Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle [alpha]^sub 2C^-adrenoceptors through the actin-binding protein filamin-2

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 α...-adrenoceptors (α...-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α...-ARs and also increased translocation of perinuclear α...-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 α...-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 α...-ARs, but not α...-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α...-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 α...-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... Together, these studies extend our previous findings to show that functional rescue of α...-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α...-AR trafficking and physiological function. (ProQuest: ... denotes formulae/symbols omitted.)