title: Cholesterol depletion inhibits epidermal growth factor receptor transactivation by angiotensin II in vascular smooth muscle cells: role of cholesterol-rich microdomains and focal adhesions in angiotensin II signaling.
abstract: Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF-R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMCs). Cholesterol and sphingomyelin-enriched lipid rafts are plasma membrane microdomains that concentrate various signaling molecules. Caveolae are specialized lipid rafts that are organized by the cholesterol-binding protein, caveolin, and have been shown to be associated with EGF-Rs. Angiotensin II stimulation promotes a rapid movement of AT(1) receptors to caveolae; however, their functional role in angiotensin II signaling has not been elucidated. Here we show that cholesterol depletion by beta-cyclodextrin disrupts caveolae structure and concomitantly inhibits tyrosine phosphorylation of the EGF-R and subsequent activation of protein kinase B (PKB)/Akt induced by angiotensin II. Similar inhibitory effects were obtained with other cholesterol-binding agents, filipin and nystatin. In contrast, EGF-R autophosphorylation and activation of Akt/PKB in response to EGF are not affected by cholesterol depletion. The early Ang II-induced upstream signaling events responsible for transactivation of the EGF-R, such as the intracellular Ca(2+) increase and c-Src activation, also remain intact. The EGF-R initially binds caveolin, but these two proteins rapidly dissociate following angiotensin II stimulation during the time when EGF-R transactivation is observed. The activated EGF-R is localized in focal adhesions together with tyrosine-phosphorylated caveolin. These findings suggest that 1) a scaffolding role of caveolin is essential for EGF-R transactivation by angiotensin II and 2) cholesterol-rich microdomains as well as focal adhesions are important signal-organizing compartments required for the spatial and temporal organization of angiotensin II signaling in VSMCs.
authors.affiliation: Department of Medicine, Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322, USA. mfukai@emory.edu
authors.forename: M
authors.initials: M
authors.lastname: Ushio-Fukai
authors.forename: L
authors.initials: L
authors.lastname: Hilenski
authors.forename: N
authors.initials: N
authors.lastname: Santanam
authors.forename: P L
authors.initials: PL
authors.lastname: Becker
authors.forename: Y
authors.initials: Y
authors.lastname: Ma
authors.forename: K K
authors.initials: KK
authors.lastname: Griendling
authors.forename: R W
authors.initials: RW
authors.lastname: Alexander
mesh_terms: D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000494:pharmacology; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000502:physiology; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D008297:Male; D008854:Microscopy, Electron; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D015533:Transcriptional Activation; D014443:Tyrosine / Q000378:metabolism; D047392:beta-Cyclodextrins
chemical_list: C495537:Cav1 protein, rat; D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D011518:Proto-Oncogene Proteins; D047392:beta-Cyclodextrins; D000804:Angiotensin II; D014443:Tyrosine; D004815:Epidermal Growth Factor; D002784:Cholesterol; D066246:ErbB Receptors; C494920:Akt1 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C031215:betadex