The pancreatic islet hormones somatostatin (SRIF), insulin and glucagon are synthesized as larger precursors. Our long term goal is to elucidate the function of polypeptide hormone precursors in mediating intracellular transport, post-translational processing and secretion of the mature hormone. PreproSRIF is a useful model for these studies since it is one of the simplest peptide hormone precursors. I. In vitro Reconstitution of Prohormone Processing and Sorting. Little is known about the molecular mechanisms whereby endocrine cells discriminate between proteins destined for the constitutive and regulated secretory pathways. To investigate prohormone processing and sorting in the Trans Golgi Network (TGN), we will use a cell-free system derived from GH3 cells expressing proSRIF. We propose to: (i) Characterize this in vitro system in detail; (ii) Investigate proSRIF cleavage in the TGN and the concomitant packaging of mature SRIF and endogenous GH into nascent secretory granules; (iii) Prepare in vitro systems from cells deficient in prohormone processing and by mixing experiments identify cell-specific components involved in prohormone processing and sorting. II. Identification of a Monobasic- residue Specific Prohormone Converting Enzyme. In many prohormones the bioactive peptide is flanked by pairs of basic amino acids, however in several precursors the hormone sequence is cleaved at single basic residues. We identified a novel proteolytic activity in yeast cells which cleaves heterologously expressed proSRIF-II correctly at a sequence the gene encoding this enzyme; (ii) Exploit the yeast gene to identify its mammalian equivalent by screening a pancreatic islet cDNA library; (iii) Co-express the protease cDNA and proSRIF-II in GH3 cells, where proSRIF-II is degraded intracellularly, to determine if the prohormone is then cleaved to SRIF-28. Our goal is not only to identify a novel prohormone processing enzyme(s) but also to understand the molecular basis whereby cells discriminate between prohormones which are substrates for proteolytic processing from those targeted for intracellular degradation. III. Structure-Function Studies on Topogenic Domains in Peptide Hormone Precursors. The SRIF propeptide functions in mediating intracellular transport and correct proteolytic processing. To identify structural features that effect precursor sorting and processing, we have over- expressed several proSRIFs in E.coli. We will: (i) Characterize conserved domains in different species of proSRIF by identifying protease-resistant and -sensitive regions of the precursors; (ii) Purify the proSRIFs to homogeneity, prepare crystals of the purified polypeptides and determine their X-ray crystallographic structure. These studies will enable us to identify structural """"""""domains"""""""" which function in mediating intracellular transport. There is now evidence that defects in proinsulin processing lead to certain forms of familial hyperinsulinemia. Therefore understanding the biosynthesis and sorting of the islet hormones has important implications concerning the etiology of diabetes.
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