Human growth hormone (GH) receptor is characterized as the 134-kilodalton tyrosine-phosphorylated protein activated by GH treatment in IM-9 cells.
Activation of the GH receptor (GHR) results in tyrosine phosphorylation of cellular proteins, and this tyrosine phosphorylation is believed to be important in GH action. We have shown previously that GH rapidly stimulates the tyrosine phosphorylation of 134-, 120-, and 93-kilodalton (kDa) proteins in the human IM-9 lymphocyte line. We now provide three lines of evidence indicating that the 134-kDa tyrosine-phosphorylated protein, not the 120- or 93-kDa proteins, is the GHR. 1) A monoclonal antibody that interacts with the extracellular domain of the GHR (Mab263) immunoprecipitated a 134-kDa phosphotyrosine-containing protein, but not a 120- or 93-kDa protein, from GH-treated IM-9 cells. 2) The GHR contains N-linked carbohydrates. When total cell lysates from GH-treated IM-9 cells were treated with endoglycosidases to remove these carbohydrates, the majority of the 134-kDa phosphotyrosine-containing protein was no longer detected. Furthermore, the immunoprecipitated 134-kDa protein was completely deglycosylated and resulted in a single band of approximately 100 kDa. Neither the 120- nor 93-kDa tyrosine-phosphorylated proteins were affected by endoglycosidase treatment. 3) The Mab263 antibody immunoprecipitated a 134-kDa phosphotyrosine-containing protein from GH-treated 293 cells (human embryonic kidney cell line) that stably express the full-length rabbit GHR. This protein was not detected in control cells expressing the neomycin resistance gene alone. We conclude that the 134-kDa protein that is tyrosine phosphorylated upon GH stimulation of IM-9 cells is the GHR.
Silva
C M
CM
Department of Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Day
R N
RN
Weber
M J
MJ
Thorner
M O
MO
D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002236:Carbohydrate Conformation; D002460:Cell Line; D004622:Embryo, Mammalian; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D013006:Growth Hormone / Q000494:pharmacology*; D006801:Humans; D007163:Immunosorbent Techniques; D007668:Kidney; D008970:Molecular Weight; D010766:Phosphorylation; D019000:Phosphotyrosine; D011986:Receptors, Somatotropin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000494:pharmacology; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D011986:Receptors, Somatotropin; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D013006:Growth Hormone; D006026:Glycoside Hydrolases