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

Authors


  • 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.

  • E-mail:gunzburg@curie.fr

  • 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.

Abstract

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.

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