CHARACTERIZATION OF GONADAL RECEPTORS AND GONADOTROPIN BIOLOGICAL ACTIVITY

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Analysis of the genomic structure of the LH receptor revealed that the gene spans at least 75 kb and contains two potential promoter regions within the first 2kb of the 5' non-coding region, a TATA box with a CAT-like motif (-1694) and a region of SP1 binding sites (-83, -103 and -143). The coding region of the LH receptor gene consists of 11 exons separated by 10 introns, all located within the putative extracellular domain prior to the first transmembrane region. Exons 1-10 code for the extracellular region and exon 11 for the transmembrane and cytoplasmic domains. Several alternate truncated forms of the LH receptor were identified and shown to result from deletions of complete (exon 9) or partial (exon 11) exons contained within the genomic structure. Exons 2-8 approximate regions of the 14 consecutive 20 amino acid repeat motifs present in the LH, TSH and FSH receptors. Exons 1, 9, 10 and 11 were found to contain important structural elements including conserved cysteines (exons 1, 9, 10 and 11), a soybean lectin motif (exon 9), and a transmembrane domain and G-protein coupling elements (exon 11). Exon 9 may be of central importance to the configuration of the hormone binding domain and may also function as a carbohydrate binding domain for either the hormone or the receptor. The gonadotropin binding domain resides in the extracellular region of the receptor and probably encompasses the region spanning exons 1-10 and has significant tertiary structure consisting of folding and disulfide loops. The single exon (exon 11) could result from an "exon shuffling" mechanism during evolution or the introduction of a complete functional domain into the coding region of the gene. The major reduction in hCG binding to the deglycosylated purified receptor indicates that N-linked carbohydrate chain(s) on the mature receptor are necessary for optimal hormone-receptor binding. The conversion of Asn173 to Gln resulted in complete loss of hormone binding activity, and removal of a carbohydrate chain at this position is probably responsible for the observed abolition of hormone binding activity of the purified receptor upon deglycosylation. Mutants containing Asn77Gln and Asn152Gln were associated with significant decreases in the total number of cell receptors (80%). Partial retention of activity by these mutants indicates that either N-linked carbohydrate chains are not essential or other glycosylation chains can partially compensate for their functions. Mutant receptors at the remaining potential glycosylation sites (269, 277 and 291) exhibited no alteration in binding. The changes in hormone binding activity upon elimination of potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.