Abstract
Caveolin1 (Cav1) is an important component of the plasmamembrane microdomains, such as caveolae/lipid rafts, that are associated with angiotensin II type 1 (AT1) and epidermal growth factor (EGF) receptors in certain cell types. The interactions of Cav1 with other signaling molecules that mediate AT1 receptor function were analyzed in angiotensin II (Ang II)- and EGF-stimulated hepatic C9 cells. This study demonstrated that cholesterol-rich domains mediate the actions of early upstream signaling molecules such as Src and intracellular Ca2+ in cells stimulated by Ang II, but not by EGF, and that Cav1 has a scaffolding role in the process of mitogen-activated protein kinase activation. Furthermore, Cav1 phosphorylation by Ang II and EGF was regulated by intracellular Ca2+ and Src, further indicating reciprocal interactions among Cav1, Src, and intracellular Ca2+ through the AT1 receptor. Phosphorylation of Cav1 and the EGF receptor by Ang II, but not of extracellular signal-regulated kinase 1/2, was dependent on intracellular Ca2+. The phosphatidylinositol 3-kinase inhibitors, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin, differentially modulated both Cav1 and EGF receptor activation by Ang II through intracellular Ca2+. These findings further demonstrate the importance of Cav1 in conjunction with the receptor-mediated signaling pathways involved in cell proliferation and survival. It is clear that differential signaling pathways are operative in Ang II- and EGF-stimulated C9 cells and that cholesterol-enriched microdomains are essential components in cellular signaling processes that are dependent on specific agonists and/or cell types.
Footnotes
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ABBREVIATIONS: Cav1, caveolin1; C9 cells, hepatic clone 9 cells; FLIPR, fluorometric imaging plate reader; AT1, angiotensin II type 1; Ang II, angiotensin II; EGF, epidermal growth factor; AT1R, angiotensin II type 1 receptor; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal-regulated kinase 1/2; PBS, phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; RSK2, ribosomal S6 kinase 2; GPCR, G-protein-coupled receptor; siRNA, small interfering RNA; PAGE, polyacrylamide gel electrophoresis; BAPTA-2/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4,d]pyrimidine; AG1478, 4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride.
- Received May 2, 2008.
- Accepted August 7, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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