The long-term objective of this project is to identify how the interaction between the gonadotropins and their receptors results in signal transduction. Particular emphasis will be placed on developing a model of receptor structure and identifying changes in receptor structure which occur after binding of hormone agonists and antagonists.
Specific aims are: 1) to identify the disulfide bonds of the luteinizing hormone receptor (LHR) and follicle stimulating hormone receptors (FSHR), 2) to characterize changes in the extracellular N-terminus and extracellular portions of the transmembrane domain which occur following binding of human chorionic gonadotropin (hCG) and hFSH, 3) to characterize changes in the intracellular portions of the receptors caused by hormone binding, and 4) to distinguish changes in the receptors which are essential for hormone activity from those which occur on binding of antagonists.
Specific aim #1 depends on the use of mutagenesis to introduce methionine residues into various portions of the LHR and FSHR. This will permit the receptors to be cleaved with CNBr into known fragments. The locations of the disulfide bonds will be determined from the molecular weights of the fragments as determined in Western blots.
Specific aim #2 depends on incorporation f radioiodine into specific tyrosine residues which are on the extracellular surface of the receptor. These will be identified by immunoprecipitation of the receptors, cleaving the receptors with CNBr, and monitoring radioactivity in specific peptide fragments. Studies are planned to determine if the presence of hormone alters the incorporation of radioiodine into these tyrosines. Procedures for specific aim #3 are similar to those of aim #2 except that the cytoplasmic surface of the receptor will be studied.
In specific aim #4 the influences of deglycosylated hormones and other antagonists on tyrosine radiolabeling will be compared with those of the native hormones. These studies should improve knowledge of the manner in which the glycoprotein hormones initiate their biological responses. Further, since the gonadotropin receptors are part of a larger class of similar proteins which participate in signal transduction, information learned should be widely applicable.
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