The long-term objective of this research is to understand how information is transferred from the outside to the inside of the cell and how it is processed within the cell. Specifically, the role of protein:lipid interactions in the processing of information by the lipid-dependent protein kinase C and the insulin receptor will be investigated. Protein:lipid interactions and protein conformation will be examined by fluorescence spectroscopy of reconstituted proteins in model membranes. In addition, the stoichiometry and specificity of protein:lipid interactions will be determined with detergent:lipid mixed micelles, where the species and number of lipids interacting with one protein can be systematically varied. The effect of phosphorylation on protein:lipid interactions will be examined, testing the hypothesis that phosphorylation alters the protein's interaction with the membrane in such a way as to transduce the extracellular signal.
Three specific aims will be addressed: 1] Regulation of Protein Kinase C by Lipid: The mechanism of the protein kinase C:membrane interaction will be investigated. The hypotheses to be tested are: a] protein kinase C cooperatively sequesters phosphatidylserine, b] diacylglycerol targets the kinase to the plasma membrane, and c] autophosphorylation releases the kinase from the membrane. 2]Regulation of Insulin Receptor by Lipid: Specificity in the insulin receptor:lipid interaction will be examined, with the goal of elucidating the role of lipid in the molecular control of the insulin receptor. The hypothesis that receptor autophosphorylation alters the receptor's interaction with specific phospholipids, and thus effector molecules such as protein kinase C, will be explored. 3] Regulation of Insulin Receptor Function by Protein Kinase C: The role of membrane environment in insulin receptor phosphorylation by protein kinase C will be determined. The hypothesis that specific lipids serve as a matrix to bring together substrate and kinase will be addressed. In addition, the possibility that alterations in the receptor:lipid interaction occur after phosphorylation by protein kinase C and induce down-regulation and receptor internalization will be investigated. Unregulated protein kinase C has been proposed to play a role in both carcinogenesis and some forms of diabetes, where its uncontrolled phosphorylation of the insulin receptor may reduce the receptor's sensitivity to incoming signals. Elucidating the role of the membrane in the mechanism of action of both protein kinase C and the insulin receptor is central to understanding how these two signalling components transduce chemical information.
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