Introduction The Rho family of small GTPases act as molecular switches to control a wide range of cellular processes in eukaryotic cells, such as normal growth, transformation, gene regulation and actin cytoskeletal organization (Hall, 1998). Each member of the Rho family has a different influence on the cytoskeletal structure and on cellular morphology. In fibroblasts, RhoA induces the formation of stress fibers associated with focal adhesions, Rac1 produces lamellipodia or membrane ruffling, and Cdc42 evokes filopodia (Nobes and Hall, 1995; Ridley et al., 1992; Ridley and Hall, 1992). In other cell types, these Rho-like GTPases trigger tissue-specific responses by their action on the actin cytoskeleton. The Rho GTPases are molecular switches that cycle between an inactive (GDP- bound) and an active (GTP-bound) conformation. They are regulated by guanine nucleotide exchange factors (GEFs) that activate them by accelerating the GDP/GTP exchange, while the GTPase-activating proteins (GAPs) stimulate GTP hydrolysis (Stam and Collard, 1999). To date, more than 30 mammalian GEFs of the Dbl family have been identified. These proteins are characterized by a Dbl homology (DH) domain of about 180 residues based on sequence homology with the central portion of the Dbl oncogene, the first identified Rho GEF active on Cdc42 and RhoA (Hart et al., 1994). In tandem with the DH domain is invariably found a pleckstrin homology (PH) domain, generally recognized as a membrane targeting module by its capacity to bind to phosphoinositides (Lemmon and Ferguson, 2000). More recently, PH domains have been shown to directly link PH-containing proteins to the actin cytoskeleton, by binding to actin (Yao et al., 1999) or to an actin-binding protein such as filamin in the case of Trio (Bellanger et al., 2000). PH domains have also been proposed to modulate the DH-mediated catalytic activity, depending on the nature of the phosphoinositides they bind (Han et al., 1998). The invariable association of a DH with a PH domain within the Rho GEF family suggests that the DH-PH tandem represents the functional unit responsible for activation of the Rho GTPases. In addition to this tandem module, Rho GEFs often present other structural or functional domains, predicted to play regulatory roles in the localization or the control of the GEF activity (Stam and Collard, 1999) and in the involvment of Rho GEFs in specific signal transduction networks. 629 The Rho small GTPases are crucial proteins involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. It has been reported that these GTPases are directly associated with cardiovascular disorders. In this context, we have searched for novel modulators of Rho GTPases, and here we describe p63RhoGEF a new Dbl-like guanine nucleotide exchange factor (GEF). P63RhoGEF encodes a 63 kDa protein containing a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to the second Rho GEF domain of Trio. Northern blot and in situ analysis have shown that p63RhoGEF is mainly expressed in heart and brain. In vitro guanine nucleotide exchange assays have shown that p63RhoGEF specifically acts on RhoA. Accordingly, p63RhoGEF expression induces RhoA-dependent stress fiber formation in fibroblasts and in H9C2 cardiac myoblasts. Moreover, we show that p63RhoGEF activation of RhoA in intact cells is dependent on the presence of the PH domain. Using a specific anti-p63RhoGEF antibody, we have detected the p63RhoGEF protein by immunocytochemistry in human heart and brain tissue sections. Confocal microscopy shows that p63RhoGEF is located in the sarcomeric I-band mainly constituted of cardiac sarcomeric actin. Together, these results show that p63RhoGEF is a RhoA-specific GEF that may play a key role in actin cytoskeleton reorganization in different tissues, especially in heart cellular morphology. Key words: GEF, RhoA, Cardiac sarcomere Summary Human p63RhoGEF, a novel RhoA-specific guanine nucleotide exchange factor, is localized in cardiac sarcomere Michel Souchet 1, *, Elodie Portales-Casamar 2, *, David Mazurais 1 , Susanne Schmidt 2 , Isabelle Léger 1 , Jean-Luc Javré 1 , Philippe Robert 1 , Isabelle Berrebi-Bertrand 1 , Antoine Bril 1 , Bernard Gout 1 , Anne Debant 2,‡ and Thierry P. G. Calmels 1,‡ 1 SmithKline Beecham Laboratoires Pharmaceutiques, Unité de Biologie Cardiovasculaire, 4 rue du Chesnay Beauregard, BP 96205, 35760 Saint- Grégoire, France 2 Centre de Recherches en Biochimie Macromoléculaire-Centre National de la Recherche Scientifique, UPR 1086, 1919 Route de Mende, 34293 Montpellier Cedex 05, France *Both authors contributed equally to this work ‡ Authors for correspondence (e-mail: t.calmels@bioprojet.com; debant@crbm.cnrs-mop.fr) Accepted 26 October 2001 Journal of Cell Science 115, 629-640 (2002) © The Company of Biologists Ltd Research Article