Correspondence to J. de Gunzburg, Unité INSERM 248, Institut Curie, Section de Recherche, 26 rue d'Ulm, F-75231 Paris Cedex 15, France
Identification of a specific effector of the small GTP-binding protein Rap2
Article first published online: 25 DEC 2001
European Journal of Biochemistry
Volume 252, Issue 2, pages 290–298, March (I) 1998
How to Cite
Janoueix-Lerosey, I., Pasheva, E., de Tand, M.-F., Tavitian, A. and de Gunzburg, J. (1998), Identification of a specific effector of the small GTP-binding protein Rap2. European Journal of Biochemistry, 252: 290–298. doi: 10.1046/j.1432-1327.1998.2520290.x
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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 ( EC188.8.131.52).
- Issue published online: 25 DEC 2001
- Article first published online: 25 DEC 2001
- (Received 22 September/8 December 1997)
- Cited By
- two-hybrid system;
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.