The long range objective of this proposal is to analyze the structure and function of the receptors for vasoactive intestinal peptide (VIP) and insulin in retinal and hepatic membranes. Comparative studies will be directed at examining the binding specificities and kinetics of binding of the VIP receptors in membranes prepared from bovine retina and liver and rat retina and liver. Structural comparisons of the VIP receptor in these tissues will be accomplished with the use of affinity labeling methodologies. Affinity labeled receptors will be analyzed by SDS gel electrophoresis and autoradiography of the dried gels. Similarly, analysis of the kinetics of binding of the insulin receptor present in neural retina and retinal endothelial cells will be carried out and compared to its peripheral counterpart in liver. VIP receptor function in retina and liver homogenates and cells will be determined by measuring VIP's ability to activate adenylate cyclase, cause alterations in Ca2+ fluxes and modulate polyphosphoinositide turnover in these two tissues. This analysis will provide information regarding whether the VIP receptor can interact with more than one signal transduction system in the retina. Isolation and purification of the retinal VIP receptor will also be undertaken in order to gain detailed structural information about this protein. Structural analysis of neural retina insulin receptor alpha and beta-subunits will be carried out both on affinity labeled and purified subunits. Affinity labeled VIP and insulin receptors will be treated with endo-beta-glucosaminidase F to remove N-linked oligosaccharide chains in an effort to determine if the polypeptide backbones of these proteins are the same across tissues and thus modified post-translationally via glycosylation or represent different gene products. Similarly, these receptors will be treated with endo-beta-glucosaminidase H, O-glycanase and neurminidase in order to obtain information on differential glycosylation of these receptors in different target tissues and thus the potential role differential glycosylation plays in altering receptor function. In the case of the insulin receptor, functional studies will include analysis of beta-subunit autophosphorylation in response to insulin. The effect of experimental diabetes on insulin receptor expression in rat retina will also be determined. The analyses planned will be instrumental in establishing functional roles for VIP and insulin receptors in the neural retina as well as providing insight into their structural characteristics.
