Antibodies to Cytoplasmic Sequences of Cloned Liver Growth Hormone (GH) Receptors Recognize GH Receptors Associated with Tyrosine Kinase Activity*

Abstract

GH stimulates tyrosyl phosphorylation of GH receptors in 3T3-F442A fibroblasts, and highly purified GH receptor preparations exhibit tyrosine kinase activity. Paradoxically, however, the GH receptor cloned from liver exhibits no sequence similarity to receptors with known signal transduction mechanisms, including those exhibiting ligand-activated tyrosine kinase activity. These observations raise the possibility that there are two kinds of receptors for GH: the first represented by the cloned liver GH receptor, and the second by a tyrosine kinase-containing GH receptor. To inquire into the possibility of two distinct GH receptors, we determined whether the cloned liver GH receptor shares structural similarities with the tyrosine kinase-associated GH receptor. When the cloned rabbit liver GH receptor is expressed in human kidney 293 cells, it migrates with a mol wt appropriate for the tyrosine kinase-associated GH receptor, despite the calculated mol wt of the cloned GH receptor being 60,000 smaller than that of the tyrosine kinase-associated GH receptor. The recognition of tyrosine kinase-associated GH receptor by antipeptide antibodies to three different epitopes on the cytoplasmic domain of the cloned liver GH receptor was also tested. Tyrosyl phosphorylated [125I]human GH-receptor complexes were prepared by immunoprecipitation with phosphotyrosyl-binding antibody; this subpopulation of GH-receptor complexes was recognized by all three antipeptide antibodies. The antibodies also recognized similarly isolated tyrosyl phosphorylated GH-receptor complexes, which had been further phosphorylated in solution on tyrosyl residues upon addition of [gamma 32P] ATP. Furthermore, highly purified GH receptors prepared by sequential immunoprecipitation using phosphotyrosyl-binding antibody and any one of the three antipeptide antibodies incorporated 32P into tyrosyl residues upon the addition of [gamma 32P] ATP. These results provide evidence that tyrosine kinase-associated GH receptors share sequence similarity in the cytoplasmic domain with the cloned liver GH receptor. The cloned GH receptor and the tyrosine kinase-associated GH receptor, therefore, are likely to be the same receptor or related receptor isoforms.