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

doi:10.1074/jbc.270.48.28982

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

From the Institut für Pharmakologie und Toxikologie Rheinisch-Westfalische Technische Hochschule Aachen, Germany

^¶ To whom correspondence should be addressed:
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät der RWTH Aachen, Wendlingweg 2, D-52057 Aachen, FRG.
Tel.: 49-241-8089120; Fax: 49-241-8888433.

Abstract

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 and RagB) 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 RagB was detected. A long splicing variant of RagA was not detected. Recombinant glutathione S-transferase (GST) fusion proteins of RagA and RagB bound large amounts of radiolabeled GTPS in a specific and saturable manner. In contrast, GTPS binding of GST-RagB hardly exceeded that of recombinant GST. GTPS bound to recombinant RagA, and RagB 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.

Footnotes

↵^§ Contributed equally to this paper.
↵* This work was supported by Grant Jo 117/9-1 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X85183[GenBank], X85184[GenBank], X90529[GenBank], and X90530[GenBank].
↵¹ The abbreviations used are:

ARF

ADP ribosylation factor

PCR

polymerase chain reaction

GST

glutathione S-transferase

bp

base pair(s)

kb

kilobase(s)

HPLC

high performance liquid chromatography

GTPS

guanosine 5′-O-(3-thiotriphosphate).
- Received March 10, 1995.
- Revision received August 24, 1995.