The overall aims are to determine the regulatory steps involved in glycosylation of luteinizing hormone (LH) and how gonadotropin-releasing hormone (GnRH) and gonadal steroids regulate glycosylation, storage, and release of LH. There are four specific aims: (1) To determine if LH glycosylation and release are regulated via different divergent intracellular pathways. This will be done by determining the relative role of, and interaction between, the putative Ca2+ and cAMP pathways in regulating LH glycosylation and release. The effects on LH glycosylation and release of drugs known to modulate signals involved in the Ca2+ and cAMP pathways will be studied. (2) To determine if different GnRH-receptor interactions regulate glycosylation and release of LH. This will be done by determining the effects of receptor cross-linkers, GnRH-associated peptide, and GnRH antagonists on GnRH-induced LH glycosylation vs. release. (3) To test the hypothesis that both co- and post-translational modification of LH (i.e., core- and terminal glycosylation and sulfation) are regulated by GnRH and gonadal steroids. This will be done by determining (a) the effect of GnRH on addition of mannose-rich lipid-carrier core carbohydrate units to the apoprotein moiety, and (b) the effect of GnRH on addition of sulfate and terminal carbohydrates other than glucosamine to LH. (4) To determine if GnRH and gonadal steroids modulate intracellular LH degradation. This will be done by monitoring LH subunit degradation in response to hormones. Enzymatically dispersed anterior pituitary cells prepared from ovariectomized rats will be cultured in flasks or on biosupport beads in the presence or absence of GnRH, gonadal steroids, or drugs. LH synthesis will be monitored by measuring incorporation of radiolabeled precursors into the protein and oligasaccharide moieties of LH. Radiolabeled LH will be isolated by immunoprecipitation with specific antibodies. Radiolabeled LH subunits in the immunoprecipitates will be assessed by SDS gel electrophoresis. Total immunoreactive LH will be measured by radioimmunoassay. Specific drugs effects on LH synthesis and release will be distinguished from non-specific toxic effects by measuring uptake and incorporation of radiolabeled precursors into total protein and by examining the cells with vital dye at the end of incubation.
