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 (microVSM), Rap1 selectively regulates expression of G protein-coupled α_2C-adrenoceptors (α_2C-ARs) through JNK-c-jun nuclear signaling. The α_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 α_2C-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 μM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2′- O-Me-cAMP (100 μM) activated RhoA, increased α_2C-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α_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 α_2C-ARs to the cell surface (∼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.

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_2C-adrenoceptors (α_2C-ARs) that mediate vasoconstriction in small blood vessels (arterioles). The Ras-related small GTPAse Rap1A increased expression of α_2C-ARs and also increased translocation of perinuclear α_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 α_2C-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_2C-ARs, but not α_2A-AR subtype, interacted with filamin. In Rap1-stimulated human microVSM, α_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 α_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 Ser²¹¹³. Together, these studies extend our previous findings to show that functional rescue of α_2C-ARs is mediated through Rap1-filamin signaling. Perturbation of this signaling pathway may lead to alterations in α_2C-AR trafficking and physiological function.

Vascular smooth muscle α_2C-adrenoceptors (α_2C-ARs) mediate vasoconstriction of small blood vessels, especially arterioles. Studies of endogenous receptors in human arteriolar smooth muscle cells (referred to as microVSM) and transiently transfected receptors in heterologous HEK293 cells show that the α_2C-ARs are perinuclear receptors that translocate to the cell surface under cellular stress and elicit a biological response. Recent studies in microVSM unraveled a crucial role of Rap1A-Rho-ROCK-F-actin pathways in receptor translocation, and identified protein-protein interaction of α_2C-ARs with the actin binding protein filamin-2 as an essential step in the process. To better understand the molecular nature and specificity of this interaction, in this study, we constructed comparative models of human α_2C-AR and human filamin-2 proteins. Finally, we performed in silico protein-protein docking to provide a structural platform for the investigation of human α_2C-AR and filamin-2 interactions. We found that electrostatic interactions seem to play a key role in this complex formation which manifests in interactions between the C-terminal arginines of α_2C-ARs (particularly R454 and R456) and negatively charged residues from filamin-2 region between residues 1979 and 2206. Phylogenetic and sequence analysis showed that these interactions have evolved in warm-blooded animals.

Ras-related Rap1A GTPase is implicated in pancreas β-cell insulin secretion and is stimulated by the cAMP sensor Epac2, a guanine exchange factor and activator of Rap1 GTPase. In this study, we examined the differential proteomic profiles of pancreata from C57BL/6 Rap1A-deficient (Null) and control wild-type (WT) mice with nanoLC-ESI-MS/MS to assess targets of Rap1A potentially involved in insulin regulation. We identified 77 overlapping identifier proteins in both groups, with 8 distinct identifier proteins in Null versus 56 distinct identifier proteins in WT mice pancreata. Functional enrichment analysis showed four of the eight Null unique proteins, ERO1-like protein β (Ero1lβ), triosephosphate isomerase (TP1), 14-3-3 protein γ, and kallikrein-1, were exclusively involved in insulin biogenesis, with roles in insulin metabolism. Specifically, the mRNA expression of Ero1lβ and TP1 was significantly (p < 0.05) increased in Null versus WT pancreata. Rap1A deficiency significantly affected glucose tolerance during the first 15–30 min of glucose challenge but showed no impact on insulin sensitivity. Ex vivo glucose-stimulated insulin secretion (GSIS) studies on isolated Null islets showed significantly impaired GSIS. Furthermore, in GSIS-impaired islets, the cAMP-Epac2-Rap1A pathway was significantly compromised compared to the WT. Altogether, these studies underscore an essential role of Rap1A GTPase in pancreas physiological function.