Differential internalization of mammalian and non-mammalian gonadotropin-releasing hormone receptors. Uncoupling of dynamin-dependent internalization from mitogen-activated protein kinase signaling

Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.     

Homologous down-regulation of gonadotropin-releasing hormone receptors and desensitization of gonadotropes: lack of dependence on protein kinase C

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.     

Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to beta-arrestin-dependent internalization

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a beta-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize beta-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring beta-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not beta-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via beta-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and beta-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a beta-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of beta-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of beta-arrestin-dependence was observed. Visualization of beta-arrestin/GFP redistribution confirmed a loss or gain of beta-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent beta-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for beta-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by beta-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the beta-arrestin-dependent pathway.     

Heterodimerization of somatostatin and opioid receptors cross-modulates phosphorylation, internalization, and desensitization

Heterodimerization has been shown to modulate the ligand binding, signaling, and trafficking properties of G protein-coupled receptors. However, to what extent heterodimerization may alter agonist-induced phosphorylation and desensitization of these receptors has not been documented. We have recently shown that heterodimerization of sst(2A) and sst(3) somatostatin receptors results in inactivation of sst(3) receptor function (Pfeiffer, M., Koch, T., Schr?der, H., Klutzny, M., Kirscht, S., Kreienkamp, H. J., H?llt, V., and Schulz, S. (2001) J. Biol. Chem. 276, 14027-14036). Here we examine dimerization of the sst(2A) somatostatin receptor and the mu-opioid receptor, members of closely related G protein-coupled receptor families. In coimmunoprecipitation studies using differentially epitope-tagged receptors, we provide direct evidence for heterodimerization of sst(2A) and MOR1 in human embryonic kidney 293 cells. Unlike heteromeric assembly of sst(2A) and sst(3), sst(2A)-MOR1 heterodimerization did not substantially alter the ligand binding or coupling properties of these receptors. However, exposure of the sst(2A)-MOR1 heterodimer to the sst(2A)-selective ligand L-779,976 induced phosphorylation, internalization, and desensitization of sst(2A) as well as MOR1. Similarly, exposure of the sst(2A)-MOR1 heterodimer to the mu-selective ligand [d-Ala(2),Me-Phe(4),Gly(5)-ol]enkephalin induced phosphorylation and desensitization of both MOR1 and sst(2A) but not internalization of sst(2A). Cross-phosphorylation and cross-desensitization of the sst(2A)-MOR1 heterodimer were selective; they were neither observed with the sst(2A)-sst(3) heterodimer nor with the endogenously expressed lysophosphatidic acid receptor. Heterodimerization may thus represent a novel regulatory mechanism that could either restrict or enhance phosphorylation and desensitization of G protein-coupled receptors.     

Internalization rates of murine and ovine gonadotropin-releasing hormone receptors

Rates of internalization of the murine GnRH receptor fused via its C-terminus to green fluorescent protein (GnRH-R-GFP) were examined in Chinese hamster ovary cells (CHO cells) and compared to those of native murine GnRH-R in a clonal murine gonadotroph cell line (LbetaT2 cells). The resulting rates of internalization of murine receptors were then compared with those of sheep GnRH-R in ovine gonadotrophs. Cells were incubated with radioiodinated [D-Ala6]GnRH on ice for 4 h to allow binding of the ligand to GnRH-R, then cells were warmed to 37 degrees C to permit internalization. Surface-bound radioligand began to decrease as soon as the cells were warmed and had decreased significantly within 20 min. A steady-state level of surface-bound radioligand was achieved after 60 min in both CHO cells and LbetaT2 cells (38% and 41%, respectively, of initial value; P < 0.05). Internalization of radioligand began immediately after warming the cells to 37 degrees C, and a significant proportion of surface ligand had been internalized by 20 min. A steady-state maximum of internalization was reached after 60 min in both CHO cells and LbetaT2 cells (29% and 28%, respectively, of total cell-associated ligand; P < 0.05). Changes in surface-bound radioligand and internalized radioligand in sheep pituitary cells were similar to those in CHO cells and LbetaT2 cells, but the amount of radioligand internalized after 60 min (40% of total cell-associated ligand) was 1.4 times higher than in CHO cells and LbetaT2 cells (P < 0.05). In a separate experiment, the effect of estradiol on the rate of internalization of GnRH-R in ovine pituitary cells was examined. Although treatment of ovine pituitary cells with estradiol approximately doubled the number of GnRH receptors, it did not alter either the rate or extent of receptor internalization. These results show that rates of internalization of recombinant murine GnRH-R-GFP in CHO cells and native murine and ovine GnRH-R in LbetaT2 cells and in sheep pituitary cells, respectively, are similar, but amounts of ovine GnRH-R internalized are greater than those for murine GnRH-R. Further, the rate of internalization of occupied receptor is similar in gonadotroph and nongonadotroph cells, and the addition of GFP to the C-terminus of the murine GnRH-R does not alter the rate of internalization.     

Stimulation of pituitary gonadotropin release does not require internalization of gonadotropin-releasing hormone

Binding of gonadotropin-releasing hormone (GnRH, pyro-Glu1-His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-Pro9-Gly-NH210) to its plasma membrane receptor is the first step leading to the release of pituitary luteinizing hormone. As in the case of other plasma membrane receptors, patching, capping, and internalization of this hormone-receptor complex occurs rapidly following exposure of cultured pituitary cells to physiological levels of releasing hormone. In the present study we sought to determine whether gonadotropin release could occur under conditions which rigorously excluded internalization. A GnRH analog, D-Lys6-GnRH (to which a small quantity of [125I]iodoTyr5-D-Lys6-GnRH was added), was coupled by its epsilon-amino group with an N-hydroxysuccinimide ester then, through a 10-A spacer arm, to a cross-linked agarose matrix. Exposure of the product to proteases, soaps, detergents, solvents, chaotropic agents, or cell cultures resulted in dissociation of < 0.28% of biologically active releasing hormone. The apparent potency of the immobilized analog was one-fourth that of the free form and it was still capable of evoking a full luteinizing hormone secretory response. It can, therefore, be concluded that internalization of GnRH is not required for gonadotropin release.     

Differential phosphorylation paradigms dictate desensitization and internalization of the N-formyl peptide receptor.

Following activation by ligand, most G protein-coupled receptors undergo rapid phosphorylation. This is accompanied by a drastic decrease in the efficacy of continued or repeated stimulation, due to receptor uncoupling from G protein and receptor internalization. Such processing steps have been shown to be absolutely dependent on receptor phosphorylation in the case of the N-formyl peptide receptor (FPR). In this study, we report results that indicate that the mechanisms responsible for desensitization and internalization are distinct. Using site-directed mutagenesis of the serine and threonine residues of the FPR carboxyl terminus, we have characterized regions that differentially regulate these two processes. Whereas substitution of all 11 Ser/Thr residues in the carboxyl terminus prevents both desensitization and internalization, substitution of four Ser/Thr residues between 328-332 blocks desensitization but has no effect on internalization. Similarly, substitution of four Ser/Thr residues between positions 334 and 339 results in a deficit in desensitization but again no decrease in internalization, suggesting that phosphorylation at either site evokes receptor internalization, whereas maximal desensitization requires phosphorylation at both sites. These results also indicate that receptor internalization is not involved in the process of desensitization. Further analysis of the residues between 328-332 revealed that restoration either of Ser(328) and Thr(329) or of Thr(331) and Ser(332) was sufficient to restore desensitization, suggesting that phosphorylation within either of these two sites, in addition to sites between residues 334 and 339, is sufficient to produce desensitization. Taken together, these results indicate that the mechanisms involved in FPR processing (uncoupling from G proteins and internalization) are regulated differentially by phosphorylation at distinct sites within the carboxyl terminus of the FPR. The relevance of this paradigm to other G protein-coupled receptors is discussed.     

Role of receptor internalization in the agonist-induced desensitization of cannabinoid type 1 receptors

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is an important mechanism for regulating signaling transduction of functional receptors at the plasma membrane. We demonstrate here that both caveolae/lipid-rafts- and clathrin-coated-pits-mediated pathways were involved in agonist-induced endocytosis of the cannabinoid type 1 receptor (CB1R) in stably transfected human embryonic kidney (HEK) 293 cells and that the internalized receptors were predominantly sorted into recycling pathway for reactivation. The treatment of CB1 receptors with the low endocytotic agonist Δ⁹-THC induced a faster receptor desensitization and slower resensitization than the high endocytotic agonist WIN 55,212-2. In addition, the blockade of receptor endocytosis or recycling pathway markedly enhanced agonist-induced CB1 receptor desensitization. Furthermore, co-expression of phospholipase D2, an enhancer of receptor endocytosis, reduced CB1 receptor desensitization, whereas co-expression of a phospholipase D2 negative mutant significantly increased the desensitization after WIN 55,212-2 treatment. These findings provide evidences for the importance of receptor endocytosis in counteracting CB1 receptor desensitization by facilitating receptor reactivation. Moreover, in primary cultured neurons, the low endocytotic agonist Δ⁹-THC or anandamide exhibited a greater desensitization of endogenous CB1 receptors than the high endocytotic agonist WIN 55,212-2, CP 55940 or 2-arachidonoyl glycerol, indicating that cannabinoids with high endocytotic efficacy might cause reduced development of cannabinoid tolerance to some kind cannabinoid-mediated effects.     

Pituitary desensitization and the regulation of pituitary gonadotropin-releasing hormone (GnRH) receptors following chronic administration of a superactive GnRH analog and testosterone

We recently demonstrated that chronic daily administration of a superactive GnRH analog to intact rats resulted in an initial stimulation of serum LH levels with a subsequent return of LH levels to baseline at a time when testosterone levels were marked decreased. These data demonstrated pituatary desensitization following chronic GnRH analog treatment. Administration of GnRH analog with a dose of testosterone which did not markedly lower serum LH levels when administered alone prevented the stimulation of LH secretion by analog. The present studies were undertaken to determine the effects of GnRH analog and testosterone administration on the regulation of pituitary GnRH receptors. Pituitary GnRH receptor binding was increased by analog treatment alone at 20 days and returned to control levels at 40 and 60 days of treatment in parallel to the observed changes in serum LH, demonstrating that one mechanism by which chronic GnRH analog treatment leads to pituitary desensitization is down-regulation of pituitary GnRH receptors. Testosterone administration alone decreased pituitary GnRH receptor binding. Combined GnRH analog and testosterone administration prevented the increase in pituitary GnRH receptors observed with analog administration alone. These studies demonstrate that changes in pituitary GnRH receptor binding correlate with changes in serum LH and that the stimulatory effects of analog administration on LH are sensitive to inhibition by small doses of testosterone.     

Beta-arrestin mediates desensitization and internalization but does not affect dephosphorylation of the thyrotropin-releasing hormone receptor

The G protein-coupled thyrotropin-releasing hormone (TRH) receptor is phosphorylated and binds to beta-arrestin after agonist exposure. To define the importance of receptor phosphorylation and beta-arrestin binding in desensitization, and to determine whether beta-arrestin binding and receptor endocytosis are required for receptor dephosphorylation, we expressed TRH receptors in fibroblasts from mice lacking beta-arrestin-1 and/or beta-arrestin-2. Apparent affinity for [(3)H]MeTRH was increased 8-fold in cells expressing beta-arrestins, including a beta-arrestin mutant that did not permit receptor internalization. TRH caused extensive receptor endocytosis in the presence of beta-arrestins, but receptors remained primarily on the plasma membrane without beta-arrestin. beta-Arrestins strongly inhibited inositol 1,4,5-trisphosphate production within 10 s. At 30 min, endogenous beta-arrestins reduced TRH-stimulated inositol phosphate production by 48% (beta-arrestin-1), 71% (beta-arrestin-2), and 84% (beta-arrestins-1 and -2). In contrast, receptor phosphorylation, detected by the mobility shift of deglycosylated receptor, was unaffected by beta-arrestins. Receptors were fully phosphorylated within 15 s of TRH addition. Receptor dephosphorylation was identical with or without beta-arrestins and almost complete 20 min after TRH withdrawal. Blocking endocytosis with hypertonic sucrose did not alter the rate of receptor phosphorylation or dephosphorylation. Expressing receptors in cells lacking Galpha(q) and Galpha(11) or inhibiting protein kinase C pharmacologically did not prevent receptor phosphorylation or dephosphorylation. Overexpression of dominant negative G protein-coupled receptor kinase-2 (GRK2), however, retarded receptor phosphorylation. Receptor activation caused translocation of endogenous GRK2 to the plasma membrane. The results show conclusively that receptor dephosphorylation can take place on the plasma membrane and that beta-arrestin binding is critical for desensitization and internalization.     

Engineered G protein coupled receptors reveal independent regulation of internalization,desensitization and acute signaling

Background  

The physiological regulation of G protein-coupled receptors, through desensitization and internalization, modulates the length of the receptor signal and may influence the development of tolerance and dependence in response to chronic drug treatment. To explore the importance of receptor regulation, we engineered a series of G_i-coupled receptors that differ in signal length, degree of agonist-induced internalization, and ability to induce adenylyl cyclase superactivation. All of these receptors, based on the kappa opioid receptor, were modified to be receptors activated solely by synthetic ligands (RASSLs). This modification allows us to compare receptors that have the same ligands and effectors, but differ only in desensitization and internalization.     

Novel localization of NMDA receptors within neuroendocrine gonadotropin-releasing hormone terminals

About 1000 hypothalamic neurons synthesize and release gonadotropin-releasing hormone (GnRH), the master molecule of reproduction in all mammals. At the level of the median eminence at the base of the brain, where GnRH and other hypothalamic releasing hormones are secreted into the capillary system leading to the anterior pituitary gland, there is non-synaptic regulation of neurohormone release by a number of central neurotransmitters. For example, glutamate, the major excitatory amino acid in the brain, directly regulates GnRH release from nerve terminals via NMDA receptors (NMDARs). Moreover, the effects of glutamate action on GnRH secretion are potentiated by estrogens, and this relates to the physiologic control of ovulation by the hypothalamus. We sought to determine the ultrastructural relationship between GnRH neuroterminals and NMDARs, and this regulation by estradiol. Using immunofluorescent confocal microscopy, postembedding immunogold electron microscopy, fractionation, and Western blotting, we demonstrated: (i) GnRH is localized in large dense-core vesicles of neurosecretory profiles/terminals, (ii) the NMDAR1 subunit is found primarily on large dense-core vesicles of neurosecretory profiles/terminals, (iii) there is extensive colocalization of GnRH and NMDAR1 on the same vesicles, and (iv) estradiol modestly but significantly alters the distribution of NMDAR1 in GnRH neuroterminals by increasing expression of NMDAR1 on large dense-core vesicles. Western blots of fractionated median eminence support the presence of NMDAR1 in subcellular fractions containing large dense-core vesicles. These data are the first to show the presence of the NMDAR on neuroendocrine secretory vesicles, its co-expression with GnRH, and its regulation by estradiol. The results provide a novel anatomical site for the NMDAR and may represent a new mechanism for the regulation of GnRH release.     

Expression of pejerrey gonadotropin-releasing hormone in three orders of fish

Molecular variants of GnRH were characterized by reverse-phase, high-performance liquid chromatography from brain extracts of fish in three different orders: Synbranchiformes (swamp eel [Synbranchus marmoratus]), Cyprinidontiformes (platyfish [Xiphophorus maculatus] and green swordtail [X. helleri]), and Atheriniformes (Patagonia pejerrey [Odontesthes hatchery]). Also, pituitary gland extracts from the pejerrey O. bonariensis (Atheriniformes) were characterized. Eluted fractions were tested in radioimmunoassays with antisera specific to GnRH, including both antisera that detected only one form of GnRH and those that detected several forms. The results show that brain extracts obtained from all species contained the same three molecular forms of GnRH, which were immunologically and chromatographically undistinguishable from chicken GnRH-II, pejerrey GnRH (pjGnRH), and salmon GnRH. This study supports the hypothesis that expression of these three forms is common in different fish orders and that pjGnRH is the main regulator of pituitary function in these fish.     

Activation of A-type gamma-aminobutyric acid receptors excites gonadotropin-releasing hormone neurons

Gamma-aminobutyric acid (GABA), acting through GABA(A) receptors (GABA(A)R), is hypothesized to suppress reproduction by inhibiting GnRH secretion, but GABA actions directly on GnRH neurons are not well established. In green fluorescent protein-identified adult mouse GnRH neurons in brain slices, gramicidin-perforated-patch-clamp experiments revealed the reversal potential (E(GABA)) for current through GABA(A)Rs was depolarized relative to the resting potential. Furthermore, rapid GABA application elicited action potentials in GnRH neurons but not controls. The consequence of GABA(A)R activation depends on intracellular chloride levels, which are maintained by homeostatic mechanisms. Membrane proteins that typically extrude chloride (KCC-2 cotransporter, CLC-2 channel) were absent from the GT1-7 immortalized GnRH cell line and GnRH neurons in situ or were not localized to the proper cell compartment for function. In contrast, GT1-7 cells and some GnRH neurons expressed the chloride-accumulating cotransporter, NKCC-1. Patch-clamp experiments showed that blockade of NKCC hyperpolarized E(GABA) by lowering intracellular chloride. Regardless of reproductive state, rapid GABA application excited GnRH neurons. In contrast, bath application of the GABA(A)R agonist muscimol transiently increased then suppressed firing; suppression persisted 4-15 min. Rapid activation of GABA(A)R thus excites GnRH neurons whereas prolonged activation reduces excitability, suggesting the physiological consequence of synaptic activation of GABA(A)R in GnRH neurons is excitation.     

Photoaffinity labeling of parathyroid hormone receptors: comparison of receptors across species and target tissues and after desensitization to hormone

Cells derived from human giant cell tumors of bone and fibroblasts derived from human neonatal foreskin respond to parathyroid hormone (PTH) by increasing the intracellular and extracellular levels of adenosine cyclic 3',5'-phosphate (cAMP). Using photoaffinity labeling methods, we examined these cells for the presence of a PTH receptor or a binding subunit of a receptor complex. A previously designed biologically active and photolabile radioligand analogue of PTH was reacted with these intact cells. After photolysis, the cells were extracted, and the proteins were denatured, reduced, and separated by electrophoresis on sodium dodecyl sulfate (Na-DodSO4)-polyacrylamide gels followed by autoradiography. A single membrane component, Mr 70 000, was labeled specifically in intact cells cultured from skeletal and dermal tissue. By mixing, in pairs, photolabeled proteins from (a) intact human cells derived from giant cell tumors of bone, (b) intact human fibroblasts, and (c) canine renal cortical membranes, the receptors (or their binding subunits) for PTH were compared directly and found to be identical in terms of molecular size (as determined by the migration position on NaDod-SO4-polyacrylamide gels) across species (dog and human) and target tissue (bone, skin, and kidney). Preincubation of cells cultured from giant cell tumors of bone with PTH resulted in loss of the PTH-induced cAMP response (desensitization). Preincubation with PTH was accompanied by a marked decrease in photoaffinity labeling of the PTH binding component and suggests that the loss of hormone response in cells preincubated with PTH was related to a decrease in the number or availability of PTH receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of number of receptors for gonadotropin-releasing hormone on the release of luteinizing hormone

The relationship between number of receptors for gonadotropin-releasing hormone (GnRH) and the ability of the anterior pituitary gland to release luteinizing hormone (LH) was examined in ovariectomized ewes. A GnRH antagonist was used to regulate the number of available receptors. The dose of GnRH antagonist required to saturate approximately 50 and 90% of GnRH receptors in ovariectomized ewes was determined. Thirty min after intracarotid infusion of GnRH antagonist, ewes were killed and the number of unsaturated (i.e., those available for binding) pituitary GnRH receptors was quantified. Infusion of 10 and 150 micrograms GnRH antagonist over a 5-min period reduced binding of the labeled ligand to approximately 50 and 12% of controls, respectively. The effect of reducing the number of GnRH receptors on release of LH after varying doses of the GnRH agonist, D-Ala6-GnRH-Pro9-ethylamide (D-Ala6-GnRH) was then evaluated. One of four doses of D-Ala6-GnRH (0.125, 2.5, 50 and 400 micrograms) was given i.v. to 48 ovariectomized ewes whose GnRH receptors had not been changed or were reduced to approximately 50 or 12% of control ewes. In ewes with a 50% reduction in GnRH receptors, total release of LH (area under response curve) was lower than that obtained for controls (P less than 0.01) at the 0.125-micrograms dose of D-Ala (6.1 +/- 0.7 cm2 vs. 13.5 +/- 0.7 cm2) but was not different at the 2.5-, 50- or 400-micrograms doses of D-Ala6-GnRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential sensitivity of agonist- and antagonist-occupied gonadotropin-releasing hormone receptors to protein kinase C activators. A marker for receptor activation

Gonadotropin-releasing hormone (GnRH) stimulates release of pituitary gonadotropins by activating specific plasma membrane receptors. In the present studies, we have used activators of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) to probe the binding characteristics of agonist- or antagonist-occupied GnRH receptors in intact cell cultures, using a radioligand receptor assay. Specific binding of [125I-Tyr5,D-Ser(tBu)6,Pro9,NHEt]GnRH (Buserelin), a high-affinity GnRH agonist, was increased to 180% of control in the presence of 150 nM phorbol 12-myristate 13-acetate (PMA) or 100 nM phorbol 12,13-dibutyrate (PDB), and to 125% of control in the presence of 200 microM 1,2-dioctanoylglycerol, after 20 min at 23 degrees C. The PMA effects were associated with apparent increases in both binding affinity and number of binding sites. The effects of protein kinase C activators on Buserelin binding were concentration- and time-dependent and were not seen with 4 alpha-PMA or 1,2-dioctanoyl-3-Cl-glycerol, neither of which activate protein kinase C. In contrast, PMA had no measurable effects on specific binding of a GnRH receptor antagonist, Ac[D-pCl-Phe1,2,D-Trp3,125I-Tyr5,D-Lys6,D-Ala10]GnRH. When cell cultures were pretreated with 100 nM PDB in the absence of GnRH and then washed to remove the phorbol ester, no effects of prior protein kinase C activation were detected upon subsequent addition of Buserelin. However, when PDB pretreatment was carried out in the presence of 0.3 microM GnRH, residual enhancement of Buserelin binding, but not antagonist binding, was observed at either 23 or 4 degrees C. The radiolabeled agonist activated, and the antagonist blocked, GnRH receptor-mediated luteinizing hormone release and [3H]inositol phosphate production in cells preloaded with [3H]inositol. These findings suggest that the action of protein kinase C on the GnRH receptor, either direct or indirect, requires the receptor to be in an activated (agonist-occupied) state but does not require receptor internalization. The mechanism of these effects on GnRH agonist binding is not known but may involve sequestration of surface receptors, expression of new receptors, and/or modulation of GnRH receptor affinity.     

Interaction of fluorescent gonadotropin-releasing hormone with receptors in cultured pituitary cells

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)