The glycoprotein hormones regulate the functions of the thyroid and gonads. Knowledge of the initial events in their actions is important for understanding health-related problems such as hyperthyroidism, precocious puberty, and polycystic ovarian disease. The overall objective of this research is to learn how glycoprotein hormones interact with their receptors to initiate signal transduction. During the current funding period we developed a model that explains the initial events in glycoprotein hormone action. The model is based on the crystal structures of hCG and ribonuclease inhibitor, a horseshoe-shaped protein formed by leucine-rich repeats similar to those in the extracellular domains of all three glycoprotein hormone receptors. In the model, a portion of the hormone binds to the central region of the horseshoe-shaped extracellular domain and the remainder becomes located in the space between its """"""""arms."""""""" Signal transduction is initiated by steric effects of the hormone on the distance between these arms. Studies in Aim 1 ill focus on residues postulated to make high affinity hormone-receptor contacts. Those in the hormone appear to be in a large groove between the alpha-and beta-subunits. Based on predictions from molecular modeling, we will study the effects of replacing these residues on hormone binding and signal transduction.
Aim 2 is designed to verify the idea that much of the hormone projects into the space between the arms of the horseshoe-shaped extracellular domain and to test model-based predictions about the proximity of these hormone an receptor residues. We will replace selected hCG residues with cysteines and react the free-SH groups of these cysteines with thiol-specific reagents that attach biotin or radioiodinated photoreactive probes. Analogs that contain biotin will be tested for their abilities to bind to LH receptor and be recognized by radioiodinated streptavidin. This will enable us to identify portions of the hormone that do not contact the receptor. The photoreactive probes will enable us to determine the proximity of specific portions of the hormone to the arms of the receptor extracellular and/or the transmembrane domains.
Aim 3 is designed to domain influence the distance between the two arms of the extracellular domain. These contacts are also predicted to alter the positions of the oligosaccharides. The model predicts that addition of bulky substituents to deglycosylated hormones will restore their abilities to initiate signal transduction., We will test this by adding polyethylene glycol (PEG) or biotin to specific sits on the hormone and monitor the effects of these treatments on hormone efficacy. We will test the idea that the positions of the oligosaccharides are altered using antibodies that bind to hormone receptor complexes. We will compare the altered using antibodies that bind to hormone receptor complexes. We will compare the abilities of these antibodies to bind to the free and receptor bound hormones before and after various hormone N-linked glycosylation signals have been removed.
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