title: Clathrin-dependent internalization of the angiotensin II AT₁A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells.
abstract: The major effects of Angiotensin II (AngII) in vascular tissue are mediated by AngII AT1A receptor activation. Certain effects initiated by AT1A receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC), AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by β-arrestin-dependent receptor internalization and NF-κB activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization of activated AT1A receptor mediates NF-κB activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2׳s effects on AT1 receptor/GFP internalization in HEK-293 cells and p65 NF-κB nuclear localization in RASMC. Pitstop-2 significantly inhibited internalization of AT1A receptor (44.7% ± 3.1% Control vs. 13.2% ± 8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT1A receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NF-κB phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNFα signaling. Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT1A receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through β-arrestin mediated internalization of G protein-coupled receptors, such as AT1A receptors.
authors.affiliation: Division of Nephrology, Department of Medicine, Medical University of South Carolina, United States. Electronic address: morinelt@musc.edu.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Morinelli
authors.affiliation: Division of Nephrology, Department of Medicine, Medical University of South Carolina, United States.
authors.forename: Linda P
authors.initials: LP
authors.lastname: Walker
authors.affiliation: Division of Nephrology, Department of Medicine, Medical University of South Carolina, United States; Ralph H. Johnson VA Medical Center, Charleston, SC, United States.
authors.forename: Juan Carlos Q
authors.initials: JC
authors.lastname: Velez
authors.affiliation: Division of Nephrology, Department of Medicine, Medical University of South Carolina, United States; Ralph H. Johnson VA Medical Center, Charleston, SC, United States.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Ullian
mesh_terms: D000818:Animals; D001011:Aorta / Q000166:cytology*; D002966:Clathrin / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D013449:Sulfonamides / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology
chemical_list: D002966:Clathrin; D044140:Receptor, Angiotensin, Type 1; D013449:Sulfonamides; D053778:Thiazolidines; C578512:pitstop 2; D051546:Cyclooxygenase 2
keywords: AT(1A) receptor; Angiotensin II; Clathrin-mediated endocytosis; Cyclooxygenase 2; NF-κB; Vascular smooth muscle cells