 cells responded to hCG by accumulating cyclic AMP and producing progesterone. Cyclic AMP accumulation was time- and dose-dependent with a maximal accumulation occurring at approximately 0.2 nM hCG. At saturating levels of hCG, cAMP levels reached a maximum by 30 min and then declined very slowly. Adenylate cyclase activity in membranes prepared from MLTC-1 cells was stimulated by hCG, LH, NaF, cholera toxin, and guanyl-5'-ylimidodiphosphate, Additionally, choleragen was found to ADP-ribosylate a membrane protein of 54,000 daltons. This protein resembles the proposed guanine nucleotide regulatory component in both size and choleragen-dependent reactivity. These data suggest that MLTC-1 cells possess a gonadotropin-responsive adenylate cyclase system consisting of a specific hormone receptor, a regulatory component, and a catalytic subunit.     

Cholesterol modulation of membrane fluidity and VIP receptor/effector system in rat prostatic epithelial cells.

Treatment of rat prostatic epithelial cells with cholesteryl hemisuccinate (ChH) resulted in a time- and dose-dependent inhibition of the stimulatory effect of the neuropeptide vasoactive intestinal peptide (VIP) on cyclic AMP accumulation, with a 40% decrease in the response to a maximally effective VIP concentration. Cell treatment with ChH led also to a similar blocking of isoproterenol (a beta-adrenergic agonist) action but did not modify forskolin (which is assumed to act directly on the catalytic unit of adenylate cyclase) activity upon cyclic AMP levels. The levels of the transduction protein Gs were similar in membranes from both control and ChH-treated cells as suggested by experiments on cholera toxin-catalyzed ADP-ribosylation. The inhibitory effect of ChH was accompanied by an increase of membrane microviscosity as estimated by measurements of fluorescence polarization. Experiments on VIP binding indicated that increasing cholesterol concentration in the plasma membrane led to a higher VIP binding capacity without changes in the affinity of VIP receptors. These data suggest that membrane cholesterol incorporation diminishes the coupling efficiency between adenylate cyclase and the VIP-receptor complex or other receptor systems (i.e., desensitization) due to an increase of plasma membrane rigidity.     

Ammonium sulfate modifies adenylate cyclase and the chemotactic receptor of Dictyostelium discoideum. Evidence for a G protein effect

(NH4)2SO4 was found to activate adenylate cyclase in Dictyostelium discoideum membranes. The effect of (NH4)2SO4 on the enzyme was observed after pretreatment of membranes but could not be observed if the salt was added to the assay mixture. Activation was seen when membranes were pretreated with 0.16 M (NH4)2SO4 and was maximal at 0.6-1.0 M. The maximal activation of the enzyme was observed within 3 min of pretreatment and was not readily reversible. The effect was specific for the NH+4 ion since pretreatment of membranes with other NH+4 salts could activate the enzyme, whereas pretreatment with NaCl or KCl could not. Pretreatment of plasma membranes with (NH4)2SO4 eliminated the sensitivity of the enzyme to the inhibitory effect of guanine nucleotides. (NH4)2SO4 pretreatment also significantly attenuated the inhibition by guanine nucleotides of cAMP binding to its plasma membrane receptor. The effect of (NH4)2SO4 on GTP inhibition of cAMP binding to its receptor was even more dramatic when the salt was present in the binding assay. (NH4)2SO4 also increased the ADP-ribosylation by cholera toxin of a 39,000-Da membrane protein. The data support the hypothesis that (NH4)2SO4-induced changes in adenylate cyclase and the cAMP receptor are due to an alteration of a putative G protein.     

Inhibition of hormone-stimulated adenylate cyclase activity after altering turkey erythrocyte phospholipid composition with a nonspecific lipid transfer protein. Phosphatidylinositol uncouples catecholamine binding from adenylate cyclase activation

The nonspecific lipid transfer protein from beef liver was used to modify the phospholipid composition of intact turkey erythrocytes in order to study the dependence of isoproterenol-stimulated adenylate cyclase activity on membrane phospholipid composition. Incorporation of phosphatidylinositol into turkey erythrocytes inhibited isoproterenol-stimulated cyclic AMP accumulation in a linear, concentration-dependent manner. Inhibition was relatively specific for phosphatidylinositol; phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid were from 3 to 7 times less effective as inhibitors of hormone-stimulated cyclase activity. Inhibition by phosphatidylinositol was not reversible when up to 90% of the incorporated phosphatidylinositol was removed, either by incubation with phosphatidylinositol-specific phospholipase C or a second incubation with transfer protein; possibly adenylate cyclase activity depends on a small pool of phosphatidylinositol that is inaccessible to either phospholipase C hydrolysis or removal by lipid transfer protein. Phosphatidylinositol incorporation inhibits adenylate cyclase activity by uncoupling beta-adrenergic receptors from the remainder of the cyclase complex. Phosphatidylinositol incorporation had no effect on stimulation of cAMP accumulation by either cholera toxin or forskolin, indicating that inhibition occurs only at the level of receptor. Phosphodiesterase activity was not altered in phosphatidylinositol-modified cells. Inhibition of cAMP accumulation was not the result of changes in either membrane fluidity or in cAMP transport out of modified turkey erythrocytes. Phosphatidylinositol inhibition of isoproterenol-stimulated cyclase activity may serve as a useful model system for hormone-induced desensitization.     

Somatostatin pretreatment increases the number of somatostatin receptors in GH4C1 pituitary cells and does not reduce cellular responsiveness to somatostatin

The GH4C1 pituitary cell line contains specific plasma membrane receptors for the inhibitory neuropeptide somatostatin (SRIF). Unlike other peptides which bind to cell surface receptors on these cells, SRIF is not rapidly internalized via receptor-mediated endocytosis. Here we examined the effects of chronic SRIF pretreatment on the subsequent ability of GH4C1 cells to bind and respond to this hormone. Treatment of cells with 100 nM SRIF increased [125I-Tyr1]SRIF binding to a maximum value of 220% of control after 20 h. Scatchard analysis demonstrated that the number, but not the affinity, of the receptors was altered. The effect of SRIF was dose-dependent (ED50 = 2.3 +/- 0.4 nM), was not mimicked by an inactive analog, and was specific for the SRIF receptor. Furthermore, pretreatment of cells with other agents, which mimic SRIF's action to decrease intracellular cAMP and free Ca2+ concentrations, did not mimic the SRIF-induced increase in receptor number. Thus, occupancy of the SRIF receptor was required for SRIF receptor up-regulation. Inhibition of protein synthesis with cycloheximide did not prevent the SRIF-induced increase in receptors, consistent with an effect of SRIF to either reduce receptor degradation or cause slow redistribution of preexisting receptors to the plasma membrane. In contrast to the effects on receptor binding, pretreating cells with SRIF did not alter either basal cAMP levels or the potency of SRIF to inhibit cAMP accumulation (ED50 = 0.5 +/- 0.2 nM). However, the maximum cAMP produced by stimulators of adenylyl cyclase was increased. The observation that chronic SRIF exposure did not cause homologous desensitization in GH4C1 cells and increased rather than decreased SRIF receptor number is consistent with the fact that this neuropeptide is not rapidly internalized by receptor-mediated endocytosis.     

Regulation of secretion from the adrenal medulla. Evidence for adenylate cyclase activity in secretory vesicle membranes.

Adenylate cyclase activity has been found in purified secretory vesicle membranes from the adrenal medulla. Activity was detected both by formation of radioactive cAMP from [alpha-32P]ATP and by the competitive protein binding assay for cAMP. Activity was highest at pH 8.0 to 8.5, and was stimulated by sodium fluoride and GppNHp, a GTP analogue known to stimulate adenylate cyclase activity in plasma membrane preparations. The reaction rate was strongly dependent on the molar ratio of Mg2+:ATP in the system. This is the first demonstration of adenylate cyclase in a secretory vesicle membrane.     

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.