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Identification of guanine nucleotide exchange factors (GEFs) for the Rap1 GTPase. Regulation of MR-GEF by M-Ras-GTP interaction.

Although the Ras subfamily of GTPases consists of approximately 20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating or RalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.

Pubmed ID: 10934204


  • Rebhun JF
  • Castro AF
  • Quilliam LA


The Journal of biological chemistry

Publication Data

November 10, 2000

Associated Grants


Mesh Terms

  • Amino Acid Sequence
  • Blotting, Northern
  • Brain
  • Calcium
  • Catalytic Domain
  • Cell Line
  • DNA, Complementary
  • DNA-Binding Proteins
  • Diglycerides
  • Dose-Response Relationship, Drug
  • Enzyme Activation
  • Guanine Nucleotide Exchange Factors
  • Humans
  • Molecular Sequence Data
  • Mutagenesis
  • Nerve Tissue Proteins
  • Plasmids
  • Precipitin Tests
  • Protein Binding
  • Protein Structure, Tertiary
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-raf
  • Receptors, Cytoplasmic and Nuclear
  • Sequence Homology, Amino Acid
  • Tissue Distribution
  • Transcription Factors
  • Tumor Cells, Cultured
  • ets-Domain Protein Elk-1
  • rap GTP-Binding Proteins
  • rap1 GTP-Binding Proteins
  • ras Guanine Nucleotide Exchange Factors