The overall objective of this proposal is to examine the molecular mechanism(s) involved in the regulation of ovarian function by luteinizing hormone. The most critical step in the action of LH is its specific interaction with a receptor in the ovary. Through this interaction with specific receptors, LH controls the development and maturation of ovarian follicles, ovulation, corpus luteum formation and steroid production. By these means LH regulates fertility, implantation of the blastocyst and maintenance of early pregnancy. The present study is intended to examine the regulation of LH receptor in the ovary. The first specific aim will continue to examine the regulation of LH receptor expression during follicle development and during peri-ovulatory periods. Specifically, the proposed studies will extend the discovery made during the last funding period of a novel mechanism by which LH receptor mRNA expression is regulated by a trans factor that was identified as mevalonate kinase. The proposed studies will examine the regulation of LH receptor mRNA expression by modulating intracellular cholesterol and steroid metabolism, the use of SiRNA to inhibit mevalonate kinase expression and thus modulate LH receptor mRNA expression, determining the role of interacting proteins in LH receptor mRNA degradation, identified using co-immunoprecipitation and yeast 2 cell hybrid techniques and the identification of key molecules involved in the regulation of LH receptor mRNA expression using DNA microarray and proteomic approaches. The second specific aim will continue the studies on post-translational modifications on LH receptor turnover. Specific studies will address the role of palmitoylation and phosphorylation on receptor internalization and post-endocytic processing using mutagenesis, confocal microscopy and metabolic labeling studies. These studies will identify critical motifs and processes involved in the trafficking of the receptor to the cell surface and its metabolic fate after interaction with LH. The role of glycosylation of the extracellular domain on the maturation and traffickingof the receptor to the cell surface will be examined using a combination of techniques involving mutagenesis, immunoprecipitation, flow cytomery and confocal microscopy. These studies address novel questions central to reproductive endocrinology and are relevant to the diagnosis and treatment of infertility.
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