title: Sub-cellular distribution of endothelin signaling pathway components in ventricular myocytes and heart: lack of preformed caveolar signalosomes.
abstract: Stimulation of endothelin receptors (ETRs) leads to activation of the extracellular signal-regulated protein kinase (ERK) cascade. It is unclear whether compartmentalization to lipid rafts is necessary for proper endothelin signaling, as methodologies employed to isolate and study caveolae involve detergent extraction, which may induce aggregation of membrane-associated proteins. The present study was to determine if components of the endothelin-1 (ET-1) pathway leading to ERK activation localize to caveolae and constitute preformed signalosomes. Microsomes were prepared from intact ventricular myocardium, in the absence of detergents, and fractionated by differential and sucrose-density gradient centrifugation to determine if caveolins and components of the ETRs post-receptor signaling cascade were in vesicles having similar physical properties. Confocal fluorescence microscopy, followed by digital deconvolution, was employed to determine if the signaling proteins colocalized with caveolin within intact, freshly isolated adult myocytes. With the exception of ET(A)Rs, proteins from the ET-1 pathway copurified in part or entirely (Galpha(11)), with caveolin-1 and caveolin-3. In contrast, with the exception of Galpha(q/11), Galpha(i3) and Gbeta G-protein subunits, most of the proteins studied showed little colocalization with caveolin-3. Thus, although components of the ET-1 signaling pathway may exist in vesicles having similar characteristics to vesicles containing caveolin, these proteins did not associate with caveolae in intact myocytes. The lack of detectable colocalization of caveolin-3 with proteins within the endothelin post-receptor signaling system in intact myocytes argues against the presence of a preformed, caveolae-associated signalosome.
authors.affiliation: Institut de Cardiologie de Montréal, Centre de Recherche, 5000, rue Bélanger, Montréal, Québec, Canada H1T 1C8.
authors.forename: Benoit
authors.initials: B
authors.lastname: Boivin
authors.forename: Louis R
authors.initials: LR
authors.lastname: Villeneuve
authors.forename: Nada
authors.initials: N
authors.lastname: Farhat
authors.forename: Dominique
authors.initials: D
authors.lastname: Chevalier
authors.forename: Bruce G
authors.initials: BG
authors.lastname: Allen
mesh_terms: D000818:Animals; D021941:Caveolae / Q000737:chemistry* / Q000378:metabolism; D051242:Caveolin 1; D051244:Caveolin 3; D022461:Caveolins / Q000032:analysis* / Q000378:metabolism; D004285:Dogs; D019204:GTP-Binding Proteins / Q000378:metabolism; D006352:Heart Ventricles / Q000166:cytology; D007527:Isoenzymes / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000737:chemistry* / Q000378:metabolism; D054799:Phospholipase C beta; D011493:Protein Kinase C / Q000378:metabolism; D051744:Protein Kinase C-epsilon; D051381:Rats; D017466:Receptors, Endothelin / Q000032:analysis* / Q000378:metabolism; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: C495537:Cav1 protein, rat; C495549:Cav3 protein, rat; D051242:Caveolin 1; D051244:Caveolin 3; D022461:Caveolins; D007527:Isoenzymes; D017466:Receptors, Endothelin; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051744:Protein Kinase C-epsilon; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins