Selective photoinduced uncoupling of the response of adenylate cyclase to gonadotropins by 5-iodonaphthyl 1-azide

5-Iodonaphthyl 1-azide (INA) has been previously shown to selectively label, on photolysis, only those proteins in contact with the membrane lipids. Low concentrations (less than 10 microM) of INA added to rat ovarian plasma membranes induced, on photoactivation, a selective and complete loss of the response of the adenylate cyclase to stimulation by human chorionic gonadotropin (hCG) or luteinizing hormone (LH). In contrast, this treatment affected neither hCG binding to the receptor nor the stimulation of the enzyme by NaF. That the uncoupling of the receptor from the enzyme by INA occurred within the lipid bilayer can be derived from the finding that the prior presence neither of saturating concentrations of hCG nor of the aqueous nitrene-scavenger glutathione (GSH) prevented this effect. Photolysis at higher concentrations of INA (0.1-1 mM) led to the inhibition of the adenylate cyclase stimulated by fluoride. This effect was totally prevented by glutathione. A similar behavior was obtained with a water-soluble analogue of INA, namely, 5-diazonionapthyl 1-azide (DAN). On photoactivation with 30 microM DAN, the NaF-stimulated adenylate cyclase was inhibited, but this effect was completely prevented by added GSH. At low concentrations where its effects are restricted to the lipid core, INA may represent a useful tool to define receptor coupling with the adenylate cyclase. The capacity of INA at low concentrations to uncouple the hormone receptor from the adenylate cyclase is not restricted to the LH/hCG receptor. Other hormone receptors tested behaved similarly. Therefore, the reported findings appear to represent a general phenomenon.     

Hormonal regulation of pancreatic islet adenyl cyclase.

Adenyl cyclase activity of rat pancreatic islet membrane was increased by secretin, pancreozymin, and isoproterenol, while ACTH, glucagon, growth hormone, and insulin had no effect. Both secretin and isoproterenol activations were enhanced by prostaglandin E1 (PGE1) and GTP. Isoproterenol activation was additive with PGE1, as was that of secretin with PGE1, but only in the presence of GTP. Secretin activation in the presence of PGE1 and GTP was equivalent to NaF stimulation. Kinetic analysis indicated that secretin and GTP increased the maximum velocity of the adenyl cyclase and tended to decrease the apparent affinity of the enzyme for ATP. Glucagon activation of islet membrane adenyl cyclase was dependent upon prior treatment of the membrane preparation with EGTA and the use of inhibitors of proteolytic enzymes during the collagenase digestion phase of islet preparation. These results suggest that hormonal regulation of insulin secretion may be affected by PGE1 and guanine nucleotide modulation of the adenyl cyclase activation process.     

Regression of thyroid hormone induced cardiac hypertrophy: Effect on cardiac beta receptors and adenyl cyclase activity

Adrenergic mechanisms may be important in the symptomatic manifestations of hyperthyroidism. The chronic administration of thyroid hormone also results in cardiac hypertrophy and increased numbers of beta-adrenergic receptors in cardiac membranes. The roles of adrenergic mechanisms in the initiation and perpetuation of this hypertrophy has been open to speculation. Rats treated chronically with L-thyroxin were sacrificed after 7 days of treatment and 1–4 days after cessation of treatment. Hearts were removed and weighed and norepinephrine measured. In other groups of identically treated rats, membranes were prepared from the left ventricle for $\text{invitro}$ measurements of beta-adrenergic receptor characteristics and adenyl cyclase activity. Regression of cardiac hypertrophy with a decrease in receptor number to control values was seen as early as 2 days after stopping thyroxine. Cardiac norepinephrine concentrations had also returned to control values at this time. Displacement of bound [H³] dihydroalprenolol by isoproterenol was not changed from control. Basal and isoproterenol stimulated adenyl cyclase activity was not changed by thyroxine administration or its cessation.The rapid reversal of the increased beta-adrenergic receptor number and cardiac hypertrophy raises the possibility that thyroid hormone may play a regulatory role in cardiac function. Although the enhancement of myocardial contractility by thyroid hormone may be mediated through cardiac hypertrophy this effect of thyroid hormone is independent of the catecholamine sensitive adenyl cyclase system.     

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.     

Regulation of particulate guanylate cyclase by Escherichia coli heat-stable enterotoxin: receptor binding and enzyme kinetics

1. Escherichia coli heat-stable enterotoxin (ST) induces a secretory diarrhea by binding to receptors on brush borders of intestinal villus cells, activating particulate guanylate cyclase and increasing intracellular concentrations of guanosine 3',5'-cyclic monophosphate (cyclic GMP). 2. However, little is known concerning coupling of receptor-ligand interaction to enzyme activation. 3. This study compares the kinetics of toxin-receptor binding and enzyme activation to better understand this transmembrane signal cascade. 4. Toxin receptor binding was linear and saturable with 50% of maximum displacement of [125I]ST by unlabeled toxin observed at 1.1 x 10(-7) M. ST increased the maximum velocity (Vmax) of guanylate cyclase with magnesium or manganese as the cation substrate without altering the affinity of the enzyme for its substrate or its positive cooperativity. 5. The concentration of toxin yielding half-maximum stimulation of guanylate cyclase was 1.2 x 10(-6) M, 10-fold higher than the affinity of the ligand for its receptor. 6. These data are consistent with the suggestion that ST-receptor interaction is coupled to activation of particulate guanylate cyclase. 7. However, the discrepancy between the affinity of ST for its receptor and its efficacy in activating the enzyme suggests that this coupling is complex. 8. Possible mechanisms underlying this coupling are discussed.     

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.     

Glycogenolytic responsiveness to glucagon, epinephrine, vasopressin and angiotensin II in the liver of developing hypothyroid rats. A comparative study of in vitro hormonal binding and in vivo biological response

Both dose-response curves and time-courses of plasma glucose levels after single maximal doses showed that in vivo glycogenolytic responsiveness to glucagon and epinephrine was significantly higher in developing hypothyroid rats, whereas it remained unchanged after vasopressin and angiotensin II injections. In contrast with the decreased basal activity of phosphorylase(a), the glucagon-stimulated activity increased in hypothyroid rats, whereas it was only slightly modified under vasopressin stimulation. Daily thyroxine treatment abolished these abnormalities. Thus, there is a close correlation between glucose output and enzyme activation. The maximal binding capacity of [3H]vasopressin and [125I]glucagon was significantly decreased in hypothyroid rats, without changes in the apparent dissociation constant of hormone from its specific receptor. Daily thyroxine treatment also abolished this deficit, which moreover appeared to be independent of possible changes in plasma hormone levels. With respect to glucagon action, neither basal nor Gpp(NH)p-stimulated adenylate cyclase activities were affected in hypothyroid rats. Glucagon-sensitive adenylate cyclase activity and the apparent activation constant appeared to be unaffected. The apparent discrepancy between the results obtained from in vivo and in vitro experiments is discussed on the basis of different membrane transducing phenomena and related intracellular mechanisms underlying the biological response to hormonal stimulation.     

Calcitonin-sensitive adenylate cyclase in rat renal tubular membranes.

1. Renal tubular membranes from rat kidneys were prepared, and adenylate cyclase activity was measured under basal conditions, after stimulation by NaF or salmon calcitonin. Apparent Km value of the enzyme for hormone-linked receptor was close to 1 x 10(-8) M. 2. The system was sensitive to temperature and pH. pH was found to act both on affinity for salmon calcitonin-linked receptor and maximum stimulation, suggesting an effect of pH on hormone-receptor binding and on a subsequent step. 3. KCl was without effect areas whereas CoCl and CaCl2 above 100 muM and MnCl2 above 1 muM inhibited F- -and salmon calcitonin-sensitive adenylate cyclase activities. The Ca2+ inhibition of the response reflected a fall in maximum stimulation and not a loss of affinity of salmon calcitonin-linked receptor for the enzyme. 4. The measurement of salmon calcitonin-sensitive adenylate cyclase activity as a function of ATP concentration showed that the hormone increases the maximum velocity of the adenylate cyclase. GTP, ITP and XTP at 200 muM did not modify basal, salmon calcitonin- and parathyroid hormone-sensitive adenylate cyclase activities. 5. Basal, salmon calcitonin- and F- -sensitive adenylate cyclase activities decreased at Mg2+ concentrations below 10 mM. High concentrations of Mg2+ (100 mM) led to an inhibition of the F- -stimulated enzyme. 6. Salmon calcitonin-linked receptor had a greater affinity for adenylate cyclase than human or porcine calcitonin-linked receptors. There was no additive effect of these three calcitonin peptides whereas parathyroid hormone added to salmon calcitonin increased adenylate cyclase activity, thus showing that both hormones bound to different membrane receptors. Human calcitonin fragments had no effect on adenylate cyclase activity. 7. Salmon calcitonin-stimulated adenylate cyclase activity decreased with the preincubation time. This was due to progressive degradation of the hormone and not to the rate of binding to membrane receptors.     

3H] norepinephrine binding by rat glial cells in culture. Lack of correlation between binding and adenylate cyclase activation.

Subcellular fractions prepared from rat glial cells in culture (clonal line c6) were used in an attempt to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [3H] norepinephrine binding and enzyme activation were measured under identical experimental conditions. Binding sites for norepinephrine could be detected; their main characteristics were: apparant Km: 4 - 10-6 M, macimal capacity: 20 pmol/mg protein.Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3-methoxydopamine (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrennergic agonist) and the blocking agent propranolol were unable to compete with [3H] norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [3H] norepinephrine binding. As in several other systems so far studied, the presence on the glial cell's membrane of a large number of "catechol-binding sites" makes it difficult to characterize the beta-adrenergic receptor.     

The oligosaccharide moiety of the beta 1-adrenergic receptor from turkey erythrocytes has a biantennary, N-acetyllactosamine-containing structure

The turkey erythrocyte beta 1-adrenergic receptor can be purified by affinity chromatography on alprenolol-Sepharose and characterized by photoaffinity labeling with N-(p-azido-m-[125I]iodobenzyl)-carazolol. Through the use of the specific glycosidases neuraminidase and endo-beta-N-acetylglucosaminidase H and affinity chromatography on lectin-Sepharose gels, we show here that the receptor is an N-glycosyl protein that contains complex carbohydrate chains. No high-mannose carbohydrate chains appear to be present. The binding of the radiolabeled antagonist dihydroalprenolol to the receptor is affected neither by the enzymic treatments nor by the presence of lectins, suggesting that the carbohydrate moiety is not involved in the catecholamine binding site.     

Studies on the assay and activities of guanyl and adenyl cyclase of rat liver

In applying recently developed methods for measuring adenyl and guanyl cyclase activities, we found that some modifications produced much better cyclic nucleotide recovery, lower assay backgrounds, and greater reliability than previously reported. The reliability and specificity of the assay methods were confirmed by substrate and product analysis. Kinetic analysis of rat liver guanyl and adenyl cyclase was subsequently performed to investigate regulatory properties of both enzymes. The Michaelis-Menton constant of guanyl cyclase activity of a 30,000g supernatant fraction of rat liver for guanosine 5′-triphosphate (GTP) was 0.04 mm. This enzyme was competitively inhibited by adenosine 5′-triphosphate (ATP) (K_i = 0.011 mM). Guanyl cyclase was activated in vitro by secretin but unaffected by carbamylcholine, hist-amine, methoxamirie, serotonin, glucagon, and pancreozymin. Liver homogenate adenyl cyclase had a Michaelis-Menten constant for ATP of 0.2 mm. This enzyme was activated by secretin, pancreozymin, glucagon, sodium fluoride, and isoproterenol. GTP (0.005 mm) enhanced the activation by both isoproterenol and glucagon. Methoxamine had no effect on adenyl cyclase activity in the presence or absence of GTP. These results suggest that both guanyl cyclase and adenyl cyclase may be mediators of hormone action in the liver.     

Inhibition of adenylate cyclase from luteinized rat ovary by monovalent cations: roles of the stimulatory guanine nucleotide-binding regulatory component and stimulatory hormone receptor

Sodium and other monovalent cations (added as chloride salts) inhibited adenylate cyclase of luteinized rat ovary. Sodium chloride (150 mM) inhibited basal enzyme activity by 20%. Sodium chloride inhibition was enhanced to 34-54% under conditions of enzyme stimulation by guanine nucleotides (GTP and its nonhydrolyzable analog 5'-guanylyl imidodiphosphate), fluoride anion, and agonists (ovine luteinizing hormone (oLH) and the beta-adrenergic catecholamine isoproterenol) acting at stimulatory receptors linked to adenylate cyclase. Sodium chloride inhibition was dependent on salt concentration over a wide range (25-800 mM) as well as the concentrations of GTP and oLH. Inhibition by NaCl was of rapid onset and appeared to be reversible. The order of inhibitory potency of monovalent cations was Li+ greater than Na+ greater than K+. The role of individual components of adenylate cyclase in the inhibitory action of monovalent cations was examined. Exotoxins of Vibrio cholerae and Bordetella pertussis were used to determine respectively the involvement of the stimulatory and inhibitory guanine nucleotide-binding regulatory components (Ns and Ni) in NaCl inhibition. Sodium chloride inhibited cholera toxin-activated adenylate cyclase activity by 29%. Ni did not appear to mediate cation inhibition of adenylate cyclase because pertussis toxin did not attenuate inhibition by NaCl. Enzyme stimulation by agents (forskolin and Mn2+) thought to activate the catalytic component directly was not inhibited by NaCl but was instead significantly enhanced. Sodium chloride (150 mM) increased both the Kd for high-affinity binding of oLH to 125I-human chorionic gonadotropin binding sites and the Kact for oLH stimulation of adenylate cyclase by sevenfold. In contrast, NaCl had no appreciable effect on either isoproterenol binding to (-)-[125I]iodopindolol binding sites or the Kact for isoproterenol stimulation of adenylate cyclase. The results suggest that in luteinized rat ovary monovalent cations uncouple, or dissociate, Ns from the catalytic component and, in a distinct action, reduce gonadotropin receptor affinity for hormone. Dissociation of the inhibitory influence of Ni from direct catalytic activation could account for NaCl enhancement of forskolin- and Mn2+-associated activities. On the basis of these results, the spectrum of divergent stimulatory and inhibitory effects of monovalent cations on adenylate cyclase activities in a variety of tissues may be interpreted in terms of differential enzyme susceptibilities to cation-induced uncoupling of N and catalytic component functions.     

Semisynthetic D-His1,N epsilon-acetimidoglucagon: structure-function relationships

The histidine residue at the amino terminus of lysine-12 protected glucagon was replaced by its D-isomer by an established semisynthetic strategy to extend a stepwise series of replacements at this position. The product was examined for its secondary structure and its function. Circular dichroism spectra obtained at concentrations from 0.25 to 1.09 mg/ml at pH 10.2 in 0.2 M phosphate buffer were similar to those obtained with native hormone. Competitive binding assays and adenylate cyclase activation assays with partially purified rat liver plasma membranes show this D-His1 analog of glucagon to be a full agonist, causing the same maximum activation of adenylate cyclase as native hormone; but both binding and activation assays show the binding affinity to be diminished about 10-fold. The data suggest that the adjustment of the bonding of the imidazole group to the receptor to bring about transduction results in constraints on the conformation along the peptide sequence which interfere with the peptide adopting the same binding conformation achieved by the native hormone.     

Cell cycle changes in the adenylate cyclase of C6 glioma cells

The adenylate cyclase of C6 glioma cell cultures was characterized for sensitivity to the beta-adrenergic agonist isoproterenol, as well as fluoride, and GTP as a function of the cell cycle. The mitotic phase of the cell cycle was emphasized because both the basal cellular cyclic AMP level and the intact C6 cell's capacity to accumulate cyclic AMP in response to isoproterenol decreased during mitosis. Basal and stimulated adenylate cyclase activities in mitotic cells were decreased relative to the enzyme activities in the G1, S, and G2 phases of the cell cycle. Analysis of the beta-adrenergic receptor using the radioligand(-)[3H]dihydroalprenolol showed that neither ligand affinity nor receptor density changed during the cell cycle, indicating that the reduced adenylate cyclase activity of the mitotic C6 cell was not caused by alterations in this hormone receptor. The reduction in the mitotic cell's basal adenylate cyclase activity was more prominent than the decrease in isoproterenol-, fluoride, or GTP-stimulated activities suggesting that the effectiveness of these enzymes activators (i.e., the efficiency of the coupling mechanism) was not attenuated during mitosis. These studies indicate that the intrinsic catalytic capacity (not the beta-adrenergic receptor or the coupling mechanism) of the C6 adenylate cyclase complex is reduced during mitosis and contributes to the mitotic cell's inability to accumulate and maintain the cyclic AMP concentration at the interphase level.     

Glucagon antagonists: contribution to binding and activity of the amino-terminal sequence 1-5, position 12, and the putative alpha-helical segment 19-27

Glucagon binding to and recognition by its cell surface receptor is the necessary first step in the cascade of events leading to the activation of adenylate cyclase by the hormone. It has long been presumed that glucagon adopts an ordered conformation upon binding to its membrane-bound receptor. A recent model of this three-dimensional structure based on biophysical data, predicts beta-turns at positions 2-5, 10-13, and 15-18, and an alpha-helical region between residues 19-27. Our approach in the design of antagonists of glucagon was to elucidate the steric and electronic features that stabilize these secondary structures to obtain analogs that bind with high affinity to the receptor but do not activate adenylate cyclase. Nineteen glucagon analogs incorporating structural changes at the amino-terminal sequence 1-5, at positions 9 and 12, and at the carboxyl-terminal helical region were synthesized. Des-His1-[Glu9]glucagon amide was recently shown to be a competitive inhibitor. Our synthetic studies in combination with this modification have resulted in seven new glucagon antagonists. The implications for the structural and conformational properties required for binding and activity of glucagon and the glucagon peptide family are discussed.     

Activation of adenylate cyclase by beta-adrenergic receptors: Investigation of rate limiting steps by simultaneous assay of high affinity agonist binding and GDP release

We report the development and application of a novel assay for high affinity binding of the agonist [³H]hydrozybenzyl-isoproterenol simultaneously with the agonist-promoted release of membrane bound [³²P] GDP in the frog erythrocyte beta-adrenergic receptor system. We find that under various assay conditions both events occur with the same rate, ranging from 0.05 to 0.5 min^?1. Addition of the non-hydrolyzable guanine nucleotide, guanylyl-imidodiphosphate simultaneously increases the rate of high affinity agonist binding and agonist promoted GDP release. In addition, the guanine nucleotide analog decreases the steady state level of high affinity agonist binding and increases the steady state level of agonist promoted GDP release with comparable potencies of 0.5 μM and 0.1 μM, respectively. The decrement in the steady state level of high affinity agonist binding (180 fmol/mg protein) due to the guanine nucleotide analog is in the same range as the reciprocal increment in the extent of agonist-induced [³²P] GDP release (180 fmol/mg protein). The concommittant activation of adenylate cyclase, by submaximal concentrations of the agonist [³H]hydroxybenzylisoproterenol and guanylylimido-diphosphate under similar assay conditions proceeds with the same rate as for the two other measured functions of the system, i.e. ³H-agonist binding and agonist-promoted [³²P] GDP release. This represents the first attempt at comparing the time course of adenylate cyclase activation with that of agonist binding and GDP release under similar assay conditions. The results indicate that GDP is not released prior to but rather coincident with formation of the complex of the hormone receptor with the regulatory protein and that enzyme activation proceeds with the same time course as agonist binds to the receptor. It is concluded that both high affinity agonist binding and GDP release represent integral aspects of the rate limiting step in the enzyme activation mechanism.     

Beta-adrenergic receptors: stereospecificity and lack of affinity for catechols

Studies of adenylate cyclase activity in rat liver, heart and fat cell microsomal preparations and in turkey and rat erythrocyte ghosts indicate that β-adrenergic receptors exhibit very strict stereospecificity for (?)-catecholamines. (+)-Isomers of active catecholamines and inactive catechol compounds do not inhibit the β-adrenergic-mediated stimulation of adenylate cyclase and thus do not interact with specific receptors. However, very high concentrations (above 10^?4 M) of (?)- and (+)-isomers, as well as of biologically inactive non-catecholamine catechols (e.g., pyrocatechol, dihydroxymandelic acid), inhibit in a nonspecific manner the basal, hormone (catecholamine, glucagon)- and NaF-stimulated adenylate cyclase activity. Studies with propranolol suggest that the low activity (0.1 to 1%) of (+)-isomers of norepinephrine can be explained by contamination with the (?)-isomer. The activity of soterenol, a potent non-catechol β-adrenergic agonist, is uninfluenced by (+)-catecholamines or catechols. It is concluded that the binding of ³H-labeled catecholamines to a variety of cells, microsomes and membranes as described in various previous studies cannot represent specific receptor interactions. Binding to receptors must demonstrate strict stereospecificity and must not be affected by unrelated catechol substances.     

Cholesterol modulation of β-adrenergic receptor characteristics

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

Cholesterol modulation of beta-adrenergic receptor characteristics

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

The follicle-stimulating hormone (FSH) receptor in testis: interaction with FSH, mechanism of signal transduction, and properties of the purified receptor

The follicle-stimulating hormone (FSH) receptor purified from calf bovine testis membranes appears to be an oligomeric glycoprotein, consisting of 4 disulfide-linked monomers of molecular weight about 60,000 each. Polyclonal antibodies to the hormone binding sites of the receptor have been developed. FSH interaction with the receptor seems to involve multiple discrete binding regions, which include amino acids 34-37 and 49-52 of the human FSH beta subunit. The interaction between FSH and the membrane-bound receptor is reversible at low temperatures but becomes increasingly irreversible as the temperature increases. FSH interaction with the soluble receptor is reversible over a wider temperature range. The hydrophobic effect is a significant factor in the initial hormone receptor interaction in each system. FSH bound to membrane receptors on cultured immature rat Sertoli cells is internalized and degraded to the level of amino acids. Current evidence suggests that the membrane receptor may exist as free receptor, and complexed with G-protein. A functional receptor/G-protein/adenylate cyclase complex has been reconstituted in liposomes. The G-protein of testis membranes contains both high and low affinity guanosine triphosphate (GTP) binding sites. Both are capable of modulating FSH receptor binding, whereas only the high affinity sites seem to be required for activation of adenylate cyclase. Although testis membranes contain a phosphatidylinositide hydrolysis system, the latter is not directly influenced by FSH.