Cyclic AMP-Rap1A signaling mediates cell surface translocation of microvascular smooth muscle α_2C-adrenoceptors through the actin-binding protein filamin-2

Abstract

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 ␣ 2C-adrenoceptors (␣ 2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of ␣ 2C-ARs and also increased translocation of perinuclear ␣ 2C-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 ␣ 2C-AR COOH terminus to identify a novel interaction with the actin cross-linker filamin-2. Yeast ␣-galactosidase assays, site-directed mutagenesis, and coimmunoprecipitation experiments in heterologous human embryonic kidney (HEK) 293 cells and in human microVSM demonstrated that ␣ 2C-ARs, but not ␣ 2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, ␣ 2C-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 ␣ 2C-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 ␣ 2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in ␣ 2C-AR trafficking and physiological function.GPCR; yeast two hybrid; filamin-2; filamin-2 phospho-Ser 2113 ; yeast ␣-galactosidase assays; Rap1A THE G PROTEIN-COUPLED ␣ 2C -adrenoceptors (␣ 2C -ARs) are stress receptors of the vascular sympathetic system (9). These receptors have a unique mechanism of regulation compared with the other two family members, the ␣ 2A -ARs and ␣ 2B -ARs. ␣ 2C -ARs are generally retained in intracellular Golgi compartment and are silent but can translocate to the cell surface under vascular stress, including cold exposure, and elicit a functional response leading to vasoconstriction (7,8,18,20). It is therefore important to understand the mechanisms of receptor expression and trafficking for a clear understanding of ␣ 2C -AR physiology and its role in pathobiology.We recently demonstrated a role of the Ras-related small GTPase Rap1A in enhancing translocation of ␣ 2C -ARs from the perinuclear compartment to the cell surface of microvascular smooth muscle cells (microVSM; Ref. 19). In human microVSM, increased levels of intracellular cAMP or exogenous addition of the cAMP analog 8-pCPT-2=-O-cAMP activated Rap1, leading to subsequent activation of RhoA, Rhoassociated kinase (ROCK), and increase in F-actin (8,19). This increased F-actin f...

Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development

“…Supporting this functional activation of RhoA by EPAC/Rap1A, inhibition of RhoA or downstream Rho-associated protein kinase, ROCK, arrested the EPAC/Rap1A-dependent F-actin enhancement (472). A followup study searched for the relation between ␣ 2c -adrenoreceptors translocation and F-actin uncovering a specific interaction between the COOH terminus of ␣ 2cadrenoreceptors and filamin-2, an actin cross-linker, using yeast-two hybrid screening (737). Indeed, forskolin, EPAC activation, and constitutively active Rap1A led to phosphorylation of filamin-2 at Ser2113 in a RhoA/ROCK-dependent manner, and consequently translocation of sequestered ␣ 2c -adrenoreceptors (737).…”

“…A followup study searched for the relation between ␣ 2c -adrenoreceptors translocation and F-actin uncovering a specific interaction between the COOH terminus of ␣ 2cadrenoreceptors and filamin-2, an actin cross-linker, using yeast-two hybrid screening (737). Indeed, forskolin, EPAC activation, and constitutively active Rap1A led to phosphorylation of filamin-2 at Ser2113 in a RhoA/ROCK-dependent manner, and consequently translocation of sequestered ␣ 2c -adrenoreceptors (737). Therefore, EPAC/Rap1A appears to be implicated in vasoconstriction to initiate heat conservation after exposure to cold stimuli in the microvasculature.…”

“…EPAC activation is also associated with the activation of eNOS, along with PKA, in endothelial cells to produce NO and induce relaxation of nearby muscle (336,878). During stress however, EPAC/Rap1 initiates a RhoA-dependent mechanism to translocate ␣ 2c -adrenoreceptors to the cell surface and induce vasoconstriction (182,183,471,472,737). Therefore, these conclusions in vascular smooth muscle depict EPAC/Rap1 and PKA cooperatively signaling through pathways that induce vasorelaxation, except in the presence of stressful stimuli where EPAC/Rap1 switches to modulate constriction of the vessels.…”

Raynaud’s Phenomenon: A Brief Review of the Underlying Mechanisms

“…In a rather elegant and orchestrated series of events, α 2C -ARs then get in close proximity and associate with actin filaments, readying themselves for the trafficking process (Jeyaraj et al, 2012). This intimate association appears to be mediated by a direct interaction between α 2C -ARs and filamin-2, a cross-linker of actin filaments (Motawea et al, 2013). Indeed, further in silico protein-protein docking examinations confirmed that the interaction between α 2C -AR and F-actin occurs via the direct binding of α 2C -AR to filamin, the actin binding protein (Pawlowski et al, 2014).…”

In Silico Modeling of Human α2C-Adrenoreceptor Interaction with Filamin-2

“…The largest cluster had 41 structures, the 5 th best HADDOCK score (−91.2) and the 6 th best (lowest) electrostatic energy (−508.8) among all 10 clusters. However, the protein-protein interfaces among those structures did not involve any interactions between the C-terminal helix of ADRA2C and FLN2 between residues 1982 and 2183, as previously proven to occur by Motawea and coworkers [15]. Thus, among the ten most populated clusters, we searched for clusters that had receptor-filamin complexes having C-terminal helix of the receptor molecule involved in interactions with the filamin molecule.…”