This proposal has long-term goals related to understanding (a) the roles of small GTP-binding proteins in directing the intracellular traffic of proinsulin and insulin within the pancreatic beta-cell, (b) the impact of constitutive versus regulated secretory pathway switches on the processing and secretion of proinsulin and insulin, and (c) the relationship between intracellular hormone trafficking and the abnormal constitutive and regulated insulin secretion typical of early stages of Type I and diabetes and Type II diabetes.
Specific aims for the project are summarized as follows: (a) To determine the intracellular localization of rab 1 protein in normal and diabetic insulin producing cells by use of immunofluorescence and immuno-electron microscopic methods and subcellular fractionation. (b) To determine the intracellular localization of other rab proteins (rabs 2, 6 and 3) likely to play roles in the insulin secretory pathway by similar methods, using antibodies that will be generated against synthetic peptide epitopes. (c) To assess the effects of overproducing rab proteins 1, 2, 6 and 3 on secretion in insulin-producing cell lines by use of expression vectors containing appropriate DNA constructs, and B-cell systems to measure insulin secretion. (d) To assess the effects of disrupting the function of rab proteins 1, 2, 6 and 3 on secretion in insulin-producing cell lines by use of antisense and dominant negative DNA constructs and the secretion systems noted above, and (e) To define the effects of altered rab protein function and diabetes on proinsulin processing and secretion by use of biosynthetic and HPLC-based radioimmunometric methods. Since individual rab proteins play unique functional roles throughout the eukaryotic secretory machinery, and since they determine the effectiveness and accuracy of peptide secretion via constitutive and regulated pathways, we wish to determine their influence specifically on B-cell function and insulin secretion. This proposal represents a study of the B-cell/rab protein system at a fundamental level at this stage, and considers the potential importance of rab protein function with respect to the faulty insulin secretion characteristic of diabetes mellitus.
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