Gonadal Receptors/mechanisms Of Action--Peptide Hormones

Child Health And Human Development

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Abstract

1) The LH receptor: The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that plays an essential role in gonadal development and differentiation. Our previous studies demonstrated regulation of the Sp1/Sp3-driven TATAless promoter of the human LH receptor by the orphan receptors EAR2 and EAR3/COUP-TF1 (inhibitory) and TR4 (stimulatory). These orphans receptors bound competitively and with high affinity to an imperfect direct-repeat motif composed of a estrogen response element half-site and a second degenerate half-site (DR). Current studies investigated the differential binding of orphan receptors to rat and human LHR promoters, and their modulation of LHR transcription in rat granulosa cells differentiated in culture by hormone treatment. This process resembles the induction of the LHR gene in granulosa cells of the human ovary and permits analysis of the role of orphan receptors during gonadal cell differentiation from early stages to luteinization. These studies demonstrated repression of rat LHR gene transcription by EAR2 and EAR3/COUP-TFI through their binding to a DR domain (albeit with 2-fold lower affinity than the human). No binding or activation was found for TR4 due to a single nucleotide difference at the second half-site of the DR. The lower binding affinity for EAR2 and EAR3 in the rat was associated with a smaller inhibitory effect than that observed in the human and was attributable to species differences in the adjacent 3-prime sequences. hCG treatment markedly reduced the inhibition of the rat LHR in granulosa cells and also decreased EAR2 and EAR3 protein levels. Abolition of the orphan receptor-mediated inhibition of the rLHR upon hCG treatment via derepression may contribute to the elevated LHR expression required for progression of granulosa cell maturation. 2) Gonadotropin regulation: Treatment with high doses of gonadotropins causes LHR-independent negative regulation of steroidogenic enzymes (steroidogenic desensitization) and up-regulation of a novel gonadotropin regulated RNA-helicase (GRTH). A previously unidentified protein that is constitutively present in Leydig cells and down-regulated by gonadotropin was recently cloned and characterized as a gonadotropin-regulated long chain acyl CoA synthetase (GR-LACS). The 79-kDa cytoplasmic protein is expressed in the pubertal and adult Leydig cells of the rat testis and shares sequence identity with two conserved regions of the LACS and luciferase families, but displays low overall amino acid similarities (23-28%). The expressed protein present in the cytoplasm of transfected cells displayed acyl CoA synthetase activity for long chain fatty acid substrates. In addition to its potential contributions to energy production and testicular steroidogenesis, GR-LACS could provide long chain acyl-CoA esters with regulatory effects on enzyme activity, membrane function, and gene expression. 3) Prolactin Receptors: Previous studies in this laboratory have mapped and resolved the genomic structure of the human prolactin gene of over 100 Kb . The studies demonstrated that the gene has a complex structure and is amenable to alternative splicing, and reported the presence of 10 exons (multiple non-coding Exons 1 and the common non-coding exon-2 and exon 3-10 coding for the long form of the receptor). In addition, a novel exon-11 of the human prolactin receptor was recently found to be distinct from its rodent counterpart, and two novel forms of the human prolactin receptor (S1a and S1b, which are derived from alternative splicing of exons 10 and 11) were identified. These new forms of the human prolactin receptor resemble the conventional receptor in having similar extracellular and transmembrane domain, but differ in having unique truncated intracellular domains. These short forms, which were found in several normal tissues and in breast cancer cell lines, are expressed as cell surface receptors and possess binding affinities comparable to the long form. However, unlike the long form, neither of the short forms mediates the prolactin induced-activation of the beta-casein gene promoter that is exhibited by the long form of the receptor. In contrast, these forms act as dominant negative repressors of the function of the long and intermediate receptor isoforms that mediated cell growth responses in human breast cancer cells. Due to the marked difference in the cellular levels of the two forms, which results from the more rapid turnover of S1a, the S1b form was a more effective inhibitor. These short forms with unique C-termini may exhibit distinct signaling pathways, in addition to modulating signaling from the long form of the receptor. These new receptors may have important roles in the diversified actions of prolactin in human tissues, and are of potential therapeutic relevance to the control of mammary cancer cell growth and immunoregulation. Other studies have addressed the consequences of disruption of the growth hormone receptor gene on testicular function. This work has provided an in vivo demonstration in GH receptor knockout mice that LH action on testosterone secretion is significantly impaired, due to a decrease in the number of testicular LH receptors. This is accompanied by diminished responsiveness of testicular steroidogenesis and decreased ability to convert androstenedione to testosterone. These changes are probably attributable to the absence of circulating IGF-I in the GH-deficient mice. These studies have indicated that IGF-I has a major role in the regulation of testicular endocrine function.