Somatotropin
growth hormone receptor signaling pathway
Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase.
Growth hormone (GH) has recently been shown to activate the GH receptor (GHR)-associated tyrosine kinase JAK2. In the present study, regions of the GHR required for JAK2 association with GHR were identified. GH-dependent JAK2 association with GHR was detected in Chinese hamster ovary (CHO) cells expressing wild-type GHR (GHR1-638) or GHR truncated at amino acid 454 (GHR1-454) or 380 (GHR1-380). JAK2 did not associate with GHR in cells expressing GHR truncated at amino acid 294 (GHR1-294) or when amino acids 297-311 containing a proline-rich motif were deleted (GHR delta P) or prolines 300, 301, 303, and 305 in the proline-rich motif were mutated to alanines (GHR4P-->A). Cross-linking 125I-human GH to GHR demonstrated that GHR mutants migrated with the appropriate molecular weight, with the exception of GHR4P-->A which migrated as a protein similar in size to GHR1-294. In studies performed in CHO and RIN-5AH cells, the ability of JAK2 to associate with the mutated GHR was found to correlate with GH-dependent activation of JAK2, tyrosyl phosphorylation of GHR (in the case of GHR1-638 and GHR1-454), and the ability of the GHR to copurify with tyrosine kinase activity. In CHO cells expressing mutated GHR, GH-dependent tyrosyl phosphorylation of cellular proteins (p121, p97, p42, and p39) was dependent on the ability to activate JAK2. No proteins showed increased tyrosyl phosphorylation in CHO cells expressing GHR1-294, GHR4P-->A, or GHR delta P. Deletion of the C-terminal half (amino acids 455-638) of the GHR ablated GH-dependent tyrosyl phosphorylation of p97. Taken together, these results provide strong evidence that the N-terminal quarter of the cytoplasmic domain of GHR and within this region, the proline-rich motif, is required for association of JAK2 with GHR and GH-dependent activation of JAK2, and that tyrosines in the N-terminal half of the cytoplasmic domain of the GHR are phosphorylated by JAK2. The finding that a specific interaction with the C-terminal half of GHR appears to be necessary for p97 phosphorylation indicates that while JAK2 activation may be necessary for a full biological response to GH, it appears not to be sufficient.