title: A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: impact on GH signaling and GHR proteolysis.
abstract: 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, 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.
authors.affiliation: University of Alabama at Birmingham, 1530 3rd Avenue South, BDB 861, Birmingham, Alabama 35294-0012, USA.
authors.forename: Jing
authors.initials: J
authors.lastname: Jiang
authors.forename: Xiangdong
authors.initials: X
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: He
authors.forename: Xin
authors.initials: X
authors.lastname: Li
authors.forename: Changmin
authors.initials: C
authors.lastname: Chen
authors.forename: Peter P
authors.initials: PP
authors.lastname: Sayeski
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Waters
authors.forename: Stuart J
authors.initials: SJ
authors.lastname: Frank
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology*; D002460:Cell Line; D019281:Dimerization; D013006:Growth Hormone / Q000037:antagonists & inhibitors* / Q000494:pharmacology / Q000502:physiology; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000494:pharmacology; D047468:Immunoprecipitation; D053614:Janus Kinase 2; D018716:Molecular Mimicry / Q000276:immunology; D009154:Mutation / Q000235:genetics; D010703:Phorbol Esters / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors; D011817:Rabbits; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D010703:Phorbol Esters; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin; C032974:12-O-retinoylphorbol-13-acetate; D013006:Growth Hormone; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2