The role of prohormones in mediating intracellular sorting, processing and secretion

Abstract

Somatostatin (SRIF) is a 14 amino acid peptide hormone that is synthesized as a larger precursor: preproSRIF. The goals of this project were: (i) To determine if cells which synthesize hormones that are not posttranslationally cleaved have processing enzymes capable of efficiently cleaving foreign prohormones; (ii) Investigate the role of the SRIF proregion in mediating ER to Golgi transport and targeting to the regulated secretory pathway; (iii) Determine the specificity of endoproteolytic cleavage using site-directed mutagenesis to alter the paired-basic residues in the processing site.;cDNA encoding preproSRIF was introduced into two mammalian cell lines, GH{dollar}\sb3{dollar} (rat anterior pituitary cell which synthesized growth hormone) and 3T3 (mouse fibroblasts) using a recombinant retrovirus. Clonal lines expressing preproSRIF were established and the proteolytic processing efficiency, specificity and kinetics of secretion determined. In GH{dollar}\sb3{dollar} cells, preproSRIF was efficiently processed to the mature hormone as determined by HPLC analysis and microsequencing. Surprisingly, mature SRIF was stored more efficiently than the endogenous growth hormone, 55% versus 5%.;Using in vitro site-directed mutagenesis, the paired-basic amino acids were changed to ArgArg and LysArg. Microsequencing showed that the mutant cleavage sites were accurately processed in GH{dollar}\sb3{dollar} cells, indicating either a relative lack of cleavage specificity or the presence of multiple amino acid-specific processing enzymes.;The role of the proregion in mediating intracellular transport and sorting was tested directly by expressing two fusion proteins: a chimera of the SRIF preproregion and ape {dollar}\alpha{dollar}-globin and ape {dollar}\alpha{dollar}-globin fused to an N-terminal signal sequence. The signal peptide of the latter chimera was accurately cleaved but globin was not secreted and rapidly turned over intracellularly (t{dollar}\sb{lcub}1/2{rcub}{dollar} = 5 min). In contrast, 30% of the newly synthesized proregion-globin was transported through the secretory pathway. Most significantly, 40% of the proSRIF-globin was proteolytically processed at the native cleavage site generating mature globin which was secreted. These results demonstrate that the propeptide can act as a universal transport sequence. (Abstract shortened with permission of author.).