Stimulation by growth hormone (GH) of GH receptor-associated tyrosine kinase activity
| Autor: | Susan E. Stred, Christin Carter-Su, J. R. Stubbart, F Talamantes, Lawrence S. Argetsinger, W C Smith, Jules A. Shafer |
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| Rok vydání: | 1992 |
| Předmět: |
inorganic chemicals
medicine.medical_specialty Blotting Western Growth hormone receptor Biology environment and public health Mice Endocrinology Internal medicine medicine Animals Phosphorylation Receptor Cells Cultured Kinase Phosphorus Receptors Somatotropin Fibroblasts Protein-Tyrosine Kinases Precipitin Tests In vitro enzymes and coenzymes (carbohydrates) Cell culture Growth Hormone Autoradiography Electrophoresis Polyacrylamide Gel Signal transduction Phosphorus Radioisotopes Tyrosine kinase hormones hormone substitutes and hormone antagonists Signal Transduction |
| Zdroj: | Endocrinology. 130:1626-1636 |
| ISSN: | 1945-7170 0013-7227 |
| DOI: | 10.1210/endo.130.3.1537311 |
| Popis: | We have shown previously that GH receptors (GHRs) become phosphorylated on tyrosyl residues when GH-responsive cells are exposed to GH. In this work we investigate the molecular mechanism by which GH binding stimulates tyrosyl phosphorylation of GHR. To test whether in the presence of GH, GHR and the tyrosine kinase responsible for GHR phosphorylation are tightly associated in a complex or form a transient enzyme-substrate complex, the rate of in vitro phosphorylation of GHR was determined as a function of receptor concentration. GH-GHR complexes were purified from 3T3-F442A fibroblasts by immunoadsorption to and elution from immobilized phosphotyrosyl-binding antibody. The rate of in vitro tyrosyl phosphorylation of GH-GHR complexes was not substantially reduced when the GHR preparation was diluted 1:10 or 1:100 before phosphorylation, consistent with a tight association between kinase and substrate (GHR). When cells were labeled metabolically with 35S, and GHRs containing phosphorylated tyrosyl residues were isolated by immunoadsorption to and elution from phosphotyrosyl-binding antibody, the ability to immunoprecipitate 35S-labeled GHR with GHR antibodies was only evident when the cells had been incubated with GH. This indicates that in vivo, GHRs are phosphorylated on tyrosyl residues only when GH is bound. Additionally, when anti-GHR antibodies were used to immunoprecipitate GHR from solubilized cells, only GHRs isolated from GH-treated cells were phosphorylated when subjected to an in vitro kinase assay. The increased tyrosyl phosphorylation of GHR detected after incubation of cells with GH is consistent either with GH increasing tyrosine kinase activity associated with the GHR or with GH inducing a conformational change in GHR that renders it susceptible to tyrosyl phosphorylation. To discern whether GH binding increased GHR-associated kinase activity, we tested the ability of anti-GHR antibody immunoprecipitates to phosphorylate the synthetic substrate poly(Glu4,Tyr). GH treatment of cells resulted in a 2-fold increase in the rate of phosphorylation of poly(Glu4,Tyr). The increase in kinase activity was dose dependent, with half-maximal stimulation between 15-20 ng/ml GH. These results provide strong evidence that GH actually increases tyrosine kinase activity associated with the GHR. This would be consistent with GH-dependent complex formation between a constitutively activated kinase and GHR and/or activation of a constitutively associated or intrinsic kinase. |
| Databáze: | OpenAIRE |
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