Identification of a specific effector of the small GTP-binding protein Rap2


  • Correspondence to J. de Gunzburg, Unité INSERM 248, Institut Curie, Section de Recherche, 26 rue d'Ulm, F-75231 Paris Cedex 15, France

  • Fax: +33 1 42 34 66 50.


  • Abbreviations. DBD, DNA-binding domain; RACE, rapid amplification of cDNA ends; GST, glutathione S-transferase; PKA, cAMP-dependent protein kinase; GAP, GTPase-activating protein; RPIP, Rap2-interacting protein; GDS, guanine-nucleotide dissociation stimulator; RGL, Ral-GDS−like protein; Rlf, Ral-GDS−like factor.

  • Enzyme. Glutathione transferase ( EC2.5.1.18).

  • Note. The novel nucleotide sequence data published here have been submitted to the Genbank database and are available under accession numbers U73941 (mouse sequence) and U93871 (human sequence) respectively.


Rap2 is a small GTP-binding protein that belongs to the Ras superfamily and whose function is still unknown. To elucidate Rap2 function, we searched for potential effectors by screening a mouse brain cDNA library in a yeast two-hybrid system using as a bait a Rap2A protein bearing a mutation of Gly to Val at position 12. This strategy lead to the identification of a protein that interacts specifically with Rap2A complexed with GTP, and requires an intact effector domain of Rap2A for interaction; we designated this protein Rap2-interacting protein 8 (RPIP8). Biochemical data obtained from in vitro studies with purified proteins confirmed the genetic results. Mouse RPIP8 consists of 446 amino acids, bears a coiled-coil domain between residues 265 and 313, and is expressed principally in brain. Its human counterpart, of 400 amino acids, exhibits 93.7 % identity in their common region. A search for similar sequences in expressed-sequence-tags databanks revealed the presence in human and rodents of mRNAs encoding the 400-residue and 446-residue forms of RPIP8. Furthermore a doublet of 45−50 kDa, corresponding to the 400-residue and 446-residue forms of the protein, was detected by western blotting of mouse brain extracts and lysates from pheochromocytoma PC12 cells and the pancreatic β-cell lines HIT-T15 and RIN-m5F. Using transient transfections of HIT-T15 cells it was possible to demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunoprecipitated with RPIP8. These data therefore argue for RPIP8 being a specific effector of the Rap2 protein in cells exhibiting neuronal properties.