Biosynthesis of human growth hormone-releasing hormone (hGRH) in the pituitary of hGRH transgenic mice.
We have studied the posttranslational processing of prohuman GH-releasing hormone (pro-hGRH) to the mature hormones, hGRH(1-44)-NH2 and hGRH(1-40)-OH, and its carboxyl-terminal peptide (hGCTP) in pituitary cells from transgenic mice bearing a metallothionein-hGRH fusion gene after incubation with [35S]methionine. After separation on HPLC, 35S-labeled and unlabeled hGRH in medium and cell extracts were characterized by RIA and immunoprecipitation with antisera against hGRH and against hGCTP. After a 4-h pulse, unlabeled and [35S]pro-hGRH, hGRH(1-44)-NH2, and hGRH(1-40)-OH were identified in medium and cell extracts by both RIA and immunoprecipitation with anti-hGRH serum. In cell extracts, after a 0.5-h pulse, [35S]pro-hGRH and hGRH(1-44)-NH2 but not [35S]hGRH(1-40)-OH were detectable. After a 0.5-h chase, however, 35S-labeled hGRH(1-40)-OH, pro-hGRH, and [35S]hGRH(1-44)-NH2 were all measurable. After a 4-h chase, comparable levels of [35S]hGRH(1-44)-NH2 and hGRH(1-40)-OH were present, and very little intracellular 35S-pro-hGRH remained. A progressive decrease in the ratio of immunoprecipitable pro-hGRH to mature hGRH peptides and an increase in the ratio of hGRH(1-40)-OH to hGRH(1-44)-NH2 was observed in the two chase periods. In medium, [35S]hGRH(1-44)-NH2 was detectable at all times, whereas only minimal amounts of [35S]hGRH(1-40)-OH were present. Labeled and unlabeled pro-hGRH in cell extracts was also detected with anti-hGCTP serum, and another peak, which coeluted with synthetic hGCTP, was also identified. The low molar ratio of intracellular immunoreactive hGCTP to hGRH (less than 0.02) suggests a more rapid turnover rate of hGCTP than of hGRH. These results demonstrate the processing of hGRH prohormone to both mature forms of hGRH and provide evidence that hGRH(1-40)-OH is derived from hGRH(1-44)-NH2.
Brar
A K
AK
Department of Internal Medicine, University of Cincinnati College of Medicine, Ohio 45267.
Downs
T R
TR
Heimer
E P
EP
Felix
A M
AM
Frohman
L A
LA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D013007:Growth Hormone-Releasing Hormone / Q000096:biosynthesis* / Q000378:metabolism; D006801:Humans; D007163:Immunosorbent Techniques; D008715:Methionine / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D010446:Peptide Fragments / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational
D010446:Peptide Fragments; D011498:Protein Precursors; C062302:pro-growth hormone releasing hormone, mouse; D013007:Growth Hormone-Releasing Hormone; D008715:Methionine