Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.6.1.1 (adenylate cyclase)
19,190 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The maturation of ovarian granulosa cells is dependent upon the pituitary gonadotropin FSH, the actions of which are mediated via specific plasma membrane receptors. To study the regulation of ovarian FSH receptor expression at the mRNA level, we used a specific cRNA probe to evaluate changes in FSH receptor transcripts in cultured granulosa cells. Granulosa cells obtained from immature estrogen-treated rats contained two predominant FSH receptor mRNA transcripts (7.0 and 2.5 kilobases), the levels of which declined in a time-related manner during a 2-day culture period. However, inclusion of FSH (30 ng/ml) in the culture medium prevented the decline in FSH receptor mRNA levels. Compared to controls, treatment of granulosa cells for 48 h with FSH (1-100 ng/ml) increased FSH receptor mRNA levels in a dose-dependent manner (ED50, 4.5 ng/ml), with a maximal 5.9 +/- 0.7-fold increase observed in response to 30 ng/ml FSH. The stimulatory actions of FSH were mimicked by the adenyl cyclase activator forskolin (0.1-30 microM), suggesting the involvement of cAMP in FSH receptor gene transcription and/or mRNA stability. Incubation of granulosa cells for 48 h with epidermal growth factor (EGF; 0.3-10 ng/ml), basic fibroblast growth factor (bFGF; 1-30 ng/ml), or insulin-like growth factor-I (IGF-I; 1-30 ng/ml) did not affect basal FSH receptor mRNA levels, whereas the highest doses of EGF and bFGF, but not IGF-I, completely suppressed the stimulatory effects of FSH (30 ng/ml) on its own receptor mRNA levels. Similarly, GnRH (10-1000 nM) attenuated the actions of FSH on its receptor mRNA levels in a dose-dependent manner (ID50, 8 nM). The inhibitory effects of GnRH (100 nM) were reversed by cotreatment with a GnRH antagonist ([Ac-D-Phe1,D-pCl-Phe2,D-Trp3,6]GnRH; 100 nM), indicating that the actions of GnRH are mediated via specific GnRH receptors. These data indicate that treatment of granulosa cells with FSH increases the levels of two FSH receptor mRNA transcripts. However, this positive feedback system, which may lead to an amplification of FSH action, is tightly regulated by the inhibitory actions of EGF, bFGF, and GnRH. Thus, the use of cultured rat granulosa cells provides a model system to analyze the hormonal regulation of FSH receptor gene expression in the ovary.
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PMID:Hormonal regulation of follicle-stimulating hormone receptor messenger ribonucleic acid levels in cultured rat granulosa cells. 131 Dec 35

The ligand specificity and biochemical properties of the human (h) FSH receptor are poorly characterized due to the low abundance of these receptors and the limited availability of human tissues. Using a fragment of rat FSH receptor cDNA, we screened a human testicular cDNA library and obtained a FSH receptor cDNA covering the entire amino acid-coding region. After transfection of a human fetal kidney cell line (293) with the hFSH receptor cDNA, radioligand receptor analysis revealed the presence of high affinity (Kd, 1.7 x 10(-9) M) FSH-binding sites on the plasma membrane. Both recombinant and wild-type hFSH displaced [125I]hFSH binding, with ED50 values of 25 and 70 ng/ml, respectively, whereas hLH, hCG, and hTSH were ineffective. Although human, rat(r), and ovine FSH as well as equine CG competed for rat testicular FSH receptor binding, only hFSH and rFSH interacted effectively with the recombinant hFSH receptor, suggesting that species-specific ligand recognition exists between human and rodent receptors. After incubation of transfected cells with hFSH, but not recombinant hLH or hCG, a dose-dependent increase (ED50, 10 ng/ml) in extracellular cAMP accumulation was observed, indicating a functional coupling of the expressed human receptor with the endogenous adenyl cyclase. In cells cotransfected with the FSH receptor expression plasmid and a luciferase reporter gene driven by the promoter of a cAMP-responsive gene, treatment with hFSH, but not hCG, resulted in a dose-dependent increase in luciferase activity. Northern blot analysis using a cRNA probe derived from the human receptor cDNA indicated the presence of multiple FSH receptor mRNA transcripts (7.0, 4.2, and 2.5 kilobases) in RNA prepared from human follicular phase ovary, but not from human corpus luteum or placenta. Additionally, two FSH-binding sites of 76 and 112 kilodaltons were detected in transfected 293 cells after ligand/receptor cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. These results demonstrate the expression of functional hFSH receptor with unique ligand specificity and provide new data on the biochemical properties of the human receptor at the mRNA and protein levels.
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PMID:Expression of recombinant human follicle-stimulating hormone receptor: species-specific ligand binding, signal transduction, and identification of multiple ovarian messenger ribonucleic acid transcripts. 132 83

We have previously described FSH receptor-mediated influx of 45Ca++ in cultured Sertoli cells from immature rats and receptor-enriched proteoliposomes via activation of voltage-sensitive and voltage-independent calcium channels. We have further shown that this effect of FSH does not require cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding protein or activation of adenylate cyclase. In the present study, we have identified regions of human FSH-beta-subunit which appear to be involved in mediating calcium influx. We screened 11 overlapping peptide amides representing the entire primary structure of hFSH-beta-subunit for their effects on 45Ca++ flux in FSH receptor-enriched proteoliposomes. hFSH-beta-(1-15) and hFSH-beta-(51-65) induced uptake of 45Ca++ in a concentration-related manner. This effect of hFSH-beta-(1-15) and hFSH-beta-(51-65) was also observed in liposomes lacking incorporated FSH receptor, suggesting that the peptide amides may act as ionophores or channel-formers. Reducing membrane fluidity by incubating liposomes (containing no receptor) with hFSH-beta-(1-15) or hFSH-beta-(51-65) at temperatures lower than the transition temperatures of their constituent phospholipids resulted in no significant (P greater than 0.05) difference in 45Ca++ uptake. The effectiveness of the calcium ionophore A23187, however, was abolished. Ruthenium red, a voltage-independent calcium channel antagonist, was able to completely block uptake of 45Ca++ induced by hFSH-beta-(1-15) and hFSH-beta-(51-65) whereas nifedipine, a calcium channel blocker specific for L-type voltage-sensitive calcium channels, was without effect. These results suggest that in addition to its effect on voltage-sensitive calcium channel activity, interaction of FSH with its receptor may induce formation of transmembrane aqueous channels which also facilitate influx of extracellular calcium.
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PMID:Synthetic peptides corresponding to human follicle-stimulating hormone (hFSH)-beta-(1-15) and hFSH-beta-(51-65) induce uptake of 45Ca++ by liposomes: evidence for calcium-conducting transmembrane channel formation. 164 50

We have previously reported that FSH stimulates flux of 45Ca2+ into cultured Sertoli cells from immature rats via voltage-sensitive and voltage-independent calcium channels. In the present study, we show that this effect of FSH does not require cholera toxin (CT)- or pertussis toxin (PT)-sensitive guanine nucleotide binding (G) protein or activation of adenylate cyclase (AC). Significant stimulation of 45Ca2+ influx was observed within 1 min, and maximal response (3.2-fold over basal levels) was achieved within 2 min after exposure to FSH. FSH-stimulated elevations in cellular cAMP paralleled increases in 45Ca2+ uptake, suggesting a possible coupling of AC activation to 45Ca2+ influx. (Bu)2cAMP, however, was not able to enhance 45Ca2+ uptake over basal levels at a final concentration of 1000 microM, although a concentration-related increase in androstenedione conversion to estradiol was evident. Exposure of Sertoli cells to CT (10 ng/ml) consistently stimulated basal levels of androstenedione conversion to estradiol but had no effect on basal levels of 45Ca2+ uptake. Similarly, CT had no effect on FSH-induced 45Ca2+ uptake, but potentiated FSH-stimulated estradiol synthesis. PT (10 ng/ml) augmented basal and FSH-stimulated estradiol secretion without affecting 45Ca2+ influx. The adenosine analog N6-phenylisopropyladenosine, which binds to Gi-coupled adenosine receptors on Sertoli cells, inhibited FSH-stimulated androgen conversion to estradiol in a dose-related (1-1000 nM) manner, but FSH-stimulated 45Ca2+ influx remained unchanged. Our results show that in contrast to FSH-stimulated estradiol synthesis, the flux of 45Ca2+ into Sertoli cells in response to FSH is not mediated either directly or indirectly by CT- or PT-sensitive G protein, nor does it require activation of AC. Our data further suggest that the FSH receptor itself may function as a calcium channel.
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PMID:Follicle-stimulating hormone receptor-mediated uptake of 45Ca2+ by cultured rat Sertoli cells does not require activation of cholera toxin- or pertussis toxin-sensitive guanine nucleotide binding proteins or adenylate cyclase. 216 27

The cAMP outputs by granulosa cells from 3-4.5 mm diameter (medium) follicles of Booroola FF ewes were similar to those by cells from greater than or equal to 5 mm diameter (large) follicles of ++ ewes with respect to time or dose of FSH, cholera toxin or forskolin. Likewise, the cAMP outputs by cells from 1-2.5 mm diameter (small) FF follicles were similar to those by cells from small and medium ++ follicles with respect to time or dose of FSH, cholera toxin or forskolin. At FSH, cholera toxin or forskolin doses of 1 microgram/ml, 0.5 microgram/ml and 10(-4) M respectively, the granulosa cell cAMP outputs of medium FF or large ++ follicles were approximately 2-fold (P less than 0.05) higher than in the respective small FF and medium ++ follicles. The effects of cholera toxin plus forskolin or FSH plus forskolin were additive irrespective of genotype or follicle size, with significant differences (P less than 0.05) observed between follicle sizes but not genotype. No differences were noted between cholera toxin plus forskolin or FSH plus forskolin on granulosa cell cAMP output. For the FSH and forskolin treatments, increased mean cAMP outputs were evident after 10 min, whereas after cholera toxin treatment they were not evident until after 20 min incubation. For all treatments the rate of cAMP production tended to slow down after 40-60 min. Pre-incubation of granulosa cells with pertussis toxin subsequently resulted in a significantly greater (P less than 0.05) FSH-induced output of cAMP relative to the untreated controls irrespective of follicle size. However, no gene-specific differences were noted when the cAMP outputs of cells from medium or small FF follicles were compared with cells from large or small-medium ++ follicles respectively. These results indicate that the activity (or composition) of the regulatory and catalytic components of adenylate cyclase in the FF granulosa cells change in a manner similar to those observed in ++ cells with the only difference being that the increases in cyclase in FF ewes occurs as follicles enlarge from 1-2.5 to 3-4.5 mm in diameter, whereas in ++ ewes they occur as follicles enlarge from 3-4.5 to greater than or equal to 5 mm in diameter. No evidence was found to link the F gene to the granulosa cell cAMP response independently of follicle size. It is suggested that the association between the F gene and the size-specific difference in follicle maturation may be unrelated to the FSH receptor/cAMP generating system.
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PMID:Effects of follicle stimulating hormone, cholera toxin, pertussis toxin and forskolin on adenosine cyclic 3',5'-monophosphate output by granulosa cells from Booroola ewes with or without the F gene. 216 31

We have raised polyclonal antibodies in rabbits against the FSH receptor, purified from calf testis and isolated the IgG fraction from the immune serum (immune IgG) by protein A affinity chromatography. When the immune IgG was incubated with purified, radioiodinated FSH receptor, the resulting complex could be immunoprecipitated by goat anti-rabbit gamma globulin. The immunoprecipitate, after dissociation of receptor from antibody, separation by SDS-PAGE under reducing conditions, and autoradiography, showed the presence of a approximately 60 kDa protein previously identified as a component of the FSH receptor. Binding of 125I-hFSH to membrane-bound receptors was inhibited in a concentration-dependent manner by immune IgG (Ed50 = 90 micrograms/ml). Nonimmune serum or IgM/IgA fractions from immune serum had no effect. 125I-labeled immune IgG bound specifically to testis membranes and the binding could be inhibited in a concentration-dependent manner by ovine FSH. These results suggest that the FSH-binding site and the antibody-binding site on the receptor are proximate or identical. Immune IgG mimicked the ability of FSH to stimulate basal adenylate cyclase activity and conversion of androstenedione to estradiol in cultured immature rat Sertoli cells. Stimulatory but submaximal effects of FSH were augmented by immune IgG. Rat Sertoli cells treated with IgG fractions from immune serum showed an intense fluorescent staining of plasma membrane receptor. No fluorescent staining of receptor was seen when preimmune IgG was used or in the presence of excess ovine FSH. These observations suggest that the polyclonal receptor antibody capable of recognizing FSH receptor behaved as an FSH binding competitor, but was also active as an agonist producing the biological effect of FSH in vitro. The effectiveness of antibodies against FSH receptor in stimulating estradiol synthesis suggests that the information needed for FSH signal transduction resides in the membrane receptor rather than in the hormone molecule. Such antibodies may offer a useful probe for further study of FSH receptor structure and mechanism of hormone action.
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PMID:Polyclonal antibodies against follitropin (FSH) receptor interfere with hormone binding, but mimic the effects of FSH. 240 17

Aprotinin (bovine pancreatic trypsin inhibitor), a serine protease inhibitor, caused a dose-dependent inhibition of [125I]human FSH ([ 125I]hFSH) binding to 1) an FSH receptor-enriched light membrane fraction prepared from bovine calf testes homogenates, 2) Triton X-100-solubilized FSH receptor, and 3) proteoliposomes containing incorporated FSH receptor-G-protein-adenylate cyclase (AC) complexes. Equilibrium binding studies with the solubilized receptor indicated that the effect of aprotinin on [125I]hFSH binding was due to a decrease in the Ka of the receptor, with no change in FSH-binding capacity. The rate of association of [125I]hFSH with its receptor was reduced by 50% in the presence of aprotinin, but no effect on dissociation of FSH-receptor complexes was evident. Aprotinin, at a concentration (250 microM) that inhibited binding of [125I]hFSH to the membrane receptor by 25%, completely inhibited basal, fluoride-stimulated and FSH-stimulated AC activity. However, aprotinin, at a concentration (50 microM) that had little effect on [125I]hFSH binding, markedly enhanced basal AC activity (3.4-fold) to the level of fluoride and FSH stimulation. Aprotinin did not inhibit [3H]5'-guanylylimidodiphosphate binding to FSH receptor-enriched membranes, suggesting that its effects on the affinity of the receptor for FSH and on AC activation were not mediated through an interaction with FSH receptor-associated G-protein. No serine protease activity could be detected in any of the receptor or hormone preparations used in this study. The ability of aprotinin to inhibit binding of [125I]hFSH to the Triton X-100-solubilized receptor and to the soluble receptor incorporated into proteoliposomes as well as to the FSH receptor-enriched membrane fraction, all of which are free of serine protease activity, is consistent with the notion that aprotinin may directly interact with the FSH receptor to sterically hinder binding of FSH. Furthermore, the apparent direct effect of aprotinin on basal as well as FSH-stimulated AC activity suggests its general usefulness in studies on the mechanism of signal transduction for ligands thought to operate via the cAMP second messenger system.
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PMID:The effects of aprotinin on follicle-stimulating hormone binding and signal transduction in bovine calf testis. 247 67

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.
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PMID:The follicle-stimulating hormone (FSH) receptor in testis: interaction with FSH, mechanism of signal transduction, and properties of the purified receptor. 249 20

We have previously reported incorporation of Triton X-100-solubilized bovine calf testis membrane protein into liposomes. The resulting proteoliposomes responded to FSH by exchange of bound GDP for [3H]5'-guanylyl imidodiphosphate ([3H]Gpp(NH)p) and by activation of adenylate cyclase (AC) (Grasso, P., Dattatreyamurty, B. and Reichert, L.E., Jr. (1988) Mol. Endocrinol. 2, 420-430). This model system was utilized to study the effects of FSH on the quaternary structure of FSH receptor-associated GTP-binding protein by comparing the gel filtration profiles of proteoliposomes solubilized with Triton X-100 after exposure to [3H]Gpp(NH)p in the presence or absence of FSH. FSH caused a redistribution of radioactivity (due to bound [3H]Gpp(NH)p) from a high molecular weight fraction (Mr greater than 100,000) to a fraction of much lower molecular weight (Mr approximately 23,000). These results are interpreted to reflect an FSH-induced dissociation of [3H]Gpp(NH)p-bound G protein from its receptor-associated complex. The apparent Mr of approximately 23,000 for the FSH receptor-associated GTP-binding protein suggests that it may represent yet another member of a family of low molecular weight GTP-binding proteins, possibly a ras gene product, recently identified in various mammalian tissues.
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PMID:Follicle stimulating hormone (FSH) induces G protein dissociation from FSH receptor-G protein complexes in reconstituted proteoliposomes. 250 56

It has been suggested that resident ovarian macrophages may play a role in the regulation of ovarian function through local paracrine secretion of regulatory molecule(s). It is the objective of the in vitro studies reported herein to evaluate the potential ovarian relevance of one such macrophage product, tumor necrosis factor alpha (TNF-alpha). To this end, use was made of a primary culture system of rat ovarian granulosa cells, the functional status of which was monitored by the acquisition of estrogen, progestin, and proteoglycan biosynthetic capacity. Whereas treatment with the gonadotropin follicle-stimulating hormone (FSH), a potent functional regulator, resulted in a substantial increase in the extent of aromatization (conversion of androgenic steroid precursors to estrogens), concomitant exposure to TNF-alpha (10 ng/ml) produced significant (p less than 0.05), yet reversible inhibition (71 +/- 7%) of this FSH effect. This specific activity of TNF-alpha was characterized by a projected minimal effective dose of less than 0.1 ng/ml, an apparent median inhibitory dose of 0.56 +/- 0.14 ng/ml, and a minimal time requirement of 48 h. Significantly, the direct effect of TNF-alpha could not be accounted for by a decrease in cellular viability or plating efficiency, nor by a decrease in the number of cells or their DNA content. Instead, TNF-alpha inhibited FSH hormonal action at the level of stimulatable adenylate cyclase activity, exerting no apparent effect either at the level of the FSH receptor or at site(s) of action distal to cAMP generation. The effect of TNF-alpha was not limited to the attenuation of estrogen biosynthesis, exerting qualitatively similar effects on FSH-supported progestin and proteoglycan biosynthetic capacity. As such, these findings are in keeping with the notion that subnanomolar concentrations of TNF-alpha, possibly of ovarian macrophage origin, may comprise the signal of a paracrine loop designed to attenuate gonadotropin action thereby playing a potential role in the development and/or demise of the ovarian follicle.
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PMID:Tumor necrosis factor alpha inhibits gonadotropin hormonal action in nontransformed ovarian granulosa cells. A modulatory noncytotoxic property. 254 76


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