title: Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α2C-adrenoceptors through the actin-binding protein filamin-2.
abstract: The second messenger cyclic AMP (cAMP) plays a vital role in vascular physiology, including vasodilation of large blood vessels. We recently demonstrated cAMP activation of Epac-Rap1A and RhoA-Rho-associated kinase (ROCK)-F-actin signaling in arteriolar-derived smooth muscle cells increases expression and cell surface translocation of functional α2C-adrenoceptors (α2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α2C-ARs and also increased translocation of perinuclear α2C-ARs to intracellular F-actin and to the plasma membrane. This study examined the mechanism of translocation to better understand the role of these newly discovered mediators of blood flow control, potentially activated in peripheral vascular disorders. We utilized a yeast two-hybrid screen with human microvascular smooth muscle cells (microVSM) cDNA library and the α2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast α-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that α2C-ARs, but not α2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α2C-ARs colocalized with filamin on intracellular filaments and at the plasma membrane. Small interfering RNA-mediated knockdown of filamin-2 inhibited Rap1-induced redistribution of α2C-ARs to the cell surface and inhibited receptor function. The studies suggest that cAMP-Rap1-Rho-ROCK signaling facilitates receptor translocation and function via phosphorylation of filamin-2 Ser(2113). Together, these studies extend our previous findings to show that functional rescue of α2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.
authors.affiliation: Center for Cardiovascular and Pulmonary Research, The Research Institute at Nationwide Children's Hospital, Columbus, Ohio;
authors.forename: Hanaa K B
authors.initials: HK
authors.lastname: Motawea
authors.forename: Selvi C
authors.initials: SC
authors.lastname: Jeyaraj
authors.forename: Ali H
authors.initials: AH
authors.lastname: Eid
authors.forename: Srabani
authors.initials: S
authors.lastname: Mitra
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Unger
authors.forename: Amany A E
authors.initials: AA
authors.lastname: Ahmed
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Flavahan
authors.forename: Maqsood A
authors.initials: MA
authors.lastname: Chotani
mesh_terms: D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D064448:Filamins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D021381:Protein Transport; D034622:RNA Interference; D034741:RNA, Small Interfering; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D015398:Signal Transduction; D014661:Vasoconstriction; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D064448:Filamins; C430803:RAP1A protein, human; D034741:RNA, Small Interfering; D018341:Receptors, Adrenergic, alpha-2; D000242:Cyclic AMP; D020827:rap1 GTP-Binding Proteins
keywords: GPCR; Rap1A; filamin-2; filamin-2 phospho-Ser2113; yeast two hybrid; yeast α-galactosidase assays