title: Corticotropin-releasing factor induces phosphorylation of phospholipase C-gamma at tyrosine residues via its receptor 2beta 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-gamma via CRF receptors. Using novel primers in reverse transcriptase-polymerase chain reaction, we determined the CRF receptor type to be that of 2beta. The levels of the CRF receptor type 2beta were not altered in cells treated with activators of protein kinase C, Ca2+ 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-beta3, -gamma1, or -gamma2 isoforms (which have molecular weights around 150 kDa) followed by Western blotting using an anti-phosphotyrosine antibody showed that only phospholipase C-gamma1 and -gamma2 were phosphorylated. The increase in phospholipase C-gamma phosphorylation was concentration-dependent with an EC50 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 2beta that is not coupled to pertussis toxin-sensitive G-proteins. Treatment with 1,2-bis(2iminophenoxy)ethane-N,N,N',N'-tetraacetic acid attenuated the phospholipase C-gamma phosphorylation. In summary, CRF induces phospholipase C-gamma phosphorylation at tyrosine residues, which depends on Ca2+ and is mediated by activation of protein tyrosine kinases via the CRF receptor type 2beta.
authors.affiliation: Department of Clinical Physiology, Division of Medicine, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA. dr._juliann_kiang@wrsmtp-ccmail.army.mil
authors.forename: J G
authors.initials: JG
authors.lastname: Kiang
authors.forename: X Z
authors.initials: XZ
authors.lastname: Ding
authors.forename: I D
authors.initials: ID
authors.lastname: Gist
authors.forename: R R
authors.initials: RR
authors.lastname: Jones
authors.forename: G C
authors.initials: GC
authors.lastname: Tsokos
mesh_terms: D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D003346:Corticotropin-Releasing Hormone / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004789:Enzyme Activation; D000078404:Epidermal Cells; D004817:Epidermis / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D037342:Pertussis Toxin; D051966:Phospholipase C gamma; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D018019:Receptors, Corticotropin-Releasing Hormone / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D010738:Type C Phospholipases / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: C101531:CRF receptor type 2; D018840:HSP70 Heat-Shock Proteins; D007527:Isoenzymes; D012333:RNA, Messenger; D018019:Receptors, Corticotropin-Releasing Hormone; D010566:Virulence Factors, Bordetella; D014443:Tyrosine; D003346:Corticotropin-Releasing Hormone; D037342:Pertussis Toxin; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma; D002118:Calcium