Evidence for association of the cloned liver growth hormone receptor with a tyrosine kinase.
The ability of the cloned liver growth hormone (GH) receptor, when expressed in mammalian cell lines, to copurify with tyrosine kinase activity and be tyrosyl phosphorylated was examined. 125I-human growth hormone-GH receptor complexes isolated from COS-7 cells transiently expressing high levels of the cloned liver GH receptor bound to anti-phosphotyrosine antibody, suggesting that the cloned GH receptor is tyrosyl phosphorylated in vivo. GH-GH receptor complexes purified from transfected COS-7 cells using anti-GH antibody incorporated 32P when incubated with [gamma-32P]ATP, indicating association of tyrosine kinase activity with cloned liver GH receptor. The level of phosphorylation of the GH receptor was very low, as compared with the endogenous GH receptor in 3T3-F442A cells, suggesting that tyrosine kinase activity is not intrinsic to the cloned GH receptor but rather resides with a kinase present at low levels in the COS-7 cells. To test whether a higher level of GH receptor phosphorylation would be observed when the GH receptor was expressed in a different cell line, GH receptor cDNAs were stably transfected into mouse L and CHO cells, which have few or no endogenous GH receptors, and RIN5-AH cells, which do express endogenous GH receptors. In vivo tyrosyl phosphorylation of the cloned GH receptor in mouse L cells and in vitro phosphorylation of the cloned GH receptor in both L and CHO cells were higher than in transfected COS-7 cells but still substantially lower than in untransfected 3T3-F442A cells. Significantly increased 32P incorporation into tyrosyl residues in GH receptors in the in vitro kinase assay was demonstrated for GH receptors isolated from the transfected RIN5-AH cells. These studies show that the cloned liver GH receptor can be tyrosyl phosphorylated when expressed in a variety of cell types. The finding that the level of phosphorylation of GH receptor appears to vary with cell type is consistent with the cloned liver GH receptor being a substrate for an associated tyrosine kinase and with the amount of such a GH receptor-associated tyrosine kinase being cell type-specific.
Wang
X
X
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109.
Uhler
M D
MD
Billestrup
N
N
Norstedt
G
G
Talamantes
F
F
Nielsen
J H
JH
Carter-Su
C
C
D016475:3T3 Cells; D000818:Animals; D000906:Antibodies; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D013006:Growth Hormone / Q000378:metabolism; D006801:Humans; D007739:L Cells; D051379:Mice; D010766:Phosphorylation; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015183:Restriction Mapping; D014162:Transfection; D014443:Tyrosine / Q000031:analogs & derivatives / Q000032:analysis / Q000276:immunology
D000906:Antibodies; D011986:Receptors, Somatotropin; D019000:Phosphotyrosine; D014443:Tyrosine; D013006:Growth Hormone; D011505:Protein-Tyrosine Kinases