GHR residues 271-390, corresponding to the membrane-proximal region of the receptor's cyto-plasmic domain, can interact in vitro with JAK2 in cell extracts, as demonstrated by binding experiments using GHR-glutathione ^-transferase (GST) fusion proteins that incorporate the Box 1 element of GHR, 298ILPPVPVP305. GHR Box 1 sequences are both necessary and sufficient for GHR to interact with JAK2, insofar as a GST fusion protein containing the Box 1 region alone is sufficient for weak, but specific, interaction with JAK2. Similarly, internal deletion of the Box 1 sequence prevents JAK2 activation and downstream JAK2-dependent signaling in cell-based reconstitution and transfection assays. JAK2 also requires Box 1 sequences for its physical and functional association with other cytokine receptor family members, such as the prolactin and erythropoietin receptors. A role for more distal cytoplasmic domain regions of GHR for stabilizing the receptor's interaction with JAK2 is, however, also apparent. The precise nature of the interaction of JAK2 with GHR Box 1 is unclear. Although Box 1 is proline-rich, it does not correspond to a typical proline-rich binding domain. Moreover, SH3 domains, which bind proline-rich sequences, have not been found in JAK2 kinase.
Coimmunoprecipation experiments demonstrate that interactions between GHR and JAK2 can be detected in the basal state, that is, in the absence of GH treatment and in the absence of tyrosine phosphorylation of either protein; however, the binding interaction is strengthened upon stimulation of cells with GH. The region of JAK2 most responsible for physical and functional interaction with the receptor is in the N-terminal ~20% of the molecule. The corresponding region is also important for JAK2' s interaction with other cytokine receptors that it binds, such as the GM-CSF receptor P-chain and interferon-y receptor, as well as for the interaction of the related JAK3 kinase with the interleukin-2 (IL-2) receptor yc chain.
Although JAK2 is the tyrosine kinase most tightly coupled to GHR activation, the participation of other tyrosine kinases in GH signaling is, by analogy to other cytokine receptors, quite possible. For example, GH induces a relatively low level of JAK1 tyrosine phosphoryation in certain cell types. However, since JAKl-deficient cells that express JAK2 are not impaired regarding GH-induced STAT and Shc tyro-sine phosphorylation, while JAK2-deficient cells are unable to support these GH responses, JAK1 might not play a physiologically significant role in GH action. Recent evidence indicates that GH, like pro-lactin, can activate src and fyn kinases, two members of the src-family of nonreceptor tyrosine kinases. Details of this activation process and the significance of these kinases in GH signaling are important areas for future research.
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