IDENTIFICATION OF A SPECIFIC EFFECTOR OF THE SMALL GTP-BINDING PROTEIN RAP2

Rap2 is a small GTP-binding protein that belongs to the Ras superfamil y and whose function is still unknown. To elucidate Rap? function, we searched for potential effecters by screening a mouse brain cDNA libra ry in a yeast two-hybrid system using as a bait a Rap2A protein bearin g a mutation of Gly to Val at position 12. This strategy lead to the i dentification of a protein that interacts specifically with Rap?A comp lexed with GTP, and requires an intact effector domain of Rap2A for in teraction; we designated this protein Rap2-interacting protein 8 (RPIP 8). Biochemical data obtained from in vitro studies with purified prot eins 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 a cids, exhibits 93.7% identity in their common region. A search for sim ilar sequences in expressed-sequence-tags databanks revealed the prese nce in human and rodents of mRNAs encoding the 400-residue and 446-res idue 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 pheochro mocytoma PCI? cells and the pancreatic beta-cell lines HIT-T15 and RIN -m5F. Using transient transfections of HIT-T15 cells it was possible t o demonstrate that [Val12]Rap2 and wild-type Rap2 could be immunopreci pitated with RPIP8. These data therefore argue for RPIP8 being a speci fic effector of the Rap2 protein in cells exhibiting neuronal properti es.