Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

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Abstract
Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. 125I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained an Mr 134000 125I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of the fibroblast form. O-Phosphotyrosine prevented 125I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with [32P]Pi, GH was shown to stimulate formation of a 32P-labeled protein which was bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. The Mr 114000 phosphorylated protein could be immunoprecipitated with anti-GH antibody, indicating that GH remained noncovalently bound to this protein during absorption to an elution from the immobilized phosphotyrosyl binding antibody. Phosphoamino acid analysis after both limited acid hydrolysis and extensive base hydrolysis of the Mr 114000 phosphoprotein confirmed the presence of phosphotyrosyl residues. These observations provide strong evidence that binding of GH to its receptor stimulates phosphorylation of tyrosyl residues in the GH receptor.