Stimulation of tyrosine phosphorylation in human cells by activation of the growth hormone receptor.
To investigate the possibility that tyrosine phosphorylation of cellular proteins might play a role in GH receptor signaling, we have studied tyrosine phosphorylation induced by GH and by GH mutants in the human lymphocyte line IM-9, a homologous cell system which is known to respond to GH by increased proliferation. IM-9 cells were treated with physiological concentrations of recombinant human GH (rhGH). Protein lysates from these cells were then analyzed by polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with an antibody specific for phosphotyrosine. rhGH stimulated the tyrosine phosphorylation of two proteins having M(r) of approximately 93,000 and 120,000. Tyrosine phosphorylation of these proteins was time and dose dependent. At 2 nM rhGH tyrosine phosphorylation of these two proteins was evident by 5 min, maximal at 15 min, and decreasing by 45 min of treatment. At doses of 10 and 100 nM rhGH, tyrosine phosphorylation was stimulated by 1 min of GH treatment. IM-9 cells were also treated with genetically engineered mutant forms of the GH protein. Previous biophysical analysis of these mutant GH proteins has shown that the GH protein contains two distinct binding sites which interact in a sequential manner with the extracellular domains of two distinct GH receptor molecules, thus forming a dimeric complex. By treating IM-9 cells with these same GH mutants and analyzing tyrosine phosphorylation, we found that tyrosine phosphorylation was inhibited under conditions which prevent receptor dimerization, thus providing evidence that formation of a dimeric GH:(GH receptor)2 complex may be important for intracellular signaling by the GH receptor.
Silva
C M
CM
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Weber
M J
MJ
Thorner
M O
MO
D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D013006:Growth Hormone / Q000008:administration & dosage / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D016296:Mutagenesis; D010750:Phosphoproteins / Q000378:metabolism; D019000:Phosphotyrosine; D011986:Receptors, Somatotropin / Q000187:drug effects / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D007328:Insulin; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D013006:Growth Hormone