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Biosynthesis of Human Growth Hormone-Releasing Hormone (hGRH) in the Pituitary of hGRH Transgenic Mice^*

Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Cincinnati College of Medicine Cincinnati, Ohio 45267
Peptide Research Department, Roche Research Center, Hoffmann-LaRoche, Inc. Nutley, New Jersey 07110

Address all correspondence and requests for reprints to: Dr. Lawrence A. Frohman, Division of Endocrinology and Metabolism, University of Cincinnati Medical Center, 231 Bethesda Avenue, ML 547, Cincinnati, Ohio 45267.

Abstract

We have studied the posttranslational processing of prohuman GH-releasing hormone (pro-hGRH) to the mature hormones, hGRH(l–44)-NH₂ and hGRH(l–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(l–44)-NH₂, and hGRH(l–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(l–44)-NH₂ but not [³⁵S]hGRH(l–40)-OH were detectable. After a 0.5-h chase, however, ³⁵S-labeled hGRH(l–40)-OH, pro-hGRH, and [³⁵S]hGRH(l–44)-NH₂ were all measurable. After a 4-h chase, comparable levels of [³⁵S]hGRH(l–44)-NH₂ and hGRH(l–40)-OH were present, and very little intracellular ³⁵Spro- hGRH remained. A progressive decrease in the ratio of immunoprecipitable pro-hGRH to mature hGRH peptides and an increase in the ratio of hGRH(l–40)-OH to hGRH(l–44)- NH₂ was observed in the two chase periods. In medium, [³⁵S] hGRH(l–44)-NH₂ was detectable at all times, whereas only minimal amounts of [³⁵Sxy2hGRH(l–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(l–40)-OH is derived from hGRH(l–44)-NH₂. (Endocrinology 129: 3274–3280, 1991)

Footnotes

^* This work was supported in part by USPHS Grant DK-30667.

Received August 1, 1991.