Phosphorylation of highly purified growth hormone receptors by a growth hormone receptor-associated tyrosine kinase.
We have shown previously that growth hormone (GH) promotes the phosphorylation of its receptor on tyrosyl residues (Foster, C. M., Shafer, J. A., Rozsa, F. W., Wang, X., Lewis, S. D., Renken, D. A., Natale, J. E., Schwartz, J., and Carter-Su, C. (1988) Biochemistry 27, 326-334). In the present study, we investigated the possibility that a tyrosine kinase is specifically associated with the GH receptor. GH-receptor complexes were first partially purified from GH-treated 3T3-F442A fibroblasts, a GH-responsive cell, by immunoprecipitation using anti-GH antiserum. 35S-Labeled proteins of Mr = 105,000-125,000 were observed in the immunoprecipitate from GH-treated cells labeled metabolically with 35S-amino-acids. These proteins were not observed in immunoprecipitates from cells not exposed to GH or when non-immune serum replaced the anti-GH antiserum, consistent with the proteins being GH receptors. GH receptors appeared to be phosphorylated, as evidenced by the presence of 32P-labeled bands, comigrating with the 105-125 kDa 35S-labeled proteins, in the immunoprecipitate of GH-treated cells labeled metabolically with [32P]Pi. When partially purified GH receptor preparation was incubated with [gamma-32P]ATP (7-15 microM) for 10 min at 30 degrees C in the presence of MnCl2, a protein of Mr = 121,000 was phosphorylated exclusively on tyrosyl residues. As expected for the GH receptor, this protein was not observed in immunoprecipitates when cells had not been treated with GH nor when non-immune serum replaced the anti-GH antiserum. GH-receptor complexes were also purified to near homogeneity by sequential immunoprecipitation with phosphotyrosyl-binding antibody followed by anti-GH antiserum. When cells were labeled metabolically with 35S-amino acids, the 35S label migrated almost exclusively as an Mr = 105,000-125,000 protein. This protein also incorporated 32P into tyrosyl residues when incubated in solution with [gamma-32P]ATP. These results show that highly purified GH receptor preparations undergo tyrosyl phosphorylation, suggesting that either the GH receptor itself is a tyrosine kinase or is tightly associated with a tyrosine kinase.
Carter-Su
C
C
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622.
Stubbart
J R
JR
Wang
X Y
XY
Stred
S E
SE
Argetsinger
L S
LS
Shafer
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000032:analysis; D007163:Immunosorbent Techniques; D051379:Mice; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D011986:Receptors, Somatotropin; D019000:Phosphotyrosine; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D010447:Peptide Hydrolases