title: RACK1 regulates angiotensin II-induced contractions of SHR preglomerular vascular smooth muscle cells.
abstract: The preglomerular microcirculation of spontaneously hypertensive rats (SHR) is hypersensitive to angiotensin (ANG) II, and studies have shown that this is likely due to enhanced coincident signaling between G protein subunits αq(Gαq; released by ANG II) and βγ (Gβγ; released by Gi-coupled receptors) to active phospholipase C (PLC). Here we investigated the molecular basis for the enhanced coincident signaling between Gβγ and Gαqin SHR preglomerular vascular smooth muscle cells (PGVSMCs). Because receptor for activated C kinase 1 (RACK1; a scaffolding protein) organizes interactions between Gβγ, Gαq, and PLC, we included RACK1 in this investigation. Cell fractionation studies demonstrated increased levels of membrane (but not cytosolic) Gβ, Gαq, PLCβ3, and RACK1 in SHR PGVSMCs compared with Wistar-Kyoto rat PGVSMCs. In SHR PGVSMCs, coimmunoprecipitation demonstrated RACK1 binding to Gβ and PLCβ3, but only at cell membranes. Pertussis toxin (which blocks Gβγ) and U73122 (which blocks PLC) reduced membrane RACK1; however, RACK1 knockdown (shRNA) did not affect membrane levels of Gβ, Gαq, or PLCβ3In a novel gel contraction assay, RACK1 knockdown in SHR PGVSMCs attenuated contractions to ANG II and abrogated the ability of neuropeptide Y (which signals via Gβγ) to enhance ANG II-induced contractions. We conclude that in SHR PGVSMCs the enlarged pool of Gβγ and PLCβ3recruits RACK1 to membranes and RACK1 then organizes signaling. Consequently, knockdown of RACK1 prevents coincident signaling between ANG II and the Gipathway. This is the first study to implicate RACK1 in vascular smooth muscle cell contraction and suggests that RACK1 inhibitors could be effective cardiovascular drugs.
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania.
authors.forename: Xiao
authors.initials: X
authors.lastname: Zhu
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania edj@pitt.edu.
authors.forename: Edwin K
authors.initials: EK
authors.lastname: Jackson
mesh_terms: D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D004195:Disease Models, Animal; D044387:GTP-Binding Protein beta Subunits / Q000378:metabolism; D044388:GTP-Binding Protein gamma Subunits / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006973:Hypertension / Q000201:enzymology* / Q000503:physiopathology; D007606:Juxtaglomerular Apparatus / Q000098:blood supply*; D008297:Male; D055806:Microvessels / Q000201:enzymology / Q000503:physiopathology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000201:enzymology; D009478:Neuropeptide Y / Q000378:metabolism; D054799:Phospholipase C beta / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D034622:RNA Interference; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D000075370:Receptors for Activated C Kinase; D015398:Signal Transduction / Q000187:drug effects; D014162:Transfection; D014661:Vasoconstriction / Q000187:drug effects*; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
chemical_list: C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D009478:Neuropeptide Y; C550931:RACK1 protein, rat; D000075370:Receptors for Activated C Kinase; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; D054799:Phospholipase C beta; C518041:Plcb3 protein, rat; D019204:GTP-Binding Proteins
keywords: RACK1; angiotensin; hypertension; kidney; signal transduction