SummaryRho/Rac proteins constitute a subgroup of the Ras superfamily of GTP hydrolases. Although originally implicated in the control of cytoskeletal events, it is currently known that these GTPases coordinate diverse cellular functions, including cell polarity, vesicular trafficking, the cell cycle and transcriptomal dynamics. In this review, we will provide an overview on the recent advances in this field regarding the mechanism of regulation and signaling, and the roles in vivo of this important GTPase family.

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca

The aim of this work was to investigate the coupling of human urotensin II (hU-II) to RhoA activation and regulation of RhoA-dependent functions. The use of the Rho-kinase inhibitor Y-27632 and the development of a membrane-permeant RhoA inhibitor (TAT-C3) allowed us to demonstrate that hU-II induced arterial smooth muscle contraction, actin stress fiber formation, and proliferation through the activation of the small GTPase RhoA and its downstream effector Rho-kinase.T he human homologue of the fish dodecapeptide urotensin II (hU-II) has been recently cloned. 1 Prepro-U-II mRNA was highly expressed in spinal cord but also found in the adrenal glands, kidney, and spleen. 1,2 hU-II-like immunoreactivity was detected in the vasculature and a diffuse staining was observed in the heart. 3 hU-II induced vasoconstriction of arteries from both rat and human. [3][4][5] With a potency Ϸ6-to 28-fold greater than endothelin-1 in nonhuman primate arteries, hU-II is the most potent mammalian vasoconstrictor identified so far. 3 hU-II has been defined as the ligand for the orphan receptor GPR14, 2,3 predominantly expressed in cardiovascular tissues. 3 Recombinant GPR14 coupled to Ca 2ϩ mobilization, and hU-II has been reported to produce a phospholipase C-dependent increase in inositol phosphates. 6 However, the intracellular signaling pathways of hU-II are not fully established.The small GTPase RhoA is now recognized as a major regulator of smooth muscle (SM) contraction involved in the control of arterial tone. 7 Thus, we postulate that hU-II should activate RhoA and regulate RhoA-dependent functions in vascular smooth muscle cells (SMCs). Materials and Methods Tension MeasurementsWistar rats (Janvier, France) were stunned and then killed by cervical dislocation. Isometric tension of endothelium-denuded arterial rings of thoracic aorta from the 2-cm portion proximal to the carotid bifurcation and pulmonary artery was measured as previously described. 8 Measurement of RhoA DistributionEndothelium-denuded aortic rings were stimulated with 0.1 mol/L hU-II. When maximal tension was raised, rings were rapidly frozen in liquid nitrogen then homogenized in lysis buffer. Membrane and cytosolic fractions were prepared and analyzed by Western blot using a mouse monoclonal anti-RhoA antibody (Santa Cruz Biotechnology) as previously described. 8 All experiments were approved by the local ethics committee. Plasmid Constructions and TAT-C3 Protein PurificationcDNA encoding for Clostridium botulinum C3 exoenzyme was cloned in frame, in the C-terminal of the HIV TAT protein transduction domain (AA 47-57) in vector pTAT-HA (kindly provided by S. Dowdy, Washington University, St. Louis, Mo). 9 Recombinant TAT-C3 protein was produced in Escherichia coli and purified as previously described. 9 SMC Culture and Actin StainingRat SMCs from the proximal segment of thoracic aorta were isolated by enzymatic dissociation and cultured as previously described. 8 Polymerized (F) actin was stained with FITC-conjugated phalloidin (5 g/mL) and Texas Red-la...

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