The thromboxane A2 receptor activates mitogen-activated protein kinase via protein kinase C-dependent Gi coupling and Src-dependent phosphorylation of the epidermal growth factor receptor.
The mitogen-activated protein kinase signaling cascade is used by many G protein-coupled receptors to initiate functional events. In this study, activation of the Gq/G11-coupled thromboxane A2 (TxA2) receptor (TP) by the TxA2 mimetic IBOP in ECV304 cells was found to induce extracellular regulated kinase (ERK) phosphorylation and tyrosine phosphorylation of the epidermal growth factor receptor (EGFR), which were inhibited by the TP antagonist SQ29548, the EGFR kinase inhibitor AG1478, the Src family kinase inhibitor PP1, the Gi/o protein inhibitor pertussis toxin (PTX), or the protein kinase C (PKC) inhibitor calphostin C. TP activation also increased Src kinase activity, which was blocked by PTX, PP1, and calphostin C, but not by AG1478, indicating that Src activation occurs before phosphorylation of EGFR. Blockade of Src activity by expression of dominant negative mutant of Src inhibits mitogen-activated protein kinase (MAPK) activation induced by TxA2. ERK activation induced by the PKC activator phorbol myristate acetate was inhibited by PTX, PP1, AG1478, and calphostin C. In contrast, activation of ERK by lysophosphatidic acid, a Gi-coupled receptor activator, was inhibited by PTX, PP1, and AG1478, but not by calphostin C. Thus, TP-stimulated ERK activation requires Gi, which in turn requires PKC activation. Immunoprecipitation of Galphai showed increased association of Galphai with TPalpha following PKC activation. In conclusion, TPalpha is directly coupled to the Gi protein by a PKC-regulated mechanism; Gi coupling causes Src-dependent transactivation of the EGFR, which is the dominant pathway in TP-mediated ERK activation.
Gao
Y
Y
Department of Medicine, Cardiovascular Division, Albert Einstein College of Medicine of Yeshiva University, Bronx, New York 10461, USA.
Tang
S
S
Zhou
S
S
Ware
J A
JA
D002460:Cell Line; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D016391:Genes, src / Q000235:genetics / Q000502:physiology*; D006801:Humans; D007202:Indicators and Reagents; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D017482:Receptors, Thromboxane / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D014404:Tubulin / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D007202:Indicators and Reagents; D017482:Receptors, Thromboxane; D014404:Tubulin; D066246:ErbB Receptors; D019061:src-Family Kinases; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go