Agonist-induced internalization of the P2Y2 receptor

The Gq/phospholipase C-linked human P2Y2 receptor was tagged at its amino terminus with the hemagglutinin A (HA) epitope sequence (P2Y2-HA) and stably expressed in 1321N1 human astrocytoma cells. Neither the pharmacological selectivity nor the signaling properties of the receptor were altered by the presence of the epitope. An enzyme-linked immunosorbent assay was developed to quantify cell surface levels of P2Y2-HA receptors using an anti-HA antibody. Incubation of cells with P2Y2 receptor agonists resulted in a concentration of agonist- and time-dependent decrease in cell surface immunoreactivity. Methodology for indirect immunofluorescence confocal microscopy was developed and applied to demonstrate that the agonist-promoted decreases in cell surface immunoreactivity paralleled increases in intracellular immunoreactivity. Agonist-induced internalization of P2Y2 receptors was demonstrated directly by prelabeling P2Y2-HA receptors with antibody before agonist challenge and then quantifying the movement of receptors from a cell surface to intracellular localization in the presence of agonist. Removal of agonist from the medium resulted in recovery of cell surface immunoreactivity to control levels within approximately 1 hr. Incubation of P2Y2-HA receptor-expressing cells with P2Y2 receptor agonists also resulted in receptor-specific desensitization of nucleotide-promoted inositol phosphate accumulation. This loss of responsiveness occurred more rapidly and to a greater extent than did the agonist-promoted loss of surface receptors. Inhibition of receptor internalization by reduction of temperature to 16 degrees had no effect on the capacity of nucleotides to induce P2Y2 receptor-specific desensitization. These results illustrate that the P2Y2 receptor undergoes agonist-promoted movement to an intracellular compartment. This receptor internalization is not required for agonist-induced desensitization.     

Contrasting internalization kinetics of human and chicken gonadotropin-releasing hormone receptors mediated by C-terminal tail

The chicken gonadotropin-releasing hormone receptor (GnRH-R) is notable for having a cytoplasmic C-terminal tail, which is not present in the mammalian GnRH-Rs. We report here that the cytoplasmic tail mediates rapid agonist-promoted receptor internalization. The chicken GnRH-R mediated internalization of gonadotropin-releasing hormone (GnRH) agonist (125I[His5-D-Tyr6]GnRH) at a rate of 11.3%.min-1, compared with only 0.71 %.min-1 for the human GnRH-R. To determine whether the presence of the cytoplasmic tail was responsible for the more rapid internalization kinetics of the chicken GnRH-R we truncated the tail after the Ile336 residue (S337stop). Receptor-mediated internalization of GnRH agonist by the S337stop-chicken GnRH-R was much slower than in the wild-type chicken receptor, and was similar to the wild-type human GnRH-R (0.55 %.min-1). These data indicate that rapid agonist-promoted internalization of the chicken GnRH-R is mediated through elements in the cytoplasmic C-terminal tail, distal to or including Ser337 and suggests that elimination of the C-terminal tail during evolution of mammalian GnRH-Rs may be related to its effects on internalization.     

Quantitation of neurokinin 1 receptor internalization and recycling in guinea-pig myenteric neurons

Agonist-induced endocytosis and recycling of G protein-coupled receptors contributes to desensitization and resensitization of the receptors. In this study, we have used fluorescence immunohistochemistry, confocal microscopy and digital image analysis to quantify the proportion of receptor in the cytoplasm and on the surfaces of nerve cells in the guinea-pig ileum. With these methods we examined the dynamics of internalization of the neurokinin 1 receptor in response to agonist, return of receptor to the cell membrane and its capacity to be re-internalized in response to further exposure to agonist. The basal level of neurokinin 1 receptor immunoreactivity in the cytoplasm was 12-15% of total cellular immunoreactivity. Concentration-response relations were generated for neurokinin 1 receptor internalization after incubation of isolated ileum with 10(-11) to 10(-6) M substance P at 4 degrees C and warming to 37 degrees C for 20 min. The threshold concentration for cytoplasmic receptor to exceed baseline was 10(-11) M and the proportion of receptor in the cytoplasm increased with increasing substance P concentration. The effect of two exposures to agonist was studied using 10(-8) M and 10(-6) M substance P. After equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C with no substance P, neurokinin 1 receptor immunoreactivity in the cytoplasm increased significantly from 12% to 36+/-3% for incubation with 10(-8) M and to 64+/-3% for 10(-6) M. When return of receptor to the surface was blocked with monensin (10(-5) M), 90% of the receptor was in the cytoplasm after 1 h at 37 degrees C following exposure to 10(-6) M substance P. After 60 min without substance P and no monensin, receptor in the cytoplasm decreased to 19+/-2% (10(-8) M) and 38+/-4% (10(-6) M). A second period of equilibration with substance P at 4 degrees C for 1 h followed by 20 min at 37 degrees C, without substance P, resulted in a second wave of endocytosis; the fractions of receptor in the cytoplasm were 47+/-2% (10(-8) M) and 70 2% (10(-6) M). These results indicate that most of the receptors on the cell surface are available for internalization and that the receptors that return to the cell surface after endocytosis rapidly regain their ability to bind ligand and undergo endocytosis.     

Mutation analysis of the gonadotropin-releasing hormone receptor gene in idiopathic hypogonadotropic hypogonadism

The chemical signals of mammalian skin that stimulate the secretion of acetabular gland contents of Schistosoma mansoni cercariae were determined by exposing cercariae to fractions of human and pig skin surface obtained by thin-layer chromatography. Postacetabular gland secretion was stimulated by hydrophilic skin extracts but was often combined with a secretion of preacetabular glands. Secretion of preacetabular glands, which contain enzymes for skin lysis, could be selectively stimulated with skin surface lipids. Two different mechanisms of lipid-stimulated preacetabular gland release could be distinguished. First, secretion in combination with penetration behavior and probably tegument transformation was stimulated by the fraction of free fatty acids. Second, secretion independent of penetration behavior and tegument transformation was exclusively stimulated by glucosylceramides and phospholipids, probably phosphatidylcholines. The secretion mechanisms seem to allow a continuous lysis of epidermal macromolecules during the skin passage of the cercariae. Free fatty acids occur in the uppermost skin layers and may stimulate the combination of the first response; phospholipids and glucosylceramides are restricted to deeper epidermal layers and may stimulate the enzyme secretion there. An active preacetabular gland release was also stimulated by toxic chemicals, which could suggest an emergency penetration program for impaired cercariae.     

Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion

The GnRH receptor (GnRHR) is a G protein-coupled receptor expressed by gonadotropes in the anterior pituitary gland. In the past several years, much has been learned about the structure-function relationships that exist in this receptor with regard to ligand binding and signal transduction. However, the lack of specific antibodies has precluded any analyses of the behavior of the unbound form of this receptor. We have constructed a functional GnRHR in which enhanced green fluorescent protein is fused to the carboxyl-terminus of the murine GnRHR. This fusion receptor was expressed diffusely throughout the cell, with approximately 38% of the fusion receptors colocalized with a plasma membrane marker in the gonadotrope-derived alphaT3 cell line, and approximately 82% of the fusion receptors colocalized with a membrane marker in Chinese hamster ovary cells. Furthermore, the fusion receptor displayed a Kd of 0.8 nM for iodinated des-Gly10,D-Ala-6-GnRH N-ethyl amide in Chinese hamster ovary cells, which was similar to the Kd of the native GnRHR expressed in alphaT3 cells. The surface mobility of the fusion protein was examined by fluorescence photobleaching recovery methods. In the unbound state the majority of the receptors were laterally mobile and displayed a lateral diffusion rate of 1.2-1.6 x 10(-9) cm2/sec. Binding of GnRH reduced the rate of lateral diffusion over 3-fold and reduced the fraction of mobile receptors from approximately 76-91% to 44-61%. Like GnRH, the competitive GnRH antagonist antide slowed the rate of receptor diffusion approximately 3-fold. In contrast to GnRH, antide had no effect on the fraction of mobile receptors. Thus, an intrinsically fluorescent GnRHR is trafficked to the plasma membrane of mammalian cells, is capable of ligand binding and signal transduction, and allows direct observation of the GnRHR in the nonligand-bound state. Furthermore, fluorescence photobleaching recovery analysis of the GnRHR-green fluorescent protein fusion reveals fundamental differences in the membrane dynamics of the GnRHR induced by the binding of an agonist vs. that induced by the binding of an antagonist.     

The effect of inhibitors of receptor internalization on the desensitization and resensitization of three Gs-coupled receptor responses

Many G protein-coupled receptors (GPCRs) are known to internalize following agonist exposure, however the relative importance of this mechanism for the desensitization and resensitization of different GPCRs is unclear. In the present study, we have pretreated NG108-15 cells with hypertonic sucrose or concanavalin A (con A), to investigate the effects of these inhibitors of internalization on the agonist-induced desensitization and subsequent resensitization of three Gs-coupled receptor responses. Incubation of cells with sucrose or con A did not affect subsequent acute agonist stimulation of the A2A adenosine receptor or the agonist-induced desensitization of this receptor response. However, the resensitization of the A2A adenosine receptor response following agonist removal was abolished in the presence of sucrose or con A. Sucrose or con A treatment affected neither the desensitization nor resensitization of IP-prostanoid receptor responsiveness. On the other hand con A but not sucrose reduced the agonist-induced desensitization of secretin receptor responsiveness. However, secretin receptor responsiveness did not resensitize within the time period studied whether or not inhibitors of internalization were present. These results indicate that receptor internalization appears to subserve different functions for different GPCRs.     

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor receptor. Role of cytoplasmic tail phosphorylation in each process

Agonist-induced sequestration, recycling, and resensitization of platelet-activating factor (PAF) receptor were characterized in transfected Chinese hamster ovary cells. Exposure of the cells to PAF led to rapid sequestration of the receptors into the intracellular compartment and desensitization of the response to PAF. The sequestration was inhibited by pretreatments that perturbed the clathrin-mediated pathway. Subsequent removal of PAF by washing with receptor antagonists led to rapid recycling of the sequestered receptors to the cell surface accompanied by resensitization to PAF. To evaluate the potential role of phosphorylation in the receptor cytoplasmic tail during these processes, mutant receptors in which the tails were truncated or substituted, so as to lack serine/threonine residues, were created. PAF phosphorylated the wild-type receptor rapidly and strongly, but the mutants did not. The maximal extent of sequestration of each mutant was lower than that of the wild-type, and one of the substituted mutants showed no sequestration. Furthermore, the sequestration-defective mutant showed evidence of desensitization after agonist stimulation but not resensitization after agonist removal. Thus, agonist-induced phosphorylation of the cytoplasmic tail facilitates but is not essential for receptor sequestration, and sequestration/recycling appears important in receptor resensitization.     

Agonist-induced phosphorylation of the luteinizing hormone/chorionic gonadotropin receptor expressed in a stably transfected cell line

Much of the definitive work on G-protein-coupled receptor phosphorylation and its impact on receptor function has been performed with the catecholamine receptors. Evidence for receptor phosphorylation is lacking, however, for G-protein-coupled receptors that bind larger ligands, such as LH/CG. Using immunoprecipitation techniques and a clonal cell line stably transfected with the LH/CG receptor, we show here for the first time that exposure of cells to hCG induces phosphorylation of its cognate receptor. The hCG-induced increase in receptor phosphorylation requires receptor activation because it cannot be elicited with a hCG antagonist and is mediated at least in part by the cAMP second messenger system. This hypothesis is supported by the finding that the hCG-induced receptor phosphorylation is greatly reduced (but not abolished) in a cell line that overexpresses cAMP phosphodiesterase and that receptor phosphorylation can be induced by activation of endogenous cAMP synthesis with prostaglandin E2 or by addition of 8-bromo-cAMP. Last, we show that LH/CG receptor phosphorylation can be induced with a phorbol ester, but not with a calcium ionophore. We also examined a potential correlation between LH/CG receptor phosphorylation and uncoupling of the receptor from its effector. Although the phorbol ester-induced phosphorylation of the LH/CG receptor can be correlated with uncoupling, other experiments indicate that hCG-induced uncoupling of the LH/CG receptor can occur under conditions where the cAMP-mediated receptor phosphorylation is greatly reduced (or abolished).     

Linkage of the ubiquitin-conjugating system and the endocytic pathway in ligand-induced internalization of the growth hormone receptor

The major function of the ubiquitin-conjugating system is the targeting of cytosolic and nuclear proteins for degradation by the proteasome. Recently, ubiquitin conjugation has been implicated in the downregulation of signalling receptors such as the mammalian growth hormone receptor (GHR) and the alpha-factor receptor in yeast. By examining truncated receptors, the internalization-deficient receptor mutant F327A and conditions under which clathrin-mediated GHR endocytosis is inhibited, we show here that GHR ubiquitination and ligand-induced GHR internalization are coupled events. Previously, we had shown that GHR endocytosis is dependent on an intact ubiquitination system. Here we present evidence that GHR ubiquitination depends on an intact endocytic pathway. Our data indicate that the ubiquitin-conjugating system and the endocytic pathway interact at the cytoplasmic tail of the GHR at the plasma membrane, where they cooperate to regulate internalization of the GHR.     

Mediation of cyclic AMP signaling by the first intracellular loop of the gonadotropin-releasing hormone receptor

The gonadotropin-releasing hormone (GnRH) receptor, which is a unique G protein-coupled receptor without a C-terminal cytoplasmic domain, activates both inositol phosphate (InsP) and cAMP signaling responses. The function of the highly basic first intracellular (1i) loop of the GnRH receptor in signal transduction was evaluated by mutating selected residues located in its N and C termini. Replacements of Leu58, Lys59, Gln61, and Lys62 at the N terminus, and Leu73, Ser74, and Leu80 at the C terminus, caused no change in binding affinity. The agonist-induced InsP and cAMP responses of the Q61E and K59Q,K62Q receptors were also unaffected, but the L58A receptor showed a normal InsP response and an 80% decrease in cAMP production. At the C terminus, the InsP response of the L73R receptor was normal, but cAMP production was reduced by 80%. The EC50 for GnRH-induced InsP responses of the S74E and L80A receptors was increased by about one order of magnitude, and the cAMP responses were essentially abolished. These findings indicate that cAMP signaling from the GnRH receptor is dependent on specific residues in the 1i loop that are not essential for activation of the phosphoinositide signaling pathway.     

Actin and fimbrin are required for the internalization step of endocytosis in yeast

In Saccharomyces cerevisiae, alpha-factor is internalized by receptor-mediated endocytosis and transported via vesicular intermediates to the vacuole where the pheromone is degraded. Using beta-tubulin and actin mutant strains, we showed that actin plays a direct role in receptor-mediated internalization of alpha-factor, but is not necessary for transport from the endocytic intermediates to the vacuole. beta-tubulin mutant strains showed no defect in these processes. In addition, cells lacking the actin-binding protein, Sac6p, which is the yeast fimbrin homologue, are defective for internalization of alpha-factor suggesting that actin filament bundling might be required for this step. The actin dependence of endocytosis shows some interesting similarities to endocytosis from the apical membrane in polarized mammalian cells.     

Regulation of rat pituitary gonadotropin-releasing hormone receptor mRNA levels in vivo and in vitro

The recent isolation of cDNAs encoding the rat pituitary gonadotropin-releasing hormone receptor (GnRHR) allows studies of the regulation of the synthesis of the GnRHR and its relationship to reproductive function. Analyses of the regulation of GnRHR mRNA levels in the rat pituitary in vivo revealed a progressive increase in levels to 2.0 +/- 0.2-fold after ovariectomy (OVX) and 5.2 +/- 1.3-fold after castration (CAST) (21 days post-operative), compared to intact adult female and male controls, respectively. Replacement therapy with 17 beta-estradiol benzoate in 21-day post-OVX female rats resulted in a marked decrease in GnRHR mRNA levels by 7 days, compared to controls. In contrast, therapy with testosterone propionate in 21-day post-CAST male rats resulted in only a modest decrease in GnRHR mRNA levels. Thus, manipulation of the reproductive endocrine system in vivo results in alterations in GnRHR synthesis at the pretranslational level, which parallel known changes in cell surface gonadotropin-releasing hormone (GnRH) binding activities. The treatment of superfused primary monolayer cultures of rat pituitary cells with hourly pulses of GnRH (10 nM, 6 min/h) resulted in a marked increase in GnRHR mRNA levels (12.8 +/- 4.3-fold compared to untreated cells). In contrast, treatment of cultured cells with continuous GnRH caused no change in GnRHR mRNA levels. These in vitro data show homologous regulation of GnRHR gene expression by GnRH, and suggest that the changes in GnRHR gene expression observed in vivo may be attributable at least in part to changes in the pattern of hypothalamic GnRH secretion.     

Effect of gonadotropin-releasing hormone on estrogen receptor messenger ribonucleic acid level in perifused pituitary cells

Porphyria cutanea tarda is a disorder of heme biosynthesis resulting from a defect or deficiency in the enzyme uroporphyrinogen decarboxylase. Heme precursors accumulate in the blood, urine, stool, and skin, where exposure to sunlight results in the clinical manifestations. Porphyria cutanea tarda has been described in adult hemodialysis patients. The pathogenesis of porphyria cutanea tarda in this population is thought to be related to the inability of hemodialysis to adequately clear porphyrin precursors, resulting in increased precursor serum levels, precursor skin deposition, and subsequent clinical manifestations. A proper diagnosis of porphyria cutanea tarda in hemodialysis patients requires fractionation of serum porphyrins. Normalization of the porphyrin profile and reversal of the dermal manifestations require the withdrawal of hepatotoxic agents and the reversal of hepatic iron overload. A case of porphyria cutanea tarda in an adult ESRD patient treated with continuous ambulatory peritoneal dialysis is described. In this patient, the disease was related to elevated serum levels of phenytoin, which had been administered for seizure disorder.     

The negative feedback actions of progesterone on gonadotropin-releasing hormone secretion are transduced by the classical progesterone receptor

Progesterone (P) powerfully inhibits gonadotropin-releasing hormone (GnRH) secretion in ewes, as in other species, but the neural mechanisms underlying this effect remain poorly understood. Using an estrogen (E)-free ovine model, we investigated the immediate GnRH and luteinizing hormone (LH) response to acute manipulations of circulating P concentrations and whether this response was mediated by the nuclear P receptor. Simultaneous hypophyseal portal and jugular blood samples were collected over 36 hr: 0-12 hr, in the presence of exogenous P (P treatment begun 8 days earlier); 12-24 hr, P implant removed; 24-36 hr, P implant reinserted. P removal caused a significant rapid increase in the GnRH pulse frequency, which was detectable within two pulses (175 min). P insertion suppressed the GnRH pulse frequency even faster: the effect detectable within one pulse (49 min). LH pulsatility was modulated identically. The next two experiments demonstrated that these effects of P are mediated by the nuclear P receptor since intracerebroventricularly infused P suppressed LH release but 3alpha-hydroxy-5alpha-pregnan-20-one, which operates through the type A gamma-aminobutyric acid receptor, was without effect and pretreatment with the P-receptor antagonist RU486 blocked the ability of P to inhibit LH. Our final study showed that P exerts its acute suppression of GnRH through an E-dependent system because the effects of P on LH secretion, lost after long-term E deprivation, are restored after 2 weeks of E treatment. Thus we demonstrate that P acutely inhibits GnRH through an E-dependent nuclear P-receptor system.     

Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase

The diagnostic usefulness of measuring plasma D-dimers using the ELISA method and the latex agglutination test has been prospectively evaluated in 117 patients hospitalized for suspicion of acute venous thrombo-embolism (AVTE): pulmonary embolism was suspected in 80 patients and the remaining 37 had a suspicion of deep vein thrombosis of the lower limbs. The diagnosis of AVTE was confirmed in 50% of the patients, all of whom underwent gold standard invasive investigation i.e. pulmonary angiography and/or contrast venography. The sensitivity, specificity, negative predictive value and positive predictive value of a D-dimers plasma concentration exceeding 500 ng/ml for the diagnosis of AVTE were respectively 98, 58, 97 and 70% when using the ELISA method, and 86, 71, 84 and 75% when using the latex assay. In 47 patients whose lung scans yielded abnormalities of indeterminate probability of pulmonary embolism, the sensitivity of the ELISA method was very high (94%), but that of latex assay was low (67%). Our results demonstrate that measuring the plasma D-dimers by the latex assay should not be used in the diagnosis of AVTE. On the other hand, the ELISA method might be of great interest in the diagnostic strategy of AVTE, as a normal concentration of D-dimers rules out almost definitely the diagnosis of AVTE, and hence, spares from performing invasive investigations.     

Coupling of gonadotropin-releasing hormone receptor to Gi protein in human reproductive tract tumors

The signaling pathway by which GnRH acts in peripheral tumors is distinct from that in the anterior pituitary. We attempted to identify the guanosine triphosphate (GTP)-binding protein (G protein) subtypes linked to GnRH receptor in the genital tract tumor membranes. Surgically removed ovarian carcinomas and uterine leiomyosarcomas were screened for GnRH receptor expression before plasma membrane isolation. The G alpha i was detected by immunoblotting of membrane extracts with specific antibody and pertussis toxincatalyzed ADP-ribosylation from nicotinamide adenine dinucleotide. Membrane phosphotyrosine phosphatase activity was determined as a GnRH-sensitive membrane event using synthetic substrate p-nitrophenyl in a spectrophotometric assay. Pertussis toxin, but not cholera toxin, brought about ADP-ribosylation of an immunodetected G alpha i of 41 kDa in the GnRH receptor-positive tumor membrane. Incubation with a GnRH analog and GTP decreased the ADP-ribosylation activity in a dose-dependent manner; a half-maximal effect occurred with 30 nmol/L buserelin (P < 0.01). The apparent inhibition by GnRH of the ADP-ribosylation demonstrated that GnRH resolved the alpha-subunit of the Gi to GTP-bound form in the membranes. The action of GnRH was neutralized by a competitive antagonist, antide. Pretreatment of the membrane with the pertussis toxin completely inhibited GnRH-sensitive phosphotyrosine phosphatase activity (P < 0.01). These data demonstrate the coupling of GnRH receptor to Gi protein subfamily. The Gi which couples GnRH receptor to the effector may define the difference of responses by peripheral tumor and the anterior pituitary.