1) Overexpression of the LH holoreceptor and extracellular hormone- binding spliced variant form B was achieved in insect cells. Overexpressed receptors are glycosylated (high mannose) and exhibit high- affinity hormone binding, these are amenable for structural studies. 2) Functional CHO chains required for hormone binding were at Asn-173 and Asn 152 and each mannose chain contributed about 2kDa. Asn-77 was not glycosylated but its mutation to Gln reduced hormone binding reflecting its location in a crucial region of the binding domain. The proximal N- acetylglucosamine linked Asn, common to the mannose (insect) and complex (mammalian) chain, is the functional sugar moiety that is necessary for the acquisition of the high-affinity conformation. This sugar residue is essential for refolding of the denatured and reduced native receptor and possibly of the nascent receptor LHR. 3) Gene transcription of LH receptor is dependent on Spl-induced promoter activation from two of four Spl binding domains Spl-2 (canonical) and Spl-4 (non-canonical) that contributes equally to transcriptional initiation. The Spl-4 binding site within Spl4 was identified as 5' GGG GTG GGG rather than to the 3' overlapping G-C box. Functional non-Spl protein(s) bind the 3' GC box and only in non-expressing cells are functional substitute for the downstream M1 regulatory domain. Removal of both transfactors may play a role in permanent silencing expression. 4) Analyses of the 5' UTRs of the prolactin receptor (PRLR) mRNAs expressed in gonadal and non-gonadal tissues, and their genomic organization, revealed three alternate first exons. Each of these exons is spliced to a common exon 2 that precedes the third exon containing the translation initiation codon. Alternate utilization of these first exons, and alternative splicing of exon 2, generate multiple 5' UTRs in PRLR transcripts. Three functional PRLR gene promoters that initiate the transcription were identified. These were found to be utilized in a tissue-specific manner both in vivo and in vitro. These findings indicate that multiple promoters control transcription of the PRLR gene, and provide a molecular basis for the differential regulation of PRLR expression in diverse tissues. 5) An homology model of the rat CY17 was derived from the alpha/betaF supersecondary structural element of the 3alpha/20beta hydroxysteroid dehydrogenase of Streptomyces Hydrogenans. 6) GHRH present in the Leydig cell is under positive control of LH and synergyzes FSH action in Sertoli cells. GHRH is also localized to the acrosomal region of early to intermediate spermatids. GHRH from Leydig and germinal cell origin may be a paracrine regulator of Sertoli cells and exert intracrine functions in the spermatids.
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