The pituitary/placental glycoprotein hormone receptor (GPHR) fancy includes follicle stimulating hormone receptor (FSHR), luteinizing hormone receptor, thyroid stimulating hormone receptor and chorionic gonadotropin receptor. Glycoprotein hormones control reproduction, sexual development and thyroid function. Studies conducted during the previous grant period focused on the structure-activity-relationships of hFSH. We have obtained direct structural information about amino acids which constitute the FSH residues involved in FSH/FSH-receptor interaction and hFSHalpha/hFSHbeta association. Together, the solved crystal structure of hCG and the hFSH mutagenesis studies have provided a better understanding of the glycoprotein hormone-receptor interaction. In this application, we turn our attention towards elucidation of the hormone binding site of the receptor and towards a better understanding of the cell biology and physiology of the receptor. A detailed knowledge of liganded receptor structure is needed and will complete understanding of the diverse physiological processes which this family of receptors control. Our major focus is to use site directed mutagenesis of FSHR to provide fine resolution of principal residues critical to stabilization of the hormone-receptor complex. Models of the GPHR are now available and will be used as a conceptual guide to assess experimental outcomes. The tertiary structure of the GPHR will also be studied by immunochemical methods which we have previously used to appraise the assembled epitopes of hFSH .Biophysical approaches will be used to study the hFSHR, identifying postranslational modifications, including disulfide bond assignments. We have previously described the cell surface distribution of hFSHR and this work will be extended using confocal microscopy to directly analyze the cell surface distribution of the receptors during the activation process. Desensitization of the GPHR is correlated with phosphorylation of cytoplasmic residues of the GPHR and the proteins involved in this mechanism of regulation of gonadal cellular responsiveness to ligand will be identified using an interaction trap (""""""""yeast two hybrid system"""""""") method.
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