We are developing an understanding of the structural basis for mechanisms of GnRH receptor binding and function. In ongoing studies, we have elucidated elements of the binding pocket for agonists and antagonists, and have developed a model of the transmembrane helix bundle. these studies have facilitated our probing of the relation of receptor structure to function and of the role of the structure and conformation of GnRH receptor ligands. We now plan to integrate these findings with studies based on a series of novel approaches designed to refine our structural and mechanistic knowledge of the receptor in its various functional states, and to investigate the properties of ligands and ligand-receptor complexes as components of the single transduction process. Towards these ends, the following aims will be pursued: 1. Probe the structure/function relations of the gnRH receptor. A. By delineating functional micrordomains of the GnRH receptor. B.By determining the relationship of the helix bundle of the GnRH receptor to that of other G-protein coupled receptors. C. By investigating the structure, conformation and interacting moieties of GnRH agonists and antagonists and seeking information about receptor mechanisms and the structural basis of activity at the GnRH receptor from the perspective of the ligands. 2. Develop, probe experimentally and refine a membrane embedded model of the GnRH receptor. A. By developing a three-dimensional computational model of the receptor including helix-connecting loop domains. B. By evaluating the receptor in a model membrane environment. C. By studying the mechanism of receptor activation. The structure/function relations of the GnRH receptor will be examined through mutagenesis and modeling studies of functional microdomains and through substituted cysteine accessibility mutagenesis. New experiments were also designed to refine our understanding of the optimal conformational structure of GnRH analogs and, concomitantly, to identify chemical groups of the ligands that are involved in interaction with the receptor, and to determine their preferred spatial organization. The resulting experimental data will be incorporated into the development of the three-dimensional computational model of the receptor that includes the helix-connecting loop domains and will be explored computationally in model membrae environments to generate hypotheses for probing the mechanisms of ligand binding and receptor activating. The evolving molecular model reflects the structural insights obtained from experimental data, serves to integrate them and plays a crucial role in delineating subsequent experiments. Al these interrelated investigations seek novel insight into the molecular mechanisms underlying the action of GnRH analogs and can provide the basis for the rational design of novel therapeutic approaches to modulate receptor activity.
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