Corticotropin-releasing factor induces phosphorylation of phospholipase C-γ at tyrosine residues via its receptor 2β in human epidermoid A-431 cells

Abstract

This laboratory previously reported that corticotropin-releasing factor (CRF) increased intracellular free calcium concentrations, cellular cAMP, inositol 1,4,5-trisphosphate, protein kinase C activity, and protein phosphorylation in human A-431 cells. The increase was blocked by CRF receptor antagonist. In this study, we identified the type of CRF receptors present and investigated whether CRF induced tyrosine phosphorylation of phospholipase C-γ via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2β. The levels of the CRF receptor type 2β were not altered in cells treated with activators of protein kinase C, Ca 2+ ionophore, or cells overexpressing heat shock protein 70 kDa. Cells treated with CRF displayed increases in protein tyrosine phosphorylation approximately at 150 kDa as detected by immunoblotting using an antibody against phosphotyrosine. Immunoprecipitation with antibodies directed against phospholipase C-β3, -γ1, or -γ2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-γ1 and -γ2 were phosphorylated. The increase in phospholipase C-γ phosphorylation was concentration-dependent with an EC 50 of 4.2±0.1 pM. The maximal phosphorylation by CRF at 1 nM occurred by 5 min. The CRF-induced phosphorylation was inhibited by the protein tyrosine kinase inhibitors genistein and herbimycin A, suggesting that CRF activates protein tyrosine kinases. Treatment of cells with CRF receptor antagonist, but not pertussis toxin, prior to treatment with CRF inhibited the CRF-induced phosphorylation, suggesting it is mediated by the CRF receptor type 2β that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2-iminophenoxy)ethane- N, N, N′, N′-tetraacetic acid attenuated the phospholipase C-γ phosphorylation. In summary, CRF induces phospholipase C-γ phosphorylation at tyrosine residues, which depends on Ca 2+ and is mediated by activation of protein tyrosine kinases via the CRF receptor type 2β.