(taken from the application) The candidate is a veterinarian who has completed three years of postgraduate clinical training and will soon complete a Ph.D. degree. The candidate's short-term goal is to obtain additional research training in an environment that will foster the skills needed to become an independent research scientist. During that time the candidate seeks a limited clinical commitment in order to maintain the clinical skills and focus that will be needed to achieve his long-term objective. Cornell's College of Veterinary Medicine is an ideal site for the candidate to pursue his immediate goals as the training environment in the Department of Molecular Medicine is superb and the clinical facilities are state-of-the-art. The candidate's long-term goal is to obtain a faculty position at a veterinary college and to develop a comprehensive research program that encompasses both basic and clinical research. In this application, the candidate proposes to investigate the effects of inhibitors of pancreatic beta cell secretion on the proteins directly involved in insulin exocytosis. Specifically, the hypothesis that inhibition of exocytosis in beta cells is due to disruption of the core complex will be tested. This unique signaling pathway will be studied using a variety of techniques to identify and to characterize the molecular interactions that produce the powerful inhibitory effect. A likely target for inhibitor action is the """"""""core complex"""""""" of proteins, composed of synaptobrevin, syntaxin, and SNAP 25. Together these proteins form a tightly bound helical structure that tethers insulin-containing granules to the islet cell membrane. The core complex is an essential intermediate in exocytosis and is regulated by a large number of cellular proteins. Inhibitor-induced changes in the core complex composition will be detected using a novel coimmunoprecipitation assay (the """"""""core complex assay""""""""). The cellular mechanisms responsible for producing the core complex effects of the inhibitors will also be characterized. First, the inhibitory GTP-binding protein involved will be identified using co-immunoprecipitation, antisense, and permeabilization techniques. Second, the core complex target for the activated G protein pathway will be determined using co-immunoprecipitation and pull-down assays, clostridial neurotoxin cleavage experiments, and over-expression studies. Finally, characterization of the essential molecular features of target proteins will be done to elucidate the direct molecular interactions.
