Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins.
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 comparison 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.
Paganini
Simona
S
Center of Excellence for Applied Biology, Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy.
Guidetti
Gianni Francesco
GF
Catricalà
Silvia
S
Trionfini
Piera
P
Panelli
Simona
S
Balduini
Cesare
C
Torti
Mauro
M
D000595:Amino Acid Sequence; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism; D020224:Expressed Sequence Tags; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D006147:Guanine / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008533:Megakaryocytes / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D013997:Time Factors; D020668:rap GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D020827:rap1 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins
D012333:RNA, Messenger; D006147:Guanine; D019204:GTP-Binding Proteins; C065562:RAP2B protein, human; C511733:Rap2C protein, human; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins