Expression and post-translational processing of gastrin in heterologous endocrine cells.
The biosynthesis of gastrin involves a complex series of post-translational processing reactions that result in the formation of a biologically active secretory product. To study the mechanisms for two specific reactions in gastrin processing, namely dibasic cleavage and amidation, we infected AtT-20, GH3, and Rin5-f cells with the retroviral expression vector, pZip-NeoSV(X), containing human gastrin cDNA. We detected gastrin and its glycine extended post-translational processing intermediates (G-gly) in the media and cell extracts of successfully infected cells. Characterization of the molecular forms of gastrin in these cell lines revealed that GH3 and Rin5-f processed gastrin in a manner similar to antral G-cells but the cleavage of the Lys74-Lys75 bond that converts G34 to G17 appeared to be suppressed in AtT-20 cells. Even after conversion of this site to Arg74-Arg75 via site-directed mutagenesis, the At-20 cells synthesized G34 predominantly. All of the infected cells amidated gastrin but the gastrin/G-gly ratio, a reflection of amidation within the cells, was enhanced in GH3 and Rin5-f cells but diminished in AtT-20 cells upon treatment with dexamethasone (10(-4) M) for 3 days. The dibasic cleavage of gastrin was uneffected by dexamethasone. Our data suggest that the activities of post-translational processing reactions responsible for the synthesis of biologically active gastrin exhibit considerable tissue and substrate specificity.
Marino
L R
LR
Department of Pediatrics, University of Michigan Medical Center, Ann Arbor 48109.
Takeuchi
T
T
Dickinson
C J
CJ
Yamada
T
T
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D004247:DNA / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D004702:Endocrine Glands / Q000166:cytology / Q000378:metabolism*; D005755:Gastrins / Q000096:biosynthesis* / Q000235:genetics; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011499:Protein Processing, Post-Translational; D051381:Rats; D012190:Retroviridae / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D005755:Gastrins; D003907:Dexamethasone; D004247:DNA