Identification of growth hormone receptor (GHR) tyrosine residues required for GHR phosphorylation and JAK2 and STAT5 activation.
To determine whether GH receptor (GHR) cytoplasmic tyrosine residue(s) and tyrosine phosphorylation are required for signal transduction, we have substituted the eight porcine (p) GHR cytoplasmic tyrosines with phenylalanine individually or in a stepwise manner from the C terminus. Conversely, the eight tyrosines were individually regenerated in a non-tyrosine-containing pGHR analog. Mutated pGHR cDNAs were transfected into mouse L cells (MLCs) and cell lines were established. Each individual tyrosine-substituted pGHR analogs was able to activate STAT5 (signal transducer and activator of transcription 5; previously termed pp95) at levels comparable to those of wild type pGHR. Analyses of these pGHR analogs revealed that a single tyrosine residue at position 487, 534, 566, or 627 is sufficient for STAT5 phosphorylation. This result suggested that a redundancy in tyrosine residue requirement may be employed in GH-mediated signal transduction. Also, we found that the requirement of tyrosine residues for STAT5 phosphorylation directly correlated with their phosphorylation status. Combining both STAT5 and GHR tyrosine phosphorylation results, we have deduced that Y332, Y487, Y534, Y566, and Y627 are pGHR tyrosine phosphorylation sites. Additionally, Janus kinase 2 was activated by GH in all pGHR tyrosine-substituted analogs, including one containing no intracellular tyrosines, which agrees with a previous report that Janus kinase 2 activation is independent of GHR tyrosine phosphorylation.
Wang
X
X
Edison Biotechnology Institute, Ohio University, Athens 45701, USA.
Darus
C J
CJ
Xu
B C
BC
Kopchick
J J
JJ
D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013006:Growth Hormone / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D053614:Janus Kinase 2; D008894:Milk Proteins; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin / Q000378:metabolism*; D050799:STAT5 Transcription Factor; D015398:Signal Transduction; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014443:Tyrosine
D004268:DNA-Binding Proteins; D008894:Milk Proteins; D011518:Proto-Oncogene Proteins; D011986:Receptors, Somatotropin; D050799:STAT5 Transcription Factor; D015534:Trans-Activators; D014443:Tyrosine; D013006:Growth Hormone; D011505:Protein-Tyrosine Kinases; C507924:JAK2 protein, human; C507926:Jak2 protein, mouse; D053614:Janus Kinase 2