Luteinizing Hormone/Choriogonadotropin-Dependent, Cholera Toxin-Catalyzed Adenosine 5′-Diphosphate (ADP)-Ribosylation of the Long and Short Forms of Gsα and Pertussis Toxin-Catalyzed ADP-Ribosylation of Giα (original) (raw)
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The LH/CG and FSH receptors: different molecular forms and intracellular traffic
Molecular and Cellular Endocrinology, 1996
Monoclonal antibodies have been raised against the LH/CG receptor [Il and have allowed to perform immunocheniical studies of the receptor in target cells. Three different forrils of the LH/CG receptor are physiologically expressed: a mature -85 kDa transnienibrane species corresponding to the full length receptor, a -68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated soluble -45-48 kDa molecular weight species corresponding to the variant messanger RNAs generated by alternative splicing. Monoclonal antibodies against the human FSH receptor were also prepared. They allow to observe the existence of two forms of the FSH receptor in the ovaries: a major -87 kDa species corresponding to the inature receptor and a niinor -81 kDa species corresponding to a high niannose rich precursor. No variant forins of the receptor corresponding to alternative mRNA transcripts were detected. The transport of hCG was examined in rat testicular microvasculature by electron microscopy and by analyzing the transfer of radiolabeled horinone and antireceptor antibodies. LH/CG receptors were present in endothelial cells and were involved in hormone transcytosis through these cells. Immunocytochemical experiments have shown that the FSH receptor has a polarized expression in the Sertoli cells of the testes whereas the LH/CG receptor is spread on the surface of thecal granulosa and luteal cells in the ovary and Leydig cells in the testes. To study the inechanism of this polarization PSH, LH and TSH receptors were expressed in polarized MDCK cells. The mechanism of basolateral localiz?tion and of transcytosis of the receptors was studied using this niodel. The effect of honlione. CAMP and agents acting on G proteins was examined.
Endocrinology, 1999
Although desensitization of most guanine nucleotide-binding (G) protein receptors is triggered by phosphorylation of the receptor, desensitization of the LH/CG receptor (-R) in porcine follicular ovarian membranes appears to be independent of LH/CG-R phosphorylation. We therefore evaluated whether desensitization of the LH/ CG-R reflected a direct inhibition of adenylyl cyclase (AC) activity by either the ␣-subunit of G i or ␥-subunits derived from any of the membrane G proteins activated in response to LH/CG-R activation or whether desensitization reflected a competition between G s and a G protein that activated phospholipase C for binding sites on the LH/ CG-R. The results showed that follicular membrane AC activity was not inhibited upon activation of the LH/CG-R despite evidence that the ACs in follicular membranes, when maximally activated by forskolin, could be inhibited when membrane G proteins were activated by guanyl-5Ј-yl imidodiphosphate, and that pertussis toxin pretreat-ment of membranes raised forskolin-stimulated AC activity, consistent with a tonic inhibition of follicular membrane AC activity. Similarly, agonist-stimulated desensitization of LH/CG-R-stimulated AC activity was not inhibited by pertussis toxin. Therefore, desensitization is not the result of inhibition of AC mediated by an inhibitory G i subunit. Follicular membrane AC was also not inhibited by G␥ subunits freed with activation of G s , G q/11 , or G 13 , based on the inabilities of exogenous G␥ to promote desensitization and of a protein that sequesters G␥ to inhibit desensitization. Desensitization was also not inhibited by a G q/11 C-terminal peptide or antiserum directed toward the C-terminus of G q/11 , nor was it reversed with the addition of G␥ to membranes exhibiting desensitized LH/CG-R, suggesting that desensitization is independent of coupling of the LH/CG-R to G q/11. These results indicate that agonist-dependent desensitization of LH/CG-R-stimulated AC activity is mediated by a unique mechanism.
PubMed, 2000
The structure-stabilizing effect of homologous and heterogeneous desensitization and albumin on rat ovarian LH/hCG receptors was analyzed by thermal perturbation technique. HCG-induced down-regulation shifted the heat inactivation profile of hCG-binding sites to a temperature lower by about 7 degrees C (T50 values). In heterogeneous desensitization, which also involves uncoupling of receptors from adenylyl cyclase system, only follicle stimulating hormone (FSH) changed the stability of ovarian LH/hCG receptors. Stimulation of other hormonal receptors, which belong to the family of membrane spanning G protein-linked receptors, i.e. beta-adrenergic, glucagon, serotonin and prostaglandin E (PGE) had no effect on the stability of the LH/hCG receptor. Reduction of the stability of the LH/hCG receptor by about 3 degrees C after PGF2alpha injection to luteinized rats may be connected with specific process of luteolysis. On the other hand, albumin had a stabilizing effect on the receptor. The receptor destabilizing action of oleic acid incorporated into ovarian membranes along with calcium stimulation of endogenous phospholipase A (PLA) activity and reversal of these effects when BSA was used as fatty acid scavenger, may indicate that free fatty acids are responsible for the thermal instability of hCG-binding sites. Fluorescence quenching studies indicated that extraction of free fatty acids by albumin elevated the accessibility of fluorophores for acrylamide, and suggest that modificated lipid-protein interactions may affect the stability of the LH/hCG receptor structure.
F1000 - Post-publication peer review of the biomedical literature, 2012
Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cellsignalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/ LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK-or AKT-pathways inhibitors. In COS-7/ LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED 50 : 107.1614.3 pM and 530.0651.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and-AKT activation was more potent and sustained by hLH versus hCG. ERK1/ 2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH-but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.
PLoS ONE, 2012
Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cellsignalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/ LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK-or AKT-pathways inhibitors. In COS-7/ LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED 50 : 107.1614.3 pM and 530.0651.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/ 2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH-but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.
Molecular Endocrinology, 1993
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 clone1 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 EP or by addition of O-bromo-CAMP. Last, we show that LH/CG receptor phosphotylation 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 experfments 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). (Molecular Endocrinology 7: 823-832,1993) be caused by changes in the functional properties of a constant number of receptors (a phenomenon that we call uncoupling), by a decrease in the density of cell surface receptors (a phenomenon that we call receptor down-regulation), and/or by changes in the functional properties of the effector system(s). All of these phenomena are likely to contribute to the diminished responsiveness of cells, and they may occur either simultaneously or sequentially. The molecular basis of agonist-induced uncoupling has been studied in detail with the &.-adrenergic recep tor and has been shown to involve phosphorylation of different serine/threonine residues catalyzed by the cAMPdependent and ,&.-adrenergic receptor kinases (reviewed in Refs. 1 and 2). Although similar studies are now being pursued with other G-protein-coupled receptors (3) it is presently unclear if the model derived from the study of ,&-adrenergic receptors is readily applicable to other members of this family. This question is particularly relevant to G-protein-coupled receptors that bind large ligands, such as LH/CG, FSH, or TSH (see Ref. 4 for a recent review).
Molecular and Cellular Endocrinology, 1995
In this study site-directed antibodies have been used to investigate the structure/activity relationships of the LH receptor in functionally active gonadal cells. Polyclonal antibodies were raised in rabbits against synthetic peptides corresponding to regions within both the extracellular N-terminal domain (antibodies 1 and 2 against residues 48-65 and 187-206, respectively) and the cytoplasmic C-terminal domain (antibody 3 against residues 622-636) of the LH receptor. Following affinity purification by chromatography on columns of immobilised peptides the antibodies were demonstrated to be peptide specific both by ELISA and by dot-blotting assays. On Western blots of membranes proteins prepared from superovulated rat ovaries, mouse I-eydig tumour (MAlO) cells, and rat testes, all three antibodies recognised a single broad band of apparent M, 95 000-100000 corresponding to the putative LH receptor. The protein of apparent M, 95000-100000 also bound "'1-hCG on ligand blots, and binding was displaced by excess unlabelled hCG. The binding of 12'I-hCG in the ligand blots was completely inhibited by excess unlabelled hCG. The two N-terminal antibodies (antibodies 1 and 2 (10 pg/ml)) also inhibited t2'I-hCG binding to a greater extent than the C-terminal antibody (antibody 3 (10 pg/ml)). Antibody l(1 and 10 pg/ml) also potently inhibited the binding of 12'I-hCG to MA10 cells. A lesser but still significant inhibition of binding was produced by antibody 2 (with 10 pg/ml), whereas at the concentrations tested antibody 3 exerted no greater inhibition than that yielded by pre-immune IgG. At 0.1 pg/ml antibody 1 significantly inhibited and at 10 pg/ml completely inhibited LH-stimulated CAMP and progesterone production by MA10 cells. With antibody 2, 10 pg/ml was required to give a significant inhibition, whereas neither antibody 3 nor pre-immune IgG had a significant effect. The antibodies had no effect on CAMP or progesterone production when added to the MA10 cells in the absence of LH. These results indicate that binding of antibody 1 and, to a lesser extent, antibody 2 interferes with ligand binding which consequently affects signal transduction. In view of the ability of the antibodies to recognise the LH receptors both in the ovary and the testis and in more than one rodent species, and their greater apparent potency than previously available antisera, the anti-peptide antibodies raised in the present study will therefore be useful to study LH receptors in normal, functionally active gonadal cells.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1984
absence of any additional stimuli. Furthermore, insulin increased the general rate of cellular protein synthesis, whereas FSH or cholera toxin each decreased it. Thus, the use of FSH or cholera toxin, without insulin, may enable one to detect the synthesis of the LH/CG receptor by metabolic labeling techniques where background protein synthesis has been lowered.
Novel biological and possible applicable roles of LH/hCG receptor
Molecular and Cellular Endocrinology, 2007
Luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptors are widely expressed in gonadal cells, however, the presence of these receptors has also been demonstrated in several other non-gonadal female and male tissues. The expression level of non-gonadal LH/hCG receptors is much lower than in gonads, although their expression is regulated by similar mechanisms and they also exert biological effects using similar signaling pathways. Hormonally regulated LH/hCG receptor expression in the oviduct suggests that LH could be involved in the regulation of its contraction, gametes/embryos transport and synchronization of the fertilization. One of the major roles of the myometrial LH/hCG receptors may also be the stimulation of growth and maintenance of the uterine relaxation during pregnancy. In pigs, LH seems to be one of the pleiotropic factors which influence the endometrial prostaglandin F 2α synthesis and initiation of the luteolysis. The LH/hCG receptor expression in several cancer cells provides new possibilities for developing new strategies for targeted cancer therapy based on lytic LH/hCG conjugates.