Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins Simona Paganini a , Gianni Francesco Guidetti a , Silvia Catricalà a , Piera Trionfini a , Simona Panelli b , Cesare Balduini a , Mauro Torti a, * a Center of Excellence for Applied Biology, Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy b Foundation Parco Tecnologico Padano, via A. Einstein, 26900 Lodi, Italy Received 18 May 2005; accepted 30 August 2005 Available online 21 September 2005 Abstract The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg 2+ -dependent. However, accurate compari- son of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg 2+ , the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins. © 2005 Elsevier SAS. All rights reserved. Keywords: GTP-binding proteins; Rap1; Rap2; GTP; GDP 1. Introduction The Rap proteins define a family of monomeric low molecular weight GTP-binding proteins that share 50–60% sequence homology with the product of the ras proto- oncogene. Four different members of this family have been identified to date: Rap1A, Rap1B, Rap2A, and Rap2B [1,2]. Rap1A and Rap1B are 95% homologous, and define a sub- family named Rap1. Similarly, a Rap2 subfamily includes Rap2A and Rap2B, which share about 90% sequence homol- ogy. Members of the Rap1 subfamily are about 70% homolo- gous with Rap2 proteins, with most aminoacid substitutions confined near the C-terminus. Many of the domains consid- ered critical for the subcellular localization and biological function are highly conserved among the Rap proteins. These include the effector domain (aminoacids 32–40), supposed to interact with downstream effectors, the nucleotide binding regions, able to mediate the interaction with GDP and GTP, and the C-terminal CAAX motif, which targets the proteins to the cell membranes [1,2]. Like other GTPases, Rap proteins are considered active when bound to GTP and inactive when associated to GDP [3,4]. Activation occurs through the release of bound GDP and the subsequent binding of GTP. This process is physi- ologically facilitated by cytosolic factors, generally indi- cated as guanine nucleotide exchange factors (GEFs). Inac- tivation occurs through the intrinsic GTPase activity, which converts bound GTP into GDP and is stimulated by regula- tory factors called GTPase activating proteins (GAPs). Sev- * Corresponding author. Tel.: +39 0382 987 238; fax: +39 0382 987 240. E-mail address: mtorti@unipv.it (M. Torti). Biochimie 88 (2006) 285–295 www.elsevier.com/locate/biochi 0300-9084/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2005.08.007