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Gonadotropin and GnRH receptors belong to the family of G protein coupled receptors. Gain of function mutations have been described, yielding constitutively active receptors. In the case of the LH receptor these dominant mutations determine familial male limited precocious puberty. Somatic mutations of this receptor may in some cases provoke Leydig-cell adenomas. The constitutive LH receptor is not associated with female precocious puberty. Inactivating mutations are recessive. Alterations in the GnRH receptor determine hypogonadotropic hypogonadism. The clinical diagnosis of this etiology of hypogonadism is extremely difficult, especially in sporadic cases. Mutations of gonadotropin receptors determine primary amenorrhea in girls, whereas in boys they are responsible for Leydig cell aplasia or hypoplasia (LH receptor) or of a variable alteration of spermatogenesis (FSH receptor). Mutations provoking only partial alterations of receptor functions are relatively more frequent, than those inducing complete receptor inactivity. They provide interesting insights into the physiology of GnRH and gonadotropin action.

Monoclonal antibodies have been raised against the LH/CG receptor [1] and have allowed to perform immunochemical studies of the receptor in target cells. Three different forms of the LH/CG receptor are physiologically expressed: a mature approximately 85 kDa transmembrane species corresponding to the full length receptor, a approximately 68 kDa high mannose containing species corresponding to a precursor which accumulates inside the cells, and truncated soluble approximately 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 approximately 87 kDa species corresponding to the mature receptor and a minor approximately 81 kDa species corresponding to a high mannose rich precursor. No variant forms 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 hormone 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 mechanism of this polarization FSH, LH and TSH receptors were expressed in polarized MDCK cells. The mechanism of basolateral localization and of transcytosis of the receptors was studied using this model. The effect of hormone, cAMP and agents acting on G proteins was examined.

The thyroid stimulating hormone receptor (TSHR) is the main autoantigen in Graves' disease. Mutations of the TSH receptor have been implicated in various thyroid diseases. In this study, we describe three polymorphic markers localised within introns 2 and 7 of the TSH receptor gene. These markers are useful for the study of genetic diseases involving the TSH receptor. They allowed us to map precisely the TSH receptor gene on chromosome 14q31 between D14S287 and D14S68. We also describe selected primers and experimental conditions for the amplification and direct genomic sequencing of the 10 exons of the receptor gene.

The complete organization of the human luteinizing hormone-choriogonadotropin (LH/CG) receptor (LH/CGR) gene and the structure of 1591 bp of its 5' flanking region have been determined. This gene spans over 70 kbp and contains 11 exons. The first ten exons and part of the last exon encode the extracellular domain of the receptor while the transmembrane and intracellular domains are encoded by the remaining part of the last exon. The gene encodes a 701 amino acids long preprotein, contrary to a previous report of 699 amino acids. Primer extension experiments and polymerase chain reaction (PCR) mapping allowed definition of the transcription initiation site, which is located 1085 bp upstream from the initiation codon. The 5' non-coding region is thus unusually long. The promoter region which is different from the murine LH/CG receptor promoter, contains two putative TATA boxes at positions -34 and -47 and a CAAT box consensus sequence at position -89. A consensus sequence corresponding to a cAMP responsive element is found at position -697. Seven API consensus sequences are also found in the 5' flanking region of the gene. Southern blot experiments demonstrated an informative biallelic polymorphism within the human LH/CG receptor gene locus using BglII endonuclease. The cloning of the human LH/CGR gene and the determination of the organization and structure of its 5' flanking region allow the study of its hormonal, developmental and tissue-specific regulation. Primers and PCR conditions are described for the direct genomic sequencing of all the exons of the gene. This information should facilitate the study of pathological mutations of the receptor.

Synergistic action of multiple steroid hormone response elements (HREs) has been proposed to be due to cooperative binding of receptors. We have studied the cooperativity of steroid hormone receptor binding to synergistic HREs in two natural genes. In the mouse mammary tumor virus long terminal repeat that contains four progesterone receptor binding sites, no cooperativity in receptor binding was observed between the single distal and the three proximal sites whereas a low level of cooperativity in receptor binding (about 2-fold) was found between the three proximal sites. This contrasted with the very strong synergism of these four HREs in stimulation of transcription. In the chicken vitellogenin II gene upstream sequences, an estrogen and a progestin response elements act synergistically. In this case again, no cooperativity of binding of the estrogen and progesterone receptors to their respective binding sites was observed. We therefore conclude that cooperative receptor binding may not always be required for synergistic action of multiple HREs.

The T47-D breast cancer cell line constitutively expresses high levels of progesterone receptor (PR). This does not appear to be related to an anomaly in the estrogen receptor (ER) as shown by cloning of the ER cDNA from T47-D cells and its insertion into the expression vector pKSV-10. When transfected into heterologous Cos-7 and L cells this receptor exerts a normal biological activity, stimulating the transcription of a reporter gene only in the presence of estrogen. Moreover, normal estrogen regulation of the transcription of the reporter gene was also observed in situ in T47-D cells. Southern blot experiments showed the presence of four copies of the progesterone receptor gene in T47-D cells. This was related to the existence of four copies of chromosome 11 in these cells. The most likely explanation of the anomalous regulation of progesterone receptor expression in T47-D cells is thus the presence of at least one copy of the PR gene bearing an anomaly in its regulatory region(s).