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)

Mono-(2-ethylhexyl)phthalate (MEHP) is one of a number of phthalate esters known to damage the rat testis with the Sertoli cell as its target. These effects can be modeled using primary testicular cell cultures. Stimulation of rat Sertoli cells by follicle-stimulating hormone (FSH) results in an increase in release of cAMP into the culture medium. However, cultures pretreated with MEHP (10(-7)-10(-4) M) showed a dose-related reduction in FSH-stimulated cAMP production (maximally greater than 50%), suggesting that MEHP is interfering with the FSH receptor-adenyl cyclase system. Detailed investigations of the system were conducted utilizing forskolin and choleratoxin, which both stimulate adenyl cyclase but bypass the FSH receptor to produce an increase in cAMP secretion. Cultures pretreated with MEHP (10(-9)-10(-5)M) showed no reduction in either forskolin- or choleratoxin-stimulated cAMP production. However, at low doses (10(-8)-10(-6)M) MEHP produced a potentiation (up to three times) of both forskolin- and choleratoxin-stimulated cAMP secretion, but gave slight inhibition at 5 X 10(-5) and 10(-4) M. These data indicate that MEHP produces a perturbation at the level of the FSH receptor, causing an inhibition of FSH action. This finding may be related to the age-dependent toxicity of the compound, since FSH is critical for the initiation of spermatogenesis in young animals, but is not necessary for the maintenance of spermatogenesis in adults. Thus an effect on FSH responsiveness in vitro may provide an indication of the mode of action of MEHP in vivo.
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PMID:Effect of mono-(2-ethylhexyl)phthalate on follicle-stimulating hormone responsiveness of cultured rat Sertoli cells. 284 63

Basal and gonadotropin stimulated adenylate cyclase activity was assessed in testicular tissues obtained from men (20-80 years). A disparity was observed in the gonadotropin responsiveness of the human testicular adenylate cyclase system to hFSH and hCG stimulation. Of the tissues analyzed, 61% were FSH responsive and 22% showed low response to hCG. Forskolin, a diterpene which activates adenylate cyclase by a receptor independent mechanism, stimulated adenylate cyclase activity in the gonadotropin unresponsive tissues. This suggests that the tissue unresponsiveness is due to an uncoupling of the catalytic subunit of the adenylate cyclase. Several functional properties of the FSH responsive human testicular adenylate cyclase were investigated. hFSH and oFSH stimulated the enzyme activity in a concentration dependent manner. However, the hormone (DG-oFSH) in which 80% of the carbohydrate residues had been removed was inactive, despite its good binding ability to the FSH receptor. hFSH stimulated adenylate cyclase activity was inhibited by DG-oFSH but not by DG-hCG (deglycosylated hCG). The data demonstrates the existence of specific FSH and LH(hCG) receptors in human testicular membranes. The FSH receptors in some tissues are coupled to adenylate cyclase. The link between the FSH receptor and adenylate cyclase may be uncoupled in the presence of the deglycosylated form of oFSH resulting in a loss of hormone response.
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PMID:Characterization of gonadotropin-sensitive adenylate cyclase activity in human testis: uncoupling of the receptor-cyclase complex by specific hormonal antagonist. 299 80

We have previously reported detergent (Triton X-100) solubilization of a follitropin (FSH) receptor-rich fraction from light membranes of bovine testis that responded to exogenous FSH by activation of adenylate cyclase (Dattatreyamurty, B., Schneyer, A., and Reichert, L. E., Jr. (1986) J. Biol. Chem. 261, 13104-13113). Upon gel filtration of the detergent-extract through Sepharose-6B, two fractions were separated. Each specifically bound [3H]guanosine 5'-imidotriphosphate (Gpp(NH)p) and had guaninetriphosphatase (GTPase) activity. Of these, one fraction (6B-Fraction-1) also bound radioiodinated human follitropin (hFSH), indicating a coelution of the nucleotide-binding protein with receptor. The other fraction (6B-Fraction-2) did not contain detectable FSH receptor activity. Several lines of evidence suggest that 6B-Fraction-1 is a complex consisting of FSH receptor and a guanine nucleotide regulatory protein, probably Ns. 1) The GTP-binding and FSH-binding activities of 6B-Fraction-1 were retained by a GTP-affinity column, and their retention by the affinity matrix could be prevented by simultaneous addition of free Gpp(NH)p. 2) When exogenous GTP was added to 6B-Fraction-1, binding of 125I-hFSH was reduced compared to controls lacking exogenous GTP. This effect of GTP was highly specific and noncompetitive, indicating that GTP did not bind to receptor. In addition, the affinity of receptor for FSH was decreased, and the rate and degree of dissociation of bound labeled FSH from receptor were increased in the presence of exogenous GTP, each in concentration-dependent manner. 3) Exposure of 6B-Fraction-1 to higher concentration of Triton X-100 reduced significantly the receptor-associated GTP-binding activity and also rendered the hormone-binding activity insensitive to GTP. 4) Treatment of highly purified testis membranes with cholera toxin plus NAD, but not pertussis toxin plus NAD, eliminated the ability of GTP to modulate the 125I-hFSH binding to receptor. 5) After cholera toxin-induced [32P]ADP-ribosylation of testis membranes, a major peak of radioactivity (presumably Ns) was coeluted with FSH receptor activity from the Sepharose-6B column. These results and the observation that the effect of GTP is noncompetitive at FSH receptor level suggest that FSH binding inhibition and the increased rate of hormone dissociation from receptor were the result of GTP interaction with a guanine nucleotide regulatory protein, probably Ns, which itself was functionally associated with the FSH receptor.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein. 311 50

An FSH receptor-enriched fraction that responds to exogenous FSH by activation of adenylate cyclase was prepared by ultrafiltration of sucrose density gradient-purified light membranes derived from bovine calf testes homogenates and solubilized with Triton X-100. To further confirm the functional nature of the detergent-solubilized FSH receptor, the extract was incorporated by lipid hydration into large multilamellar vesicles composed of dioleoyl phosphatidylcholine and cholesterol, 2:1 molar ratio. Receptor incorporation was determined by measurement of specific binding of [125I] human FSH ([125I] hFSH). Substitution of dioleoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine or increasing the cholesterol concentration of the vesicles reduced specific binding of [125I]hFSH. Under conditions favoring optimal incorporation of the receptor, specific binding of [125I]hFSH was time and temperature dependent and saturable when increasing concentrations of radioligand were added to a constant amount of proteoliposomes. Reconstituted proteoliposomes bound 1600 fmol FSH/mg protein with an affinity of 3.54 x 10(9) M-1. Inhibition of [125I] hFSH binding by hFSH was comparable to that seen with the membrane-bound receptor (ED50 = 10 ng). Equilibrium binding studies with [3H]Gpp(NH)p indicated that a single class of high affinity GTP binding sites with an association constant (Ka) of 3.33 x 10(7) m-1 which bound 2.19 fmol [3H]Gpp(NH)p/mg protein had also been incorporated into the proteoliposomes. Addition of FSH induced a 2-fold stimulation of [3H]Gpp(NH)p binding, supporting our earlier studies suggesting that the detergent-solubilized FSH receptor is complexed to the G protein. Of particular significance in the present study was the observation that both NaF and FSH stimulated cAMP production in the reconstituted system. In addition to belonging to a class of membrane receptors functionally and physically associated with G protein, this observation suggests that FSH receptors in bovine calf testicular membranes may be associated, at least in part, with adenylate cyclase as well.
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PMID:Reconstitution of hormone-responsive detergent-solubilized follicle stimulating hormone receptors into liposomes. 313 32

Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.
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PMID:Solubilization of functional and stable follitropin receptors from light membranes of bovine calf testis. 375 49

The interaction of FSH with membrane receptors from rat and calf testis has been studied in some detail. The FSH receptor has been solubilized through use of the nonionic detergent Triton X-100 and highly purified by affinity chromatography on Affigel-10 coupled to ovine FSH. Hormone binding activity of the solubilized receptor has been preserved for extended periods through use of the structure-stabilizing agent glycerol. Other components of the FSH testes receptor system including the guanyl nucleotide binding protein and adenylate cyclase have been solubilized by nonionic detergents and also found to be stabilized by glycerol. FSH binding activity has been observed in testes cytosol and represents a putative class of receptors prepared from testes in the absence of detergent. The concentration of this buffer-soluble component decreased with age and increased concomitantly with loss of membrane receptors consequent to their down-regulation after administration of exogenous FSH. Phospholipids seem involved in the interaction of FSH with membrane-bound, detergent-solubilized, and buffer-soluble FSH binding activity. Phospholipids may maintain or stabilize a particular receptor conformation necessary for interaction with the hormone. A specific role for GTP seems indicated in regulation of FSH-stimulated adenylate cyclase activity in immature rat testis. Follitropin binding to testes receptor appears modulated by a variety of factors present in serum, testes extracts, follicular fluid, and seminal plasma, which are poorly understood at present. Inhibition of FSH binding by seminal plasma best-fit by a model proposing two hormone binding sites per receptor molecule, where binding to one site decreases the affinity of the other site for FSH. As a result of studies in this and other laboratories, the molecular endocrinology of FSH interaction with testis receptors is becoming increasingly understood.
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PMID:Biochemical properties of the testicular follitropin-receptor system. 628 87

The regulation of ovarian gonadotropin-sensitive adenylate cyclase and FSH receptors was studied in hypophysectomized diethylstilbestrol-primed rats treated with FSH and/or the potent GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa). The animals were treated with 7.5 micrograms ovine FSH twice daily for 2 days, either alone or with 10 micrograms GnRHa. FSH-stimulated adenylate cyclase activity was augmented by 2.5- to 3.5-fold in the presence of 5'-guanyl-imidodiphosphate. Adenylate cyclase responses to FSH were almost completely abolished by GnRHa treatment in ovarian homogenates from control animals and rats treated with FSH. This inhibition was receptor specific, since GnRHa did not block adenylate cyclase stimulation by prostaglandin E2 or isoproterenol. No inhibition of 5'-guanyl-imidodiphosphate- or sodium fluoride-stimulated adenylate cyclase activity was noted after any hormone treatment. When GnRHa treatment was initiated at 12, 24, or 36 h during the 2-day period of FSH treatment, inhibition of both FSH- and LH-stimulated adenylate cyclase was observed in ovaries collected at 48 h. Whereas FSH treatment increased the ovarian FSH receptor concentration by more than 100%, concomitant treatment with GnRHa prevented this increase and reduced FSH receptors to 60% of the control level. Treatment with GnRHa alone caused a 65% decrease in FSH receptor levels below the untreated control values. Histological analysis of hormone-treated ovaries indicated that FSH stimulated follicle growth and antrum formation, but caused little luteinization. GnRHa did not completely prevent the effects of FSH on follicle growth, but did induce degeneration and premature cleavage of ova. GnRHa alone suppressed the diethylstilbestrol-stimulated mitotic activity, slightly increased degenerative changes in granulosa cells, and caused oocyte cleavage and premature antrum formation. These findings demonstrate that GnRHa inhibits FSH-dependent adenylate cyclase by a mechanism involving the loss of binding sites for FSH. It is also evident that only short term exposure to GnRHa is necessary for expression of the inhibitory action of the peptide upon FSH- and LH-stimulated adenylate cyclase.
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PMID:Inhibitory actions of a gonadotropin-releasing hormone agonist on ovarian follicle-stimulating hormone receptors and adenylate cyclase in vivo. 629 48

Many Sertoli functions are regulated by the receptor-mediated action of follicle-stimulating hormone (FSH). The interaction of FSH with its specific cell surface receptors leads to stimulation of a number of intracellular events, including the activation of guanine nucleotide binding protein (G protein), adenylate cyclase and the cAMP-dependent protein kinase (PKA) pathway. In addition to positive regulation of cell functions, a phenomenon of refractoriness occurs after primary exposure of target cells to the hormone. Different sites of lesion have been suggested including down-regulation of FSH receptor, uncoupling of the receptor and the G protein/adenylate cyclase complex, and stimulation of nucleotide phosphodiesterases or inhibition of PKA activity. Alterations of cell responsiveness are mediated by a combination of these different mechanisms occurring over different time-scales and hormonal concentrations.
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PMID:[Molecular mechanisms of stimulation and desensitization of Sertoli cells by follicle-stimulating hormone]. 773 57

The expression and function of gonadotropin receptors, and the secretion of steroids, transferrin, and cytokines were investigated in three immortalized (single transfection with v-myc) mouse granulosa cell lines (GRM01, GRM01L, and GRM02). A dose-dependent increase in progesterone production was obtained in GRM01 and GRM02 cells after addition of LH, FSH, modulators of the adenylate cyclase enzyme system, and cAMP analogues. The LH-induced release of progesterone was already detectable in GRM02 cells after 8 h and was related to incubation time and cell number. Both epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) induced the secretion of progesterone in GRM02 cells, while no effect was obtained with TGF beta. LH receptor concentration was highest in the GRM02 cell line. FSH receptor mRNA was visualized in GRM01 and GRM02 cells. Aromatase activity in GRM02 cells was induced by androgens and inhibited by aromatase inhibitors. Whereas all cell lines were able to secrete transferrin, only in GRM01 cells was transferrin secretion increased significantly by LH. FSH did not affect transferrin secretion in the three cell lines, in contrast to forskolin or 8-bromo-cAMP. The immortalized mouse granulosa cell lines were able to express and release several growth factors. The expression and secretion of activin, inhibin, TGF beta, EGF, TGF alpha, insulin-like growth factor II, fibroblast growth factor (acidic and basic), platelet-derived growth factor, and interleukin-6 suggest an autocrine or paracrine role for these factors in follicular differentiation and function. In conclusion, these cells, derived from mural granulosa cells and immortalized in a preovulatory state, can be used to study granulosa cell physiology or to study the role of granulosa cells and their derivatives in the process of follicular maturation, fertilization, and early embryonic development.
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PMID:Secretion of steroids, growth factors, and cytokines by immortalized mouse granulosa cell lines. 802 76

The primary sequence of FSH receptor (FSHR) is homologous to LH and TSH receptors (LHR and TSHR). This family of receptors belong to the G-protein-coupled class of membrane-bound receptors. A very large extracellular domain suggests that interaction of ligand with receptor is likely to be complex. Secondary structure analysis of the FSHR R265-S296 primary sequence, which has little homology to LHR, predicted a helix-turn-helix motif. An objective of these studies was to test directly the hypothesis that FSHR R265-S296 is accessible in FSHR and plays a role in hormone binding. Rat FSHR (rFSHR) was expressed in insect cells and used as a source of receptor for binding studies. Recombinant receptor had a Kd in the picomolar range with about 200,000 receptors/cell and appeared as two forms (180 and 75 kilodaltons) by Western blot analysis. Functional coupling of the rat FSHR to adenylate cyclase in insect cells was demonstrated. Antipeptide antibodies against FSHR R265-S296 inhibited binding of radiolabeled hFSH to insect cell rat FSHR. In contrast, neither nonimmune rabbit serum nor antipeptide antibodies against FSHR G150-L183 inhibited the binding of radiolabeled hFSH to rat FSHR in insect cells. Similar results were obtained with recombinant human FSHR in Y1 cells, measuring progesterone production as an end point. Confocal microscopy using antihuman FSHR R265-S296 demonstrated that recombinant human FSHR on Chinese hamster ovary cells existed as discrete patches on the surface. In summary, the data offer compelling evidence that portions of the peptide sequence FSHR R265-S296 are accessible to the antipeptide antibodies and may be involved in hormone binding.
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PMID:Accessibility of rat and human follitropin receptor primary sequence (R265-S296) in situ. 803 17


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