IN VITRO BIOSYNTHESIS OF INSULIN AND GLUCAGON

The pancreatic islet hormones somatostatin (SRIF), glucagon and insulin are synthesized as larger precursors. Our long term goal is to determine the role of the presursors in mediating intracellular transport, post-translational processing and secretion of the hormones. PreproSRIF is a useful model for these studies since it is one of the simplest peptide hormone precursors, containing only a single bioactive peptide and one proteolytic processing site for excision of the mature hormone. Using recombinant DNA techniques a series of mutated preproSRIF molecules will be constructed to investigate functional domains in the precursor. Site-directed mutagenesis will be used to alter individual amino acids in the proteolytic processing site; deletion mutants lacking defined regions of the propeptide will be constructed and the propeptide will be fused with non-secretory proteins. These cDNAs will be transfected into heterologous cells and the parameters of secretion investigated. Common sorting and processing domains in yeast preproalpha factor and preproSRIF will be identified by expressing native prepro-SRIF cDNAs and chimeric cDNAs encoding variable amounts of these respective precursors in yeast. cDNA encoding preproglucagons will be introduced into different cell types and their post-translational processing and secretion investigated. In contrast to preproSRIF, preproglucagons encode several glucagon-related peptides and thus represent the next order of complexity of precursor processing. Translation stop codons will be introduced into preproinsulin cDNA which will be used in a coupled transcription-translation system to generate truncated preproinsulins. These will be used to investigate the minimum size of nascent polypeptide chains that can be transfered across the ER membrane. Prohormone processing enzymes will be identified using a molecular approach: A cDNA library from cells exhibiting high levels of processing activity will be inserted into a retrovirus expression vector and recomtinant virus used to infect L cells in which preproSRIF cDNA has been stably integrated and which secrete only proSRIF, since they lack prohormone processing activity. Secretion of mature SRIF will be assayed using an antibody immunoblotting procedure; DNA will be isolated from positive cells and characterized. Identifying structural determinants that mediate processing of islet hormone presursors had direct relevance to understanding their synthesis and secretion and has important implications concerning the etiology of diabetes.