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This version published online on September 2, 2004
Molecular Endocrinology, doi:10.1210/me.2004-0102
Molecular Endocrinology Vol. 0, No. 2004 200401021-
doi:10.1210/me.2004-0102
Copyright © 2004 by the Endocrine Society.
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*Compound via MeSH
*Substance via MeSH

Submitted on March 9, 2004
Accepted on August 24, 2004

A Conformationally-sensitive Growth Hormone Receptor Antibody: Impact on GH Signaling and GHR Proteolysis

Jing Jiang, Xiangdong Wang, Kai He, Xin Li, Changmin Chen, Peter P. Sayeski, Michael J. Waters, and Stuart J. Frank*

Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Alabama at Birmingham, Birmingham, AL; Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL; Department of Pathology, University of Alabama at Birmingham, Birmingham, AL; School of Biomedical Sciences and Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia; Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL; Endocrinology Section, Medical Service, Veterans Affairs Medical Center, Birmingham, AL

* To whom correspondence should be addressed. E-mail: sjfrank{at}uab.edu.

The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven {beta} strands organized into two antiparallel {beta} sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, while subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHRext-mAb, which recognizes the rabbit (rb) and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHRext-mAb recognized a GST fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHRext-mAb failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHRext-mAb dramatically inhibited GH-induced JAK2 and STAT5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHRext-mAb only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Further, anti-GHRext-mAb prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHRext-mAb inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHRext-mAb has promise as GH antagonist and as a tool in studies of conformational changes required for GHR activation.




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