Mammalian Type I Gonadotropin-Releasing Hormone Receptors Undergo Slow, Constitutive, Agonist-Independent Internalization

Medical Research Council Human Reproductive Sciences Unit, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh EH16 4TJ, United Kingdom.
Endocrinology (Impact Factor: 4.5). 04/2008; 149(3):1415-22. DOI: 10.1210/en.2007-1159
Source: PubMed


Regulatory elements present in the cytoplasmic carboxyl-terminal tails of G protein-coupled receptors contribute to agonist-dependent receptor desensitization, internalization, and association with accessory proteins such as beta-arrestin. The mammalian type I GnRH receptors are unique among the rhodopsin-like G protein-coupled receptors because they lack a cytoplasmic carboxyl-terminal tail. In addition, they do not recruit beta-arrestin, nor do they undergo rapid desensitization. By measuring the internalization of labeled GnRH agonists, previous studies have reported that mammalian type I GnRH receptors undergo slow agonist-dependent internalization. In the present study, we have measured the internalization of epitope-tagged GnRH receptors, both in the absence and presence of GnRH stimulation. We demonstrate that mammalian type I GnRH receptors exhibit a low level of constitutive agonist-independent internalization. Stimulation with GnRH agonist did not significantly enhance the level of receptor internalization above the constitutive level. In contrast, the catfish GnRH and rat TRH receptors, which have cytoplasmic carboxyl-terminal tails, displayed similar levels of constitutive agonist-independent internalization but underwent robust agonist-dependent internalization, as did chimeras of the mammalian type I GnRH receptor with the cytoplasmic carboxyl-terminal tails of the catfish GnRH receptor or the rat TRH receptor. When the carboxyl-terminal Tyr325 and Leu328 residues of the mammalian type I GnRH receptor were replaced with alanines, these two mutant receptors underwent significantly impaired internalization, suggesting a function for the Tyr-X-X-Leu sequence in mediating the constitutive agonist-independent internalization of mammalian type I GnRH receptors. These findings provide further support for the underlying notion that the absence of the cytoplasmic carboxyl-terminal tail of the mammalian type I GnRH receptors has been selected for during evolution to prevent rapid receptor desensitization and internalization to allow protracted GnRH signaling in mammals.

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    • "A functional link between cAMP pathway activation and high GnRH pulsatility was also identified in the proestrus-specific elevation of pituitary cAMP levels typical of the rat ovarian cycle (Kimura et al., 1980; Garrell et al., 2010). On the other hand, the unique structural feature of mammalian GnRH-R, lack of a carboxy-terminal tail, is now recognized to be responsible for low level constitutive receptor internalization, as well as for its inability to undergo rapid agonist-induced internalization (Pawson et al., 2008). Thus, GnRH-R can maintain cAMP pathway activation under stimulation conditions that would induce desensitization in most G-protein coupled receptors, GPCRs (Millar et al., 2004; Cohen-Tannoudji et al., 2012). "
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    ABSTRACT: The copper-gonadotropin-releasing hormone molecule (Cu-GnRH) is a GnRH analog, which preserves its amino acid sequence, but which contains a Cu(2+) ion stably bound to the nitrogen atoms including that of the imidazole ring of Histidine(2). A previous report indicated that Cu-GnRH was able to activate cAMP/PKA signaling in anterior pituitary cells in vitro, but raised the question of which intracellular mechanism(s) mediated the Cu-GnRH-induced cAMP synthesis in gonadotropes. To investigate this mechanism, in the present study, female rat anterior pituitary cells in vitro were pretreated with 0.1μM antide, a GnRH antagonist; 0.1μM cetrorelix, a GnRH receptor antagonist; 0.1μM PACAP6-38, a PAC-1 receptor antagonist; 2μM GF109203X, a protein kinase C inhibitor; 50mM PMA, a protein kinase C activator; the protein kinase A inhibitors H89 (30μM) and KT5720 (60nM); factors affecting intracellular calcium activity: 2.5mM EGTA; 2μM thapsigargin; 5μM A23187, a Ca(2+) ionophore; or 10μg/ml cycloheximide, a protein synthesis inhibitor. After one of the above pretreatments, cells were incubated in the presence of 0.1μM Cu-GnRH for 0.5, 1, and 3h. Radioimmunoassay analysis of cAMP confirmed the functional link between Cu-GnRH stimulation and cAMP/PKA signal transduction in rat anterior pituitary cells, demonstrating increased intracellular cAMP, which was reduced in the presence of specific PKA inhibitors. The stimulatory effect of Cu-GnRH on cAMP production was partly dependent on GnRH receptor activation. In addition, an indirect and Ca(2+)‑dependent mechanism might be involved in intracellular adenylate cyclase stimulation. Neither activation of protein kinase C nor new protein synthesis was involved in the Cu-GnRH-induced increase of cAMP in the rat anterior pituitary primary cultures. Presented data indicate that conformational changes of GnRH molecule resulting from cooper ion coordination affect specific pharmacological properties of Cu-GnRH molecule including specific pattern of intracellular activity induced by complex in anterior pituitary cells in vitro.
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    • "Some species have been shown to be particularly resistant to rapid desensitization by GnRH agonists at the pituitary level because they have evolved cell surface GnRH receptors that are slow to internalize (lacking a phosphorylated tail on the receptor) making desensitization and eventual downregulation take longer or never occur [Pawson et al., 2008]. Perhaps female sea otters have this special adaptation within GnRH cell surface receptors in both the pituitary and perhaps the ovaries as well to prevent rapid desensitized to GnRH stimulation as an adaptation to protracted high reproductive hormone levels needed for effective ovulation and the maintenance of a long lived corpus luteum observed in this species [Aspden et al., 1996; Larson et al., 2003; Pawson et al., 2008]. "
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    ABSTRACT: The sea otter (Enhydra lutris) is a popular exhibit animal in many zoos and aquariums worldwide. Captive sea otters from these populations are owned by the United States Fish and Wildlife Service (USFWS). The USFWS has requested that these sea otters be prevented from breeding in order to save captive space for wild rescued animals. Sea otters are often housed in mixed sex groups, therefore a chemical contraceptive method or surgical removal of gonads must be used to prevent potential pregnancy. The contraceptive, Suprelorin® or deslorelin, has been used in many different species to effectively suppress reproduction but duration of effect may vary not only between species but also individuals. Here, we report the effects of one to several consecutive deslorelin implants on gonadal reproductive hormones found in fecal samples from six captive sea otters (two males and four females) compared to two control otters (one male and one female) housed at three zoological institutions. We documented the longitudinal hormone signatures of many stages of the contraceptive cycle including pretreatment (PT), stimulatory phase (S), effective contraception (EC), and hormone reversal (HR) that was characterized by a return to normal hormone levels. Deslorelin was found to be an effective contraceptive in sea otters and was found to be reversible documented by a live birth following treatment, however the duration of suppression in females was much longer than expected with a 6-month and a 1-year implant lasting between 3 and 4 years in females. Zoo Biol. 32:307–315, 2013. © 2012 Wiley Periodicals, Inc.
    Full-text · Article · May 2013 · Zoo Biology
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    • "Whereas these studies were rather measuring ligand internalization, investigations conducted by Millar's group, using an epitope-tagged receptor allowed to trace the receptor itself. These studies established that GnRH-R exhibits a low level of constitutive internalization and does not undergo rapid agonistdependent internalization as compared to non-mammalian GnRH or the TSH-releasing hormone receptors (Pawson et al., 2008). The refractory state of gonadotrope cells under a sustained GnRH challenge is thus believed to occur through desensitization mechanism affecting downstream signaling entities such as Gαq/11, PKC isoforms , or IP3 receptors (Willars et al., 2001; Liu et al., 2003) rather than the receptor itself. "
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    ABSTRACT: The gonadotropin-releasing hormone (GnRH) pulsatile pattern is critical for appropriate regulation of gonadotrope activity but only little is known about the signaling mechanisms by which gonadotrope cells decode such pulsatile pattern. Here, we review recent lines of evidence showing that the GnRH receptor (GnRH-R) activates the cyclic AMP (cAMP) pathway in gonadotrope cells, thus ending a long-lasting controversy. Interestingly, coupling of GnRH-R to the cAMP pathway as well as induction of nitric oxide synthase 1 (NOS1) or follistatin through this signaling pathway take place preferentially under high GnRH pulsatility. The preovulatory surge of GnRH in vivo is indeed associated with an important increase of pituitary cAMP and NOS1 expression levels, both being markedly inhibited by treatment with a GnRH antagonist. Altogether, this suggests that due to its atypical structure and desensitization properties, the GnRH-R may continue to signal through the cAMP pathway under conditions inducing desensitization for most other receptors. Such a mechanism may contribute to decode high GnRH pulsatile pattern and enable gonadotrope cell plasticity during the estrus cycle.
    Full-text · Article · Aug 2012 · Frontiers in Endocrinology
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