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 (<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.