GPR54 inactivation does not impede neuroendocrine onset of puberty; rather, it delays and slows down pubertal maturation of the gonadotropic axis. The L102P loss of function mutation in GPR54 results in a more quantitative than qualitative defect of gonadotropic axis activation.

KiSS1 is a putative metastasis suppressor gene in melanoma and breast cancer-encoding kisspeptins, which are also described as neuroendocrine regulators of the gonadotropic axis. Negative as well as positive regulation of KiSS1 gene expression by estradiol (E 2 ) has been reported in the hypothalamus. Estrogen receptor a (ERa level is recognized as a marker of breast cancer, raising the question of whether expression of KiSS1 and its G-protein-coupled receptor (GPR54) is down-or upregulated by estrogens in breast cancer cells. KiSS1 was found to be expressed in MDA-MB-231, MCF7, and T47D cell lines, but not in ZR75-1, L56Br, and MDA-MB-435 cells. KiSS1 mRNA levels decreased significantly in ERa-negative MDA-MB-231 cells expressing recombinant ERa. In contrast, tamoxifen (TAM) treatment of ERa-positive MCF7 and T47D cells increased KiSS1 and GPR54 levels. The clinical relevance of this negative regulation of KiSS1 and GPR54 by E 2 was then studied in postmenopausal breast cancers. KiSS1 mRNA increased with the grade of the breast tumors. ERa-positive invasive primary tumors expressed sevenfold lower KiSS1 levels than ERanegative tumors. Among ERa-positive breast tumors from postmenopausal women treated with TAM, high KiSS1 combined with high GPR54 mRNA tumoral levels was unexpectedly associated with shorter relapse-free survival (RFS) relative to tumors expressing low tumoral mRNA levels of both genes. The contradictory observation of putative metastasis inhibitor role of kisspeptins and RFS to TAM treatment suggests that evaluation of KiSS1 and its receptor tumoral mRNA levels could be new interesting markers of the tumoral resistance to anti-estrogen treatment.

Isolated gonadotropic deficiency or isolated hypogonadotropic hypogonadism is defined as a low sexual hormone secretion by the gonads associated with low LH and FSH plasma levels. Kallmann syndrome is defined as a congenital isolated gonadotropic deficiency associated with anosmia whereas the phenotype of the idiopathic form is limited to the gonadotropic axis. For several years, it has been known that mutations of the KAL-1 gene or loss-of-function mutations of GnRH receptor did not explain all familial cases of isolated gonadotropic deficiency with or without anosmia. Thus the existence of other genes playing a major role in the physiology of the gonadotropic axis was highly suggested. In 2003, fibroblast growth factor receptor 1 (FGFR1) and GPR54 were shown to be two of these genes. FGFR1 loss-of-function mutations were reported in Kallmann syndrome whereas inactivating mutations of GPR54 were described in the idiopathic form of the gonadotropic deficiency. These genetic studies have opened up a new chapter in the physiology and the pharmacology of the gonadotropic axis. European Journal of Endocrinology 151 U83-U88

Several molecular models of glycoprotein hormone receptor activation have been proposed. It has been suggested that ligand binding to the ectodomain (ECD) leads to major changes in intramolecular interactions between the ECD and the transmembrane domain. We studied these intramolecular modifications by generating a recombinant LH/CG receptor (LHR) bearing an intramolecular cleavage site. We did this by inserting a furin site at position 316 in the hinge region of the ECD (LHR_Fur316). Affinity for human chorionic gonadotropin (hCG) and cAMP production upon hCG stimulation was identical to those of wild-type LHR. Western blot analysis showed that the LHR_Fur316 receptor was cleaved into two subunits linked by disulfide bridges. Chemical shedding of the ECD from the transmembrane domain did not increase basal adenylate cyclase activity, indicating that the first 294 residues did not act as an inverse agonist. The truncated LHR_316 was still activated by hCG but with an EC50 higher than that for the wild-type receptor. Zero length cross-linking was used to study intramolecular interactions between the two domains of LHR_Fur316. Cross-linking efficiency was similar for the basal and activated states, which indicated that the two domains interacted closely in the basal state, and this tight interaction persisted during activation. Our data suggest that activation of the LHR results from subtle modifications of intramolecular interactions between the two domains and low-affinity binding of hCG to the extracellular loops or residues preceding the first transmembrane segment.

The TSH receptor (TSHR), a member of the large family of G protein-coupled receptors, controls both function and growth of thyroid cells; hence, mutations of this receptor result in thyroid dysfunction. Here, we took advantage of the description of a new inactivating TSHR mutation (Q489H) in two brothers with hypothyroidism, to precise maturation, intracellular trafficking, exporting pathways, and activation mechanisms of this receptor. Functional characterization of the Q489H-TSHR in transiently transfected HEK293 cells showed cell surface expression, normal TSH binding affinity, and its inability to generate intracellular cAMP in response to TSH stimulation. Western blot analysis of the whole membrane proteins or proteins expressed at the cell surface showed that Q489H-TSHR expressed in HEK293 transfected cells are restricted to mannose-rich uncleaved receptor. Analysis of the export pathway toward cell surface indicated that both Q489H and wild-type receptors followed the same intracellular route to cell surface throughout endoplasmic reticulum and Golgi apparatus. This study shows that Q489H substitution impedes complete glycosylation of TSHR extracellular domain within the Golgi apparatus and that Q489H-TSHR expressed at the cell surface is unable to undergo intramolecular cleavage as well as to switch toward an active conformation under TSH stimulation. Altogether, our results show that 1) Q489H substitution within the first extracellular loop induces a misfolding of TSHR, blocking it into an inactive conformation and impeding complete glycosylation and intramolecular cleavage, and 2) a misfolded G protein-coupled receptor can bypass endoplasmic reticulum or Golgi apparatus quality control and reach the cell surface as an immature receptor.

No abstract

No abstract

No abstract