G120R, a Human Growth Hormone Antagonist, Shows Zinc-dependent Agonist and Antagonist Activity on Nb2 Cells

doi:10.1074/jbc.270.16.9222

G120R, a Human Growth Hormone Antagonist, Shows Zinc-dependent Agonist and Antagonist Activity on Nb2 Cells (*)

From the (1) Endocrine Unit and Division of Molecular Pathology, Middlesex Hospital, London W1N 8AA, United Kingdom, the
(2) National Institute of Medical Research, Mill Hill, London NW7 1AA, United Kingdom, and the
(3) Edison Biotechnology Institute, Ohio University, Athens, Ohio 45701

^¶ To whom correspondence should be addressed:
Division of Molecular Pathology, University College London, The Windeyer Bldg., 46 Cleveland St., London W1P 6DB, UK.
Tel.: 44-071-636-8333 (ext. 3362); Fax: 44-071-380-9496.

Abstract

Substitution of arginine for glycine at position 120 in native 22-kDa human growth hormone (hGH) results in an analogue, G120R, which is unable to dimerize the GH receptor and is widely used to probe the molecular mechanism of action of hGH. When acting on human GH receptors, G120R antagonizes several biological effects of hGH, but is itself inactive as an agonist. It has been reported that this mutant also antagonizes hGH activation of the rat or human prolactin (PRL) receptor in cell-based assays, with no agonist activity. We have now tested this mutant in a sensitive MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)-ESTA (eluted stain assay) bioassay using rat PRL receptors in the Nb2 cell line. We confirm that G120R acts as an efficient antagonist of native hGH, but show that it can also act as an agonist to generate intracellular signals leading to metabolic activation and proliferation of Nb2 cells. We have demonstrated an unusual sensitivity to the presence of zinc (Zn). In the absence of added Zn, G120R shows weak but full agonist activity in the bioassay, and this can be blocked by co-incubation with recombinant hGH-binding protein. G120R can therefore be utilized to discriminate between the molecular mechanisms of hGH interactions with its somatogenic and lactogenic receptors. Future studies with G120R in the rat may need to take account of its significant agonist effects on PRL receptors.

Footnotes

↵^§ Supported by Children Nationwide and Pharmacia, Stockholm, Sweden.
↵* This work was supported by the David and Frederick Barclay Foundation and by the Special Trustees of the Middlesex Hospital. 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:

hGH

human growth hormone

PRL

prolactin

ESTA

eluted stain assay

rhGHBP

recombinant human growth hormone-binding protein

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

hPRLBP

human prolactin-binding protein.
↵²T. Wells, A. Mode, J. Kopchick, and I. Robinson, manuscript in preparation.
↵³The use of the ESTA bioassay system is the subject of an International Patent Application.