A detailed interdisciplinary program consisting of two phases is described which will prepare Jeffrey H. Periman, M.D. to be an independent basic scientist. In phase 1, he will be involved in rigorous didactic training and in experimental and theoretical studies simultaneously under close supervision of his co-sponsors. In phase 11, he will take on a more independent role in design and conduct of experiments and of theoretical analyses. Dr. Periman's progress will be evaluated by a committee of four senior scientists. Dr. Periman's training will center around a research project to define at the molecular and structural levels the binding site of the thyrotropin-releasing hormone receptor (TRH-R) using experimental and theoretical approaches in a complementary fashion. TRH is an important regulatory hormone for the anterior pituitary and an important neuromodulator in the CNS. The cloning of mouse pituitary TRH-R CDNA makes it possible to mutate the receptor and determine the effects of such changes on ligand binding. Combined with recent developments in macromolecular modeling of proteins and receptors, the TRH-R CDNA affords a special opportunity to define the binding site of the TRH-R and to initiate the construction of a three-dimensional (3-D) model of the TRH-R. Insights gained from this study should be applicable to other systems, particularly those involving small ligands. The working hypothesis is that the binding site of the TRH-R has several contact points on specific amino acid residues.
The specific aims are as follows: 1. To identify amino acid residues on the receptor that may be involved in TRH binding. To this end, existing structure-activity relationships, the Protein Data Bank, and theoretical methods will be used. 2. To determine which amino acids are involved in binding the TRH molecule. Site directed mutagenesis, binding assays, and TRH analogs will be used. 3. To construct a three-dimensional model of the TRH binding site in the TRH-R. To this end, the structure of TRH, the location of the amino acids involved in binding TRH, homology between TRH-R and bacteriorhodopsin, and helical interactions will be used to optimize the arrangement of the helices in space.
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