Aspartate 171 Is the Major Primate-specific Determinant of Human Growth Hormone
ENGINEERING PORCINE GROWTH HORMONE TO ACTIVATE THE HUMAN RECEPTOR*
- Stuart N. Behncken‡,
- Scott W. Rowlinson§,
- Jennifer E. Rowland,
- Becky L. Conway-Campbell,
- Thea A. Monks and
- Michael J. Waters
- From the Department of Physiology and Pharmacology and the Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Queensland 4072, Australia
Abstract
It has been known for more than 4 decades that only primate growth hormones are effective in primate species, but it is only with the availability of the 2.8 Å structure of the human growth hormone (hGH)·hGH-binding protein (hGHBP)2complex that Souza and co-workers (Souza, S. C., Frick, G. P., Wang, X., Kopchick, J. J., Lobo, R. B., and Goodman, H. M. (1995)Proc. Natl. Acad. Sci. U. S. A. 92, 959–963) were able to provide evidence that Arg-43 on the primate receptor is responsible. Here we have examined systematically the interaction between Arg-43 (primate receptor) or Leu-43 (non-primate receptors) and their complementary hormone residues Asp-171 (primate GH) and His-170 (non-primate hormones) in a four-way comparison involving exchanges of histidine and aspartate and exchanges of arginine and leucine. BAF/B03 lines were created and characterized which stably expressed hGH receptor, R43L hGH receptor, rabbit GH receptor, and L43R rabbit GH receptor. These were examined for site 1 affinity, for the ability to bind intact cells, and for proliferative biopotency using hGH, D171H hGH, porcine GH, or H170D porcine GH. We find that the single interaction between Arg-43 and His-170/171 is sufficient to explain virtually all of the primate species specificity, and this is congruent with the crystal structure. Accordingly, for the first time we have been able to engineer a non-primate hormone to bind to and activate the human GH receptor.
Footnotes
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↵* This work was supported by National Health and Medical Research Council and Australian Research Council grants (to M. J. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The first two authors contributed equally to this work.
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↵‡ To whom correspondence should be addressed. E-mail:behncken{at}plpk.uq.edu.au.
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↵§ Recipient of an National Health and Medical Research Council C. J. Martin fellowship. Present address: Dept. of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN 37232.
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↵1 The abbreviations used are: GHR, growth hormone receptor; GH, growth hormone; (GHBP)2, GH-binding protein; h, rb, b, and p prefixes indicate human, rabbit, bovine, and porcine, respectively; MAb, monoclonal antibody; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide.
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↵2 S. W. Rowlinson, S. N. Behncken, and M. J. Waters, unpublished observations.
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- Received May 28, 1997.
- Revision received August 5, 1997.
