Abstract
We previously reported that EphA4, a member of the Eph family of receptor tyrosine kinases, is an important modulator of growth hormone (GH) signaling, leading to augmented synthesis of insulin-like growth factor 1 (IGF1) for postnatal body growth. In the present study, we report the molecular interactions of EphA4, GH receptor (GHR), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 5B (STAT5B). EphA4 binds to GHR at both its extracellular and intracellular domains and phosphorylates GHR when stimulated with a ligand. The cytoplasmic domain of EphA4 binds to the carboxy-terminus of JAK2 in contrast to the known binding of GHR to the amino-terminus. STAT5B binds to the amino-terminal kinase domain of EphA4. Ligand-activated EphA4 and JAK2 phosphorylate each other and STAT5B, but JAK2 does not appear to phosphorylate EphA4-bound STAT5B. Ligand-activated EphA4 induces the nuclear translocation of STAT5B in a JAK2-independent manner. GHR expression is required for the activation of STAT5B signaling, even via the JAK2-independent pathway. Various ephrins that have affinity for EphA4 induce STAT5B phosphorylation. These findings suggest the molecular mechanisms by which ephrin/EphA4 signaling enhances the canonical GH-IGF1 axis.
Highlights
Growth hormone (GH) is an anterior pituitary-derived peptide hormone that exerts a diverse array of physiological actions on body growth and metabolism
We extensively examined the molecular interactions among EphA4, Growth hormone receptor (GHR), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 5B (STAT5B) using deletion mutants of EphA4, GHR, and JAK2
We previously reported that EphA4 forms a complex with the known assembly of GHR/JAK2/ STAT5B, leading to the GH- and ephrin-mediated activation of STAT5B through JAK2-dependent and JAK2-independent pathways, which in combination enhance the expression of insulin-like growth factor 1 (IGF1) mRNA [19]
Summary
Growth hormone (GH) is an anterior pituitary-derived peptide hormone that exerts a diverse array of physiological actions on body growth and metabolism. For transient eukaryotic expression in HEK293T cells, cDNAs for WT and mutant human EphA4, mouse Jak2, and mouse Ghr were incorporated into a pcDNA3.1 vector (Thermo Fisher Scientific Inc.; Product #V79020) that was modified to express proteins that were carboxy-terminally fused with different peptide tags (3Flag, HA, or 6myc).
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