Cyclic AMP-Rap1A signaling activates RhoA to induce α2c-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells

Jeyaraj SC, Unger NT, Eid AH, Mitra S, El-Dahdah NP, Quilliam LA, Flavahan NA, Chotani MA. Cyclic AMP-Rap1A signaling activates RhoA to induce alpha(2c)-adrenoceptor translocation to the cell surface of microvascular smooth muscle cells. Am J Physiol Cell Physiol 303: C499-C511, 2012. First published May 23, 2012; doi:10.1152/ajpcell.00461.2011.-Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (micro-VSM), Rap1 selectively regulates expression of G protein-coupled alpha(2C)-adrenoceptors (alpha(2C)-ARs) through JNK-c-jun nuclear signaling. The alpha(2C)-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional alpha(2C)-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 mu M) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2'-O-Me-cAMP (100 mu M) activated RhoA, increased alpha(2C)-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The alpha(2C)-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional alpha(2C)-ARs to the cell surface (similar to 4-to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.