CLONING OF A NOVEL FAMILY OF MAMMALIAN GTP-BINDING PROTEINS (RAGA, RAGB(S), RAGB(1)) WITH REMOTE SIMILARITY TO THE RAS-RELATED GTPASES

cDNA clones of two novel Ras-related GTP-binding proteins (RagA and Ra gB) were isolated from rat and human cDNA libraries, Their deduced ami no acid sequences comprise four of the six known conserved GTP-binding motifs (PM1, -2, -3, G1), the remaining two (G2, G3) being strikingly different from those of the Ras family, and an unusually large C-term inal domain (100 amino acids) presumably unrelated to GTP binding. Rag A and RagB differ by seven conservative amino acid substitutions (98% identity), and by 33 additional residues at the N terminus of RagB, In addition, two isoforms of RagB (RagB(s) and RagB(l)) were found that differed only by an insertion of 28 codons between the GTP-binding mot ifs PM2 and PM3, apparently generated by alternative mRNA splicing, Po lymerase chain reaction amplification with specific primers indicated that both long and short form of RagB transcripts were present in adre nal gland, thymus, spleen, and kidney, whereas in brain, only the long form RagB(1) was detected, A long splicing variant of RagA was not de tected, Recombinant glutathione S-transferase (GST) fusion proteins of RagA and RagB(s) bound large amounts of radiolabeled GTP gamma S in a specific and saturable manner, In contrast, GTP gamma S binding of GS T-RagB(l) hardly exceeded that of recombinant GST, GTP gamma S bound t o recombinant RagA, and RagB(s) was rapidly exchangeable for GTP, wher eas no intrinsic GTPase activity was detected. A multiple sequence ali gnment indicated that RagA and RagB cannot be assigned to any of the k nown subfamilies of Ras related GTPases but exhibit a 52% identity wit h a yeast protein (Gtr1) presumably involved in phosphate transport an d/or cell growth. It is suggested that RagA and RagB are the mammalian homologues of Gtr1 and that they represent a novel subfamily of Ras-h omologous GTP binding proteins.