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A Conformationally Sensitive GHR [Growth Hormone (GH) Receptor] Antibody: Impact on GH Signaling and GHR Proteolysis

Department of Medicine (J.J., X.W., S.J.F.), Division of Endocrinology, Diabetes, and Metabolism, Department of Cell Biology (K.H., S.J.F.), Department of Pathology (X.L.), University of Alabama at Birmingham, Birmingham, Alabama 35294-0012; School of Biomedical Sciences and Institute for Molecular Bioscience (C.C.), University of Queensland, Brisbane, Queensland, Australia; Department of Physiology and Functional Genomics (P.P.S.), University of Florida College of Medicine, Gainesville, Florida 32610; and Endocrinology Section (S.J.F.), Medical Service, Veterans Affairs Medical Center, Birmingham, Alabama 35233

Address all correspondence and requests for reprints to: Stuart J. Frank, University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012. 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 ß strands organized into two antiparallel ß sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas 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-GHR_ext-mAb, which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR_ext-mAb recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR_ext-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-GHR_ext-mAb dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR_ext-mAb only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR_ext-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-GHR_ext-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-GHR_ext-mAb has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.

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