Prosomatostatin processing in pituitary GH3 cells. Identification and secretion of the intact propeptide.
Preprosomatostatin (preproSRIF) is a peptide hormone precursor that undergoes tissue-specific processing at either a single set of paired basic residues to yield SRIF-14 or, alternatively, at a monobasic site to produce SRIF-28, an NH2 terminally extended form of SRIF-14. Mammalian preproSRIFs are a family of precursors that are remarkably conserved from rat to humans. In five species, the signal peptide and propeptides are approximately 96% identical; this high degree of sequence identity may be indicative of functional conservation. Since the propeptide is approximately five times larger than SRIF-14, we hypothesized that it would be secreted as a separate polypeptide following proSRIF proteolytic processing. To test this idea, we raised polyclonal antibodies to the entire propeptide to follow its biosynthesis and secretion. Here we demonstrate that in transfected rat anterior pituitary GH3 cells both SRIF-14 and the intact 9.5-kDa propeptide were processed coordinately from proSRIF with identical kinetics. Treatment of the cells with chloroquine, a weak base which inhibits processing to mature SRIF-14, also inhibited the appearance of the 9.5-kDa propeptide. Approximately 40% of the propeptide was targeted to the regulated secretory pathway as determined by its quantitative secretion in response to secretagogues. We also examined the secretion of the SRIF propeptide independently of SRIF-14 by expressing a truncated "propeptide" in which SRIF-14 was deleted. Significantly, the SRIF propeptide was itself efficiently transported through the secretory pathway and secreted into the culture medium. This suggests that the propeptide possesses all the topogenic information necessary for intracellular transport. The coordinate secretion of the intact propeptide with mature SRIF-14 suggests that it might function as a novel bioactive peptide.
Elgort
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Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
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D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D005399:Fishes; D006801:Humans; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D010903:Pituitary Gland, Anterior; D010911:Pituitary Neoplasms; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D013004:Somatostatin / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D011498:Protein Precursors; D013004:Somatostatin; C028456:prosomatostatin; D008715:Methionine