CONTROL OF PEPTIDE HORMONE BIOSYNTHESIS BY PC2 AND 7B2

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The generation of biologically active polypeptide hormones by neuroendocrine cells entails a complex series of biosynthetic, maturational, and transport events. One of the most crucial early steps is proteolytic processing of the large precursor polypeptide by subtilisin-like proteolytic enzymes, the prohormone convertases (PCs). Many critical questions regarding the action of the PCs remain to be answered; in particular, the mechanisms which regulate the activity of the PCs are poorly understood. We and others have recently shown that the neuroendocrine protein 7B2 represents a binding partner for PC2 and exerts a profound influence on its biosynthesis. The carboxyl terminal peptide of this protein (7B2 CT peptide) is a potent inhibitor of PC2 and of proPC2 activation, while the amino-terminal portion of this protein (21 kDa 7B2) paradoxically facilitates the intracellular transport and the activation of proPC2 to mature PC2. Major goals of this proposal are to elucidate both the mechanism for termination of inhibition of the CT peptide as well as the mechanism by which the 21 kDa protein acts to promote proPC2 activation. Specific aims include the following: 1) to use antisense and metabolic labelling experiments to precisely define the cellular roles of 7B2 in the maturation of proPC2, and to provide structure-function relationships for the interaction of these two molecules; and 2) to use purified recombinant molecules in in vitro binding experiments to determine the affinities and optimum binding conditions of the various forms of 7B2 and PC2 for each other. We will also test the hypothesis that 7B2 is required for folding and/or during proPC2 activation. Taken together, the experiments described in this proposal should provide significant information on the cell biology and biochemistry of the interaction of proPC2/PC2 with its regulatory protein, 7B2. The long-term goal of these experiments is to understand key regulatory mechanisms in the production of bioactive peptide hormones. Ultimately this understanding may provide new avenues for the therapeutic modulation of peptide hormone levels.