Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca 2؉ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of ␣T3-1 cells with GnRH reduced the spike Ca 2؉ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) by 30 -50%. Ca 2؉ -free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P 3 generation, implying that attenuation of the Ca 2؉ response underlies the Ins(1,4,5)P 3 reduction rather than vice versa. Intracellular Ca 2؉ pool depletion cannot explain desensitization of the Ca 2؉ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (halftimes 10 -20 min and 4 -6 h). Moreover, 1-h GnRH pretreatment attenuated the spike Ca 2؉ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca 2؉ -free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca 2؉ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P 3 of Ca 2؉ from an intact intracellular pool.

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been found at its highest tissue concentrations in the anterior pituitary, where it is localised in gonadotrophs. Its specific guanylyl cyclasecontaining receptor, GC-B, is also expressed on several anterior pituitary cell types, and CNP potently stimulates cGMP accumulation in rat pituitary cell cultures and pituitary cell lines. The mouse gonadotroph-derived T3-1 cell line has been shown to express CNP as well as GC-B (but not GC-A) receptors, suggesting that CNP may well be an autocrine regulator of gonadotrophs.Comparing effects of three natriuretic peptides (atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and CNP) on cGMP accumulation in four pituitary cell lines ( T3-1, TtT-GF, AtT-20 and GH 3 ) we find that CNP is most potent and effective in T3-1 cells. In these cells, CNP-stimulated cGMP accumulation was found to desensitise during a 30 min exposure to CNP.Pretreatment with CNP for up to 6 h also caused a significant reduction in the ability of CNP to subsequently stimulate cGMP accumulation. This effect was receptor specific, because pretreatment with sodium nitroprusside (an activator of nitric oxide-sensitive guanylyl cyclase), or with ANP or BNP, did not cause desensitisation of CNP-stimulated cGMP accumulation. Protein kinase C activation with phorbol esters also inhibited CNPstimulated cGMP accumulation and such inhibition was also seen in cells desensitised by pretreatment with CNP. Thus it appears that the endogenous GC-B receptors of T3-1 cells are subject to both homologous and heterologous desensitisation, that the mechanisms underlying these forms of desensitisation are distinct, and that cGMP elevation alone is insufficient to desensitise GC-B receptors.

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of GnRH-stimulated gonadotropin release, but the mechanisms involved are poorly understood. Recent studies have suggested, however, that GnRH-stimulated phosphoinositidase C (PIC) activity does not undergo rapid ( < 5 min) homologous desensitization in alpha T3-1 cells, and we have, therefore, used this cell line to address the question of whether desensitization occurs distal to PIC activity and/or in an intermediate time frame. We show that GnRH stimulates a rapid increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; maximum at 10-20 sec with a modest reduction thereafter] and that the GnRH-stimulated accumulation of [3H]IPs (in cells stimulated in the presence of LiCl) increases linearly over 5-300 sec. This clearly indicates that desensitization of PIC does not occur within this period and that the dramatic reduction in the rate of Ins(1,4,5)P3 accumulation (10-30 sec) is due to its metabolism, rather than to a reduction in Ins(1,4,5)P3 generation. Pretreatment for 60 min with 10(-7)M GnRH reduced cell surface GnRH receptor number by 48% (without measurably altering Kd). The pretreatment also reduced maximal GnRH-stimulated [3H]IP accumulation (to 66% of the control) and increased the EC50 for GnRH-stimulated [3H]IP accumulation approximately 3-fold, demonstrating that desensitization of GnRH-stimulated [3H]IP accumulation can, indeed, occur within 60 min, but that this may be attributable to receptor loss (without appreciable uncoupling of residual receptors from their immediate effector system). Pretreatment for 60 min with GnRH also caused a dose-dependent reduction in both spike and plateau phases of the GnRH effect on cytosolic Ca2+. This effect could not be overcome by stimulation with high concentrations of GnRH and appears, therefore, to reflect not only receptor loss, but, also, an additional inability of agonist-occupied GnRH receptors to elevate cytosolic Ca2+. The effect of KCl on cytosolic Ca2+ was similarly reduced by GnRH pretreatment, suggesting that desensitization of voltage-operated Ca2+ channels mediates desensitization of the plateau phase Ca2+ response to GnRH. Such a mechanism could not, however, explain desensitization of the spike phase of the Ca2+ response to GnRH seen in normal or Ca2+ -free medium. Accordingly, the data reveal a novel mechanism for homologous desensitization to GnRH in which agonist-occupied GnRH receptors are rendered unable to mobilize intracellular Ca2+ and imply that desensitization of GnRH-stimulated Ins(1,4,5)P3 production and/or action occurs.

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Mutation ofN-glycosylation sites in the mouse gonadotropin-releasing hormone receptor was previously shown to impair its expression in COS-1 cells. We therefore investigated the effects of adding an extra glycosylation site to the human gonadotropin-releasing hormone receptor, as a means for increasing its expression. Covalent labeling of the mutant receptor expressed in COS-1 cells with a gonadotropin-releasing hormone (GnRH) photoreactive analog demonstrated a shift in apparent molecular weight, indicating that the new site was in fact glycosylated. The receptor with extra glycosylation site displayed normal binding affinities for agonists buserelin and [D: -Ala(6)-Pro(9)-NHEt]-GnRH, and the antagonist antide, and a slightly increased affinity for GnRH. Receptor number was increased by 1.7-fold in membrane preparations from cells expressing the mutant receptor, compared with wild-type. Photoaffinity labeling of cell-surface receptors in intact cells demonstrated a 1.8-fold increase in binding sites on the cell surface. The GnRH receptor (GnRHR) with extra glycosylation site conferred a markedly enhanced signaling response to agonist. Dose-response curves for GnRH-stimulated inositol phosphate production were left-shifted by an average of 4.4-fold, and maximal inositol phosphate responses were increased by 1.2 fold, in cells transfected with mutant compared with wild-type receptor, indicating that the increase in binding sites represented functional receptors. These results demonstrate that addition of an extra glycosylation site enhances expression of the human GnRHR, a strategy that may be applicable to other cell-surface receptors.