Expression of a cytomegalovirus-human growth hormone-releasing hormone precursor fusion gene in transfected GH3 cells.
Pituitary GH3 cells were transfected with a human growth hormone-releasing hormone (hGRH) precursor minigene fused to the promoter region of either a cytomegalic immediate early gene (pCMV) or the mouse metallothionein-1 gene (mMT) to examine the molecular heterogeneity of the translation products. Expression of the hGRH message occurred following transfection of the cells with each fusion gene. Extracts of pCMV-hGRH-transfected GH3 cells as well as the culture medium contained detectable levels of immunoreactive (ir)-hGRH peptides. Analysis of molecular heterogeneity by reverse-phase high performance liquid chromatography and radioimmunoassay indicated that both mature forms of hGRH (hGRH(1-44)-NH2 and hGRH(1-40)-OH) were synthesized in the cells, although hGRH(1-44)-NH2 was the primary form secreted into the medium. A high molecular weight form of ir-hGRH, believed to represent the hGRH precursor (or a partially processed form of the precursor) was detected in cells and, in smaller amounts, in the medium. Several ir-hGRH peptides, presumed cleavage products of the mature forms of hGRH, were also found. The efficiency of processing of the hGRH precursor and metabolism of the mature hormonal forms in transfected cells grown in the presence of four different peptidase inhibitors varied with the inhibitor present. Transfected GH3 cells, therefore, possess all of the necessary enzymes for and are capable of processing the hGRH precursor to mature GRH and provide a model to study hGRH biosynthesis.
Brar
A K
AK
Division of Endocrinology, University of Cincinnati College of Medicine, OH 45267.
Coleman
T A
TA
Kopchick
J J
JJ
Frohman
L A
LA
D000818:Animals; D015152:Blotting, Northern; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics*; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D005814:Genes, Viral; D013007:Growth Hormone-Releasing Hormone / Q000032:analysis / Q000235:genetics*; D006801:Humans; D008668:Metallothionein / Q000235:genetics; D051379:Mice; D010902:Pituitary Gland / Q000032:analysis / Q000166:cytology*; D010911:Pituitary Neoplasms / Q000032:analysis / Q000473:pathology; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics*; D011863:Radioimmunoassay; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000032:analysis / Q000473:pathology; D014760:Viral Fusion Proteins / Q000235:genetics
D011480:Protease Inhibitors; D011498:Protein Precursors; D014760:Viral Fusion Proteins; D013007:Growth Hormone-Releasing Hormone; D008668:Metallothionein