Gastrin biosynthesis in canine G cells.
Gastrin requires extensive posttranslational processing for full biological activity. It is presumed that progastrin is cleaved at pairs of basic amino acids by a prohormone convertase to form a glycine-extended intermediate (G-Gly) that serves as a substrate for peptidyl-glycine alpha-amidating monooxygenase (PAM), resulting in COOH-terminally amidated gastrin. To confirm the nature of progastrin processing in a primary cell line, we performed [(35)S]methionine-labeled pulse-chase biosynthetic experiments in canine antral G cells. Radiolabeled progastrin reached a peak earlier than observed for G-Gly or amidated gastrin. G-Gly radioactivity accumulated in G cells and preceded the appearance of radioactivity in amidated gastrin. The conversion of G-Gly to amidated gastrin was enhanced by the PAM cofactor ascorbic acid. To determine whether one member of the prohormone convertase family (PC2) was responsible for progastrin cleavage, G cells were incubated with PC2 antisense oligonucleotide probes. Cells treated with antisense probes had reduced PC2 expression, an accumulation of radiolabeled progastrin, and a delay in the formation of amidated gastrin. Progastrin in antral G cells is cleaved via PC2 to form G-Gly that is converted to amidated gastrin via the actions of PAM.
Stepan
Vinzenz
V
Department of Pediatrics, University of Michigan, Ann Arbor, Michigan 48109-0656, USA.
Sugano
Kentaro
K
Yamada
Tadataka
T
Park
Jung
J
Dickinson
Chris J
CJ
D000818:Animals; D002478:Cells, Cultured; D004285:Dogs; D005755:Gastrins / Q000096:biosynthesis* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005998:Glycine / Q000378:metabolism; D008715:Methionine / Q000493:pharmacokinetics; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009097:Multienzyme Complexes / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D045706:Proprotein Convertase 2; D011706:Pyloric Antrum / Q000166:cytology / Q000201:enzymology*; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism; D013462:Sulfur Radioisotopes
D005755:Gastrins; D009097:Multienzyme Complexes; D016376:Oligonucleotides, Antisense; D013462:Sulfur Radioisotopes; D008715:Methionine; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D013381:Subtilisins; D045706:Proprotein Convertase 2; D005998:Glycine