Growth Hormone (GH) and a GH Antagonist Promote GH Receptor Dimerization and Internalization

doi:10.1074/jbc.271.12.6708

Growth Hormone (GH) and a GH Antagonist Promote GH Receptor Dimerization and Internalization (*)

From the Edison Biotechnology Institute, Molecular and Cellular Biology Program, and Department of Biological Sciences, Ohio University, Athens, Ohio 45701-2979

^§ To whom correspondence should be addressed. Tel.: 614-593-4534; Fax: 614-593-4795.

Abstract

It has previously been shown that a human growth hormone (hGH) analog, hGH-G120R, acts as a GH antagonist (Chen, W. Y., Wight, D. C., Wagner, T. E., and Kopchick, J. J.(1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5061-5065; Chen, W. Y., White, M. E., Wagner, T. E., and Kopchick, J. J.(1991) Endocrinology 129, 1402-1408; Chen, W. Y., Chen, N-Y., Yun, J., Wang, X. Z., Wagner, T. E., and Kopchick, J. J.(1994) J. Biol. Chem. 269, 15892-15897). In this study, we report the ability of hGH and hGH-G120R to be internalized by GH receptor expressing cells. Additionally, results of chemical cross-linking experiments revealed that both native hGH and hGH-G120R form complexes similar in size to that expected for hGH when bound to recombinant hGH-binding protein (bp). The molecular mass of the complex was determined to be approximately 280 kDa which is consistent with multiple receptors interacting with the ligand. The predominant radiolabeled band detected was a complex of approximately 140 kDa which probably represents one GH molecule bound to one GH receptor. The cross-linked complexes were not detected in the presence of excess unlabeled hGH or hGH-G120R and were not observed in cells which do not express detectable levels of GH receptors. Also, GH induced tyrosine phosphorylation of a complex of proteins of approximately 95 kDa in these cells whereas hGH-G120R did not. Thus, we have separated the hGH or hGH-G120R/GHR binding and internalization capabilities from the ability to stimulate tyrosine phosphorylation of intracellular proteins.

Footnotes

↵* This work was supported in part by the State of Ohio's Eminent Scholar Program which includes a grant from Milton and Lawrence Goll, and grants from National Research Initiative Competitive Grants Program/United States Department of Agriculture 94-37206-0939 and Sensus Inc. (to J. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

GHR

growth hormone receptor

GH

growth hormone

hGHbp

human growth hormone-binding protein

MLC

mouse L cells

DMEM

Dulbecco's modified Eagle's medium

PAGE

polyacrylamide gel electrophoresis.
↵² X. Wang and J. J. Kopchick, unpublished results.
- Received September 5, 1995.
- Revision received December 20, 1995.