Demonstration of growth hormone (GH) receptor-associated tyrosine kinase activity in multiple GH-responsive cell types

Highly purified GH-receptor preparations from 3T3-F442A fibroblasts, whose differentiation into adipocytes is promoted by GH, have been shown to contain a tyrosine kinase capable of phosphorylating GH receptors. In the current work, characteristics of the tyrosine kinase responsible for the in vitro phosphorylation of GH receptors from cultured 3T3-F442A fibroblasts were examined, and the presence of this GH receptor-associated tyrosine kinase activity was demonstrated in multiple cell types. GH-receptor complexes from GH-treated cells were partially purified by immunoprecipitation using anti-GH antibodies and then incubated as an immune complex with [gamma 32P] ATP. Incorporation of 32P into the GH receptor from 3T3-F442A fibroblasts was apparent within 1 min at 30 C after the addition of [gamma 32P]ATP (5-10 microM). A divalent cation was requisite for the phosphorylation; Mn2+ was significantly more effective than Mg2+ and Co2+; Ba2+, Ca2+, or Zn2+ had no effect. Excess unlabeled ATP, but not cytosine triphosphate, GTP, or uridine triphosphate, abolished 32P incorporation into the GH receptor and [gamma 32P]GTP could not replace [gamma 32P]ATP as a source of 32P. At 5.5 mM Mn2+, phosphorylation exhibited a biphasic dose response to ATP, with maximal phosphorylation occurring at a concentration of 10 microM ATP. At more physiological concentrations of ATP (1 mM), phosphorylation of the GH receptor was also stimulated by lower concentrations of Mn2+ (as low as 500 nM). Optimal reaction conditions determined for the phosphorylation reaction in 3T3-F442A fibroblasts were used to demonstrate incorporation of 32P from [gamma 32P]ATP into partially purified GH receptors from cultured human IM-9 lymphocytes, murine 3T3-F442A adipocytes, rat H-35 hepatoma cells, and freshly isolated rat adipocytes. The 32P was shown to be incorporated into tyrosyl residues in receptors from the two cell types tested (IM-9 lymphocytes and rat adipocytes). Cross-linked [125I] hGH-receptor complexes solubilized from the four cell types (IM-9 lymphocytes, 3T3-F442A adipocytes, H-35 hepatoma cells, and freshly isolated rat adipocytes) bound to and could be eluted from phosphotyrosyl antibody, suggesting that tyrosyl phosphorylation of GH receptors in all of these cells occurs in vivo. The presence of tyrosine kinase activity associated with GH receptors in multiple cell types from different species is consistent with tyrosine kinase activity playing a role in the actions of GH.