Expression and processing of prohormones in nonendocrine cells.
Pancreatic polypeptide (PP) and gastrin are initially synthesized as larger precursors that require posttranslational processing to produce the biologically active peptides. These steps include tryptic cleavage at paired basic residues, their subsequent removal by carboxypeptidase H, and formation of a carboxy-terminal amide moiety via the action of peptidyl-glycine alpha-amidating monooxygenase (PAM). Although these posttranslational processing reactions are presumed to occur primarily in the secretory granule of endocrine cells, nonendocrine cells that do not possess these structures nevertheless are able to posttranslationally process a wide variety of proteins destined for export. In these studies we sought to determine whether the mechanisms for prohormone processing are present in nonendocrine cell lines. We examined two fibroblast cell lines (psi-2, BHK) a hepatocyte cell line (Hepa), and an exocrine pancreatic cell line (AR42J). We used the pZIPneo(SVX) retroviral vector to express cDNA clones encoding human PP and gastrin in the nonendocrine cells. Transfected psi-2, BHK, and Hepa cells produced a precursor of PP that appeared to be secreted constitutively, with little remaining in intracellular stores. Almost no posttranslational processing of the PP precursor was evident in these cells. By contrast, AR42J cells were capable of expressing and storing fully processed and carboxy-terminally amidated PP and gastrin. These data support the notion that the sorting mechanisms in endocrine and exocrine cells are similar and that the posttranslational processing of peptide hormone precursors requires storage in secretory granules.(ABSTRACT TRUNCATED AT 250 WORDS)
Dickinson
C J
CJ
Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor 48109.
Takeuchi
T
T
Guo
Y J
YJ
Stadler
B T
BT
Yamada
T
T
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D005755:Gastrins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008099:Liver / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D010179:Pancreas / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D010191:Pancreatic Polypeptide / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000032:analysis / Q000235:genetics; D011863:Radioimmunoassay; D014162:Transfection
D005755:Gastrins; D011498:Protein Precursors; D010191:Pancreatic Polypeptide; D012313:RNA