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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The demonstration that GnRH provokes the accumulation of diacylglycerol and the redistribution of protein kinase C to the membrane fraction in gonadotropes suggests a role for this enzyme as a mediator of GnRH action. In the present work we have investigated the possibility that protein kinase C might mediate GnRH-stimulated receptor down-regulation and desensitization. Pretreatment of pituitary cells for 6 h with GnRH (10(-11) - 10(-6) M) caused a biphasic change in GnRH receptor number [the maximum binding (Bmax) for 125I-buserelin binding was increased by 10(-10) M GnRH and reduced by 10(-7) and 10(-6) M GnRH] and caused desensitization (pretreatment with 10(-9) - 10(-6) M GnRH reduced the proportion of cellular LH released in a subsequent challenge with GnRH). Pretreatment for 6 h with 0.2-200 nM phorbol myristate acetate (a protein kinase C-activating phorbol ester) did not cause desensitization, but at 200 nM, did reduce GnRH receptor number. As a further test of the requirement for protein kinase C for GnRH action, cells were depleted of all measurable protein kinase C (and rendered unresponsive to protein kinase C activators) by prior treatment with a high dose of phorbol myristate acetate (500 nM for 6 h followed by 12 h in plating medium). Depletion of protein kinase C did not alter the ability of GnRH to desensitize gonadotropes or down-regulate its own receptors. The demonstration that the effects of GnRH on receptor number and gonadotrope responsiveness are neither blocked by depletion of protein kinase C nor entirely mimicked by activation of protein kinase C suggests that these effects of the releasing hormone are not solely mediated by this enzyme.
Mol Endocrinol 1987 Jun
PMID:Homologous down-regulation of gonadotropin-releasing hormone receptors and desensitization of gonadotropes: lack of dependence on protein kinase C. 285 5

Expression of receptors for the hypothalamic regulatory peptide, gonadotrophin-releasing hormone (GnRH), was investigated by intracellular recording from Xenopus oocytes injected with poly(A)+ mRNA isolated from rat anterior pituitary glands. Membrane depolarizations were induced in oocytes in a dose-dependent fashion following the application of GnRH (10nM - 1 microM) or a GnRH superactive agonist, buserelin (1nM - 1 microM). The response was reversibly blocked by the addition of a GnRH antagonist (1 microM). TRH (10nM - 1 microM) had no effect on most of these oocytes. In contrast, some other oocytes which showed no responses to GnRH or to the GnRH agonist, displayed depolarizing responses to TRH (10nM - 1 microM). A relatively small number of oocytes responded to both ligands. Control oocytes did not respond to the GnRH analogues or to TRH. This successful expression of the GnRH receptor could provide a new approach to the study of the receptor, and serve as a means for the isolation and cloning of the encoding genes.
J Mol Endocrinol 1988 Nov
PMID:Functional expression of rat pituitary gonadotrophin-releasing hormone receptors in Xenopus oocytes. 290 32

GnRH, high potassium concentrations, and cAMP derivatives have been previously shown to increase GnRH receptor levels (GnRH-R) in cultured rat pituitary cells. However, the effect of these changes in receptor number on subsequent stimulated LH release has not been investigated. In this study pretreatment of pituitary cells with either 1 nM GnRH, 58 mM KCl, or 1 mM dibutyryl cAMP (dbcAMP) resulted in a 70-100% increase in GnRH-R 7-10 h later. Subsequent LH responses to GnRH in those cells pretreated with GnRH and KCl were markedly reduced and the dose-response characteristics altered such that the curves were non-sigmoidal. When corrected for depletion of cellular LH during the pretreatment period these GnRH response curves were similar to control, implying that hormone depletion was the explanation for apparent desensitisation. By contrast, dbcAMP and low-dose calcium ionophore (0.1 microM A23187) pretreatment, which did not deplete cellular LH, neither enhanced nor decreased subsequent sensitivity to GnRH. Thus, 4 agents which all, under these conditions, increased GnRH receptors did not sensitise gonadotrophs to GnRH. By contrast, pretreatment with 10(-9) and 10(-8) M GnRH for either 12 or 16 h rendered cells completely or partially refractory to further GnRH stimulation, despite an increase in GnRH receptors. This desensitisation could not be explained by cellular LH depletion, and was specific to the homologous ligand since dose-responses to the Ca2+ ionophore A23187 and KCl were normal when corrected for LH depletion. Non-receptor-mediated depletion of cellular LH during A23187 pretreatment (10 microM for 10 h) did not alter subsequent GnRH dose-responses, after correction for LH content. These data indicate that, under these in vitro conditions, the increased GnRH receptors are not functionally linked to the secretory apparatus of the gonadotroph. Furthermore, homologous ligand-induced desensitisation is both time- and concentration-dependent and is mediated largely by post-receptor cellular events independent of cellular LH content. Therefore, post-receptor cellular processes may be more important than changes in GnRH receptors in regulating gonadotrophin secretion. It is suggested that an increase in GnRH receptors may represent a cellular response to generalised gonadotroph activation by a variety of agents, and does not necessarily signify enhanced responsiveness to GnRH.
Mol Cell Endocrinol 1985 Jun
PMID:Increased gonadotrophin releasing hormone receptors on pituitary gonadotrophs: effect on subsequent LH secretion. 298 39

The present study was designed to test the hypothesis that there is a functional interaction between the calcium-calmodulin system, which appears to mediate the action of gonadotropin-releasing hormone (GnRH), and activators of protein kinase C, which stimulate luteinizing hormone (LH) release by a mechanism which does not require extracellular calcium. We have examined a diacylglycerol and a phorbol ester, which both activate protein kinase C and stimulate LH release. These compounds show synergistic action with calcium ionophore A23187 as secretogogues. Pimozide (a calmodulin antagonist), methoxyverapamil (a calcium ion channel inhibitor), and Ac[D-pCl-Phe1,2-D-Trp3-D-Lys6-D-Ala10]GnRH (a potent gonadotropin-releasing hormone antagonist) were used to show that the diacylglycerol and phorbol ester can stimulate LH release in a manner that is independent of both Ca2+ and calmodulin and do not work by means of a direct action on the GnRH receptor. These observations, coupled with previously published reports that extracellular Ca2+ mobilization is both necessary and sufficient for initiation and perpetuation of GnRH-stimulated LH release, indicate that activation of protein kinase C by endogenous diacylglycerols may serve as an amplifier of the GnRH-stimulated signal which appears to be mediated independently by the Ca2+-calmodulin system.
Mol Pharmacol 1985 May
PMID:Diacylglycerols and protein kinase C. Potential amplifying mechanism for Ca2+-mediated gonadotropin-releasing hormone-stimulated luteinizing hormone release. 315 62

Development of GnRH-mediated gonadotrope desensitization was examined under conditions in which membrane fluidity was altered by temperature and/or chemical means. Cultured pituitary cells were preincubated at temperatures ranging from 4 degrees C to 37 degrees C for 3 h with a desensitizing concentration of GnRH (10(-9) M) or with vehicle alone. Cells were then rinsed and responsiveness assessed by a second 3 h incubation with GnRH at 37 degrees C. As preincubation temperatures decreased from 37 degrees C to 23 degrees C, development of desensitization in gonadotropes was progressively reduced. At 23 degrees C and below, gonadotropes failed to become desensitized to GnRH. Decreases in membrane fluidity occurred over the same temperature range as measured directly by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into plasma membrane. When membrane fluidity was increased by incubating cells with the membrane mobilizing agent 2-(2-methoxy-ethoxy)-ethyl-8-(cis-2-n-octylcyclopropyl)-octanoate (A2C), low temperature blockade of GnRH-mediated gonadotrope desensitization was reversed. A2C had no measurable effects on either GnRH receptor binding or number and caused no cytotoxic effects. These studies suggest that development of gonadotropine desensitization to GnRH can be regulated by the state of membrane fluidity.
Mol Cell Endocrinol 1987 Sep
PMID:Membrane fluidity regulates development of gonadotrope desensitization to GnRH. 366 88

The regulatory actions of FSH and the GnRH agonist [D-Ala6]des-Gly10-GnRH N-ethylamide (GnRHa) upon ovarian GnRH receptors were studied in granulosa cells obtained from ovaries of hypophysectomized diethylstilbestrol-treated rats. When granulosa cells were cultured for 48 h in the presence of FSH (5-250 ng/ml) the binding of 125I-GnRHa to granulosa cell receptors was increased in a dose-dependent manner, to a maximum of 3-4 fold above the control value. Addition of FSH (100 ng) also caused a dose-dependent increase of more than 100-fold in the accumulation of cAMP and progesterone in the culture medium. In freshly prepared cells, Scatchard analysis of GnRHa binding data revealed an equilibrium constant (Ka) of 1.1 x 10(10) M-1 and GnRH receptor concentration fo 401 fmoles/mg protein. Granulosa-cell GnRH receptors decreased during culture without FSH, but were maintained in the presence of 100 ng FSH at 580 femoles/mg protein, with Ka of 0.8 x 10(10) M-1. This action of FSH on GnRH receptors was significantly reduced by 10(-8) M GnRHa. Also, GnRHa concentrations of 10(-10) and 10(-8) M caused inhibition of FSH-induced cAMP and progesterone accumulation. In cells cultured with GnRHa alone, there was a slight enhancement of GnRH receptors by GnRHa concentrations up to 10(-8) M, and a decrease below control levels with higher amounts. Also, GnRHa concentrations from 10(-8) to 10(-5) M caused a 3-4-fold increase in cAMP accumulation in the absence of FSH. These results demonstrate that FSH maintains GnRH receptors in cultured granulosa cells, and that GnRHa attenuates this effect, as well as the other actions of FSH on granulosa cell maturation. It is also evident that GnRHa itself can slightly stimulate cAMP production and partially maintain GnRH receptors, but at high concentrations causes loss of the homologous receptor sites. These findings also emphasize the ability of GnRH agonists to exert both positive and negative direct effects on rat granulosa cell function.
Mol Cell Endocrinol 1982 Jul
PMID:GnRH receptors in cultured rat granulosa cells: mediation of the inhibitory and stimulatory actions of GnRH. 628 94

Previous reports have demonstrated that chronic exposure to high concentrations of gonadotropin-releasing hormone (GnRH) induces a state of refractoriness to GnRH in the pituitary. In order to determine the role of the GnRH receptor in desensitization, we have compared the ability of GnRH to stimulate luteinizing hormone (LH) secretion with changes in GnRH binding. Cultured rat anterior pituitary cells exposed to 1 nM GnRH for 12 h became refractory to this dose of GnRH but were able to release LH in response to higher concentration of GnRH. Exposure to 1 nM or 10 nM GnRH not only caused a shift in the EC50 of GnRH to release LH from 0.28 nM to about 4.5 nM, but also produced a decrease in the maximal response which could not be fully explained by the reduced LH cell content. Examination of GnRH receptor binding to cells pretreated with similar doses of GnRH revealed no change in receptor affinity and a 10-90% increase in receptor number. This paradoxical up-regulation of GnRH receptor number occurred over a period of 6 h and was completely abolished in the presence of cycloheximide. The continuous presence of GnRH was not required for receptor up-regulation since pulses of GnRH were just as effective in increasing GnRH binding. The results indicate that changes in GnRH receptor affinity and number do not always parallel the changes in pituitary responsiveness to GnRH. Therefore, GnRH-induced desensitization cannot be fully explained by down-regulation of receptors and must involve a post-receptor mechanisms.
Mol Cell Endocrinol 1983 Apr
PMID:Desensitization of cultured pituitary cells to gonadotropin-releasing hormone: evidence for a post-receptor mechanism. 630 8

Solubilization in an active form of the pituitary plasma membrane protein carrying binding sites for gonadotropin-releasing hormone (GnRH) was possible by using as detergent CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate), which is a zwitterionic derivative of cholic acid. In contrast, the use of the non-ionic detergent Triton X-100 resulted in the loss of GnRH binding sites. CHAPS at a concentration of 5-10 mM allowed the solubilization of up to 80% of available sites, and remaining non-solubilized sites retained unaltered binding properties. Characterization of solubilized binding sites of GnRH was achieved by using as radioactive ligand and standard a stable GnRH analog. [D-Trp6,(NEt)Pro9,desGly10]-GnRH. Maximum binding of GnRH to solubilized binding sites was possible in the presence of a very low amount of CHAPS in the incubation medium. Scatchard analysis of classical saturation experiments indicated the presence of a single class of high affinity, low capacity binding sites. The mean value for the affinity constant KA was 0.36 +/- 0.07 X 10(10) M -1 (n = 5) for solubilized GnRH binding sites, when analysed in the presence of 2 mM CHAPS; when solubilized GnRH binding sites were analysed in the absence of CHAPS, significantly higher KA values were observed, ranging from 1.2 to 5.9 X 10(10) M -1. Parallel analysis of the plasma membranes prior to solubilization indicated a mean KA value of 0.53 +/- 0.13 X 10(10) M -1 (n = 5). The binding specificity of the pituitary GnRH binding sites as seen by evaluating cross-reactions with a panel of agonists and antagonists of GnRH was not altered by solubilization. Gel filtration on Sephadex G-25 of solubilized binding sites preincubated with radioiodinated GnRH analog demonstrated the presence of a large molecular weight complex. In conclusion, CHAPS appears to be quite suitable for the solubilization of the binding site moiety of the pituitary GnRH receptor with good retention of the binding characteristics, thus offering a new possibility for the purification and characterization of the GnRH receptor protein.
Mol Cell Endocrinol 1983 Jul
PMID:Solubilization of pituitary GnRH binding sites by means of a zwitterionic detergent. 630 85

In the present work we show that Ca2+ is both necessary and sufficient to evoke homologous up-regulation of the gonadotropin-releasing hormone (GnRH) receptor. Extracellular Ca2+ as well as RNA and protein synthesis were required for this event, and it was blocked by Ca2+ ion channel blockers. Drugs which stimulated increased intracellular Ca2+ levels also stimulated receptor up-regulation and enhanced responsiveness even in the absence of added GnRH. Such drugs were effective below the concentrations needed to evoke luteinizing hormone (LH) release, suggesting that enhanced levels of Ca2+ ion, rather than LH depletion, is the responsible agent. A GnRH antagonist did not evoke up- or down-regulation; however, a conjugate of this antagonist, which stimulated microaggregation of the GnRH receptor, also stimulated these biphasic actions. In contrast to up-regulation, down-regulation of the GnRH receptor appears to be Ca2+-independent and does not require RNA or protein synthesis. These data are consistent with a model in which microaggregation of the GnRH receptor is the final step in common to a branched pathway consisting of Ca2+-dependent (LH release, enhanced sensitivity, up-regulation) and Ca2+-independent (desensitization, down-regulation) events.
Mol Pharmacol 1984 Jan
PMID:Biphasic regulation of the gonadotropin-releasing hormone receptor by receptor microaggregation and intracellular Ca2+ levels. 632 52

Gonadotropin secretion of adult male rats was inhibited by one-week treatment with a GnRH antagonist analogue (N-acetyl-Ala1,D-p-Cl-Phe2,D-Trp3,6-GnRH, Ant.) or a GnRH antiserum (A/S). Since Ant. but not A/S binds to GnRH receptors (R), it was expected that comparison of the two treatment regimes would elucidate a physiological role for testicular GnRH-R. Furthermore, the effect of combined gonadotropin and prolactin deficiency was studied in rats treated with Ant. and bromocriptine (BR). Available pituitary GnRH-R were decreased by both Ant. and A/S (P less than 0.01), but not by BR. Testicular content of free GnRH-R was decreased by Ant. by 70-80% (P less than 0.01), probably owing to occupancy, but A/S and BR had no effect on these binding sites. Serum LH and FSH fell by about 75% with Ant. and A/S treatments (P less than 0.01), and Prl by 90% with BR (P less than 0.01). Intratesticular testosterone (T) decreased significantly with Ant. and A/S treatment (P less than 0.01-0.05), but was unaffected by BR. Only Ant. and BR treatments together, but neither alone, were able to suppress Leydig cell capacity to produce T in vitro. Relative blockade of C21 steroid side-chain cleavage by Ant. and A/S was suggested by elevated progesterone (P) and/or P/T ratios in the serum and testis tissues. Ant. and A/S had no effect on testicular LH and FSH-R, but both decreased testicular Prl-R by about 50% (P less than 0.01-0.05).(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Endocrinol 1984 Feb
PMID:Effect of treatment with GnRH antagonist, GnRH antiserum and bromocriptine on pituitary-testicular function of adult rats. 632 69


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