Interaction with filamin2 enables translocation of α2C‐adrenoceptors to the plasma membrane of smooth muscle cells

Abstract

α2C-Adrenoceptors (α2C-ARs) expressed by cutaneous arterial smooth muscle cells (SMCs) are sequestered in the transGolgi and are functionally silent. We have demonstrated that translocation and functional rescue of α2C-ARs to the plasma membrane can be achieved by activation of Rap1-GTPase and subsequent activation of RhoA and Rho kinase (ROCK). The goal of this study was to identify mechanism(s) involved in this response. A yeast 2 hybrid screen was performed using a SMC cDNA library, with the α2C-AR C-terminus as bait. A novel interaction was identified between α2C-ARs and the actin binding protein filamin2. Activation of Rap1-GTPase by forskolin (FSK, 10 μM) or 8-pCPT-2′-O-Me-cAMP (8-CPT, 100μM) caused redistribution of α2C-ARs from a perinuclear region to intracellular filamentous structures and to the plasma membrane of SMCs. A similar pattern of staining and translocation was observed for endogenously and exogenously-expressed α2C-ARs. Staining for filamin colocalized with α2C-ARs on the filaments and at the plasma membrane. Disruption of filamin2 expression by siRNA oligoduplexes inhibited the redistribution of α2C-ARs in response to FSK or 8-CPT. FSK or 8-CPT stimulated phosphorylation of filamin. Filamin phosphorylation and redistribution of α2C-ARs in response to 8CPT or FSK were reduced by the ROCK inhibitors H-1152 (0.1 μM), Y-27632 (0.6 μM) or fasudil (10 μM). Therefore, filamin2 plays a crucial role in the translocation and functional rescue of α2C-ARs to the cell surface of SMCs in response to Rap1-ROCK signaling. Perturbation of this signaling pathway may lead to alterations in α2C-AR trafficking and physiological function.