title: Domains of the growth hormone receptor required for association and activation of JAK2 tyrosine kinase.
abstract: 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.
authors.affiliation: Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622.
authors.forename: J A
authors.initials: JA
authors.lastname: VanderKuur
authors.forename: X
authors.initials: X
authors.lastname: Wang
authors.forename: L
authors.initials: L
authors.lastname: Zhang
authors.forename: G S
authors.initials: GS
authors.lastname: Campbell
authors.forename: G
authors.initials: G
authors.lastname: Allevato
authors.forename: N
authors.initials: N
authors.lastname: Billestrup
authors.forename: G
authors.initials: G
authors.lastname: Norstedt
authors.forename: C
authors.initials: C
authors.lastname: Carter-Su
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D013006:Growth Hormone / Q000378:metabolism*; D006801:Humans; D053614:Janus Kinase 2; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D015398:Signal Transduction; D013329:Structure-Activity Relationship
chemical_list: D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D013006:Growth Hormone; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; D053614:Janus Kinase 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases