Growth hormone (GH) is a potent growth-promoting and metabolic hormone. GH receptor (GHR) is a member of the cytokine receptor superfamily. GHR binds GH in its extracellular domain and signals via its cytoplasmic domain's regulated interaction with molecules including the tyrosine kinase, JAK2. GH promotes homodimerization of GHRs such that a 1:2 GH:GHR complex is believed to constitute the activated GHR assemblage. Relatively little is known about GHR dimerization as it occurs in cells and about influence of GHR dimerization on its association with signaling molecules. We previously described GH-induced GHR disulfide linkage that results in a covalently linked GHR dimer form. The role(s) of GHR disulfide linkage has been enigmatic, but a large fraction of activated GHRs undergo this linkage and GHRs rendered detergent-insoluble by GH are progressively accounted for by the disulfide-linked form. We recently found that cysteine-241 (an unpaired extracellular cysteine) is critical for GHR disulfide linkage and GUR detergent insolubility. In addition, this linkage is a biochemical proxy for GHR dimerization. Using this proxy, along with our new dimerization-sensitive anti-GHR extracellular domain antibody, anti-GHRext our data suggest that GH-induced dimerization augments association of GHR with JAK2. We hypothesize: 1) GH-induced GHR dimerization and/or conformational change pursuant to dimerization results in signal initiation by enhancing GHR-JAK2 interaction; 2) though noncovalent GHR dimerization is essential for signal initiation, other important aspects of GH-induced GHR function, such as its capacity for sustained signaling, its intracellular itinerary, and its degradation are influenced by GHR disulfide-linkage and detergent insolubility. To test these hypotheses, our specific aims are: 1. Systematically examine the relationships between GH-induced GHR dimerization, GHR disulfide linkage, and anti-GHRext reactivity by: using cells that express either normal GHR or GHRs mutated in the GHR dimerization interface; comparing normal GH (22K) to the 20K GH variant; and mapping the anti-GHRext epitope(s) accounting for the antibody's dimerization sensitivity. 2. Determine the influence of the GHR cytoplasmic domain on GHR dimerization and the basis for GH-induced enhancement of association between GHR and JAK2. 3. Determine roles of GH-induced GHR disulfide linkage and GHR detergent insolubility in OH-induced signaling, cell proliferation, and subcellular OHR routing, using cells that stably express wild-type vs. cysteine-241 mutant GHRs. Results will significantly impact knowledge of GH-induced GHR activation and cytokine receptor signaling. Longer-term goals made possible by these studies may include; mechanistic evaluation of drugs that activate or inhibit OH signaling; crystallographic structural analysis of the GHR-JAK2 complex; and evaluation of functions of GHR disulfide linkage in intact animals.
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