Cloning of a Novel Family of Mammalian GTP-binding Proteins (RagA, RagB^s, RagB¹) with Remote Similarity to the Ras-related GTPases *

cDNA clones of two novel Ras-related GTP-binding proteins (RagA and RagB) were isolated from rat and human cDNA libraries. Their deduced amino 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-terminal domain (100 amino acids) presumably unrelated to GTP binding. RagA 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 RagB¹) were found that differed only by an insertion of 28 codons between the GTP-binding motifs PM2 and PM3, apparently generated by alternative mRNA splicing. Polymerase chain reaction amplification with specific primers indicated that both long and short form of RagB transcripts were present in adrenal gland, thymus, spleen, and kidney, whereas in brain, only the long form RagB¹ was detected. A long splicing variant of RagA was not detected. Recombinant glutathione S-transferase (GST) fusion proteins of RagA and RagB^s bound large amounts of radiolabeled GTPγS in a specific and saturable manner. In contrast, GTPγS binding of GST-RagB¹ hardly exceeded that of recombinant GST. GTPγS bound to recombinant RagA, and RagB^s was rapidly exchangeable for GTP, whereas no intrinsic GTPase activity was detected. A multiple sequence alignment indicated that RagA and RagB cannot be assigned to any of the known subfamilies of Ras-related GTPases but exhibit a 52% identity with a yeast protein (Gtr1) presumably involved in phosphate transport and/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-homologous GTP binding proteins.