Growth hormone (GH) regulates postnatal growth and metabolism in humans and other vertebrates. GH signaling begins with its interaction with cell surface GH receptor (GHR), a transmembrane glycoprotein cytokine receptor family member. GHR encodes no intrinsic enzyme activity; rather, it couples to JAK2, a cytoplasmic tyrosine kinase whose activation is required for downstream signaling resulting in GH's regulation of cellular behavior and gene expression. GH activates the signal transducer and activator of transcription (STAT)-5, extracellular signal-regulated kinase (ERK), and phosphatidylinositol-3 kinase (PI3K) pathways. Despite intense study of GH signaling for nearly two decades, molecular mechanisms modulating GH sensitivity and differential coupling of JAK2 activity to signaling pathways are largely unclear. Our recent exciting insights into GHR and JAK2 activation and regulation, include: 1) the cellular level of JAK2 correlates with the relative abundance of mature, cell surface GHR; 2) plasma membrane GHR and JAK2 are enriched in caveolae/lipid raft compartments; and 3) important aspects of GHR function are inhibited by a monoclonal antibody that recognizes the extracellular domain in a conformationally-dependent fashion. We hypothesize: 1) JAK2 serves critical roles both in determining the level of GHR available for activation and in mediating GH-induced signal transduction. 2) Spatial localization of GHR and its signaling molecules within regions of the plasma membrane regulates critical aspects of GH signaling.
Our specific aims are: 1. Define JAK2's role in regulating availability of cell surface GHR. We will test mechanisms by which JAK2 affects GHR surface abundance in JAK2-deficient and -replete cells. We will manipulate JAK2 levels in cells endogenously expressing GHR and JAK2 to define effects on surface GHR availability and signaling. 2. Define mechanisms of caveolar/raft localization of GHR and its impact on GH signaling. We will define the GHR region(s) required for caveolae/rafts localization and the impact of upstream ERK pathway activators on caveolae/raft localization of GHR and its signaling molecules. 3. Characterize anti-GHRext.mAb as a selective GH antagonist. The epitope for this conformation- sensitive antibody will be mapped using a random PCR mutagenesis-yeast expression system and analyzed in light of the known structure of the GHR extracellular domain. We will test this antibody's ability to act in vivo as a GH antagonist. Results of these studies will significantly expand our knowledge of GH action and tools available to manipulate it. . ? ? ?
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