Processing and intracellular targeting of somatostatin

Mammalian prosomatostatin (PSST) yields two bioactive peptides SST-14 and SST-28 through C-terminal cleavage at basic residues by protein convertases (PCs). Additionally, a second cleavage, mediated by an unknown enzyme at a putative Lys13 residue at the NH2-termius generates PSST[1--10] (antrin). The function of the PSST prosegment is unknown, but its high degree of sequence homology throughout vertebrate evolution argues that it may harbor information for directing PSST towards the regulated secretory pathway (RSP). Therefore, the PSST NH2-terminal region has been extensively analyzed with the following questions asked: Does the NH2-terminus play a role in targeting of PSST to the RSP? Is PSST cleaved at the Lys13 site resulting in antrin production, and by the action of which enzyme? What is the identity of the putative receptor in the trans-Golgi involved in properly targeting PSST? To test the role of antrin in sorting function, alanine scanning and deletional mutagenesis were constructed and stably expressed in AtT-20 cells. The region Pro5 to Gln12 which constitutes an alpha-helix was identified as being important in precursor targeting with Leu7 and Leu11 being critical. Earlier morphological studies have implicated immature secretory granules as the site of prohormone processing however, blockade of PSST targeting through disruption of the helix did not hinder the ability of PSST to be processed at its C-terminus. Furthermore, I find that PSST NH2-terminal cleavage does not take place at a basic site but, most likely at the -R6-L 7-R8-Q9-F 10-L11↓ recognition motif. Overexpression experiments in COS-1 and HEK-293 cells implicate the recently cloned subtilase-kexin-isozyme 1 (SKI-1) in antrin production. Additionally, as proteins function exclusively by means of interaction with other molecules, using the PSST NH2-terminus as a bait in yeast two-hybrid screening assay I attempted to find specific components in the trans-Golgi fo