Identification of a specific effector of the small GTP-binding protein Rap2.
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 beta-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.
Janoueix-Lerosey
I
I
Unité INSERM 248, Institut Curie, Section de Recherche, Paris, France.
Pasheva
E
E
de Tand
M F
MF
Tavitian
A
A
de Gunzburg
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000502:physiology; D002352:Carrier Proteins; D003001:Cloning, Molecular; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015870:Gene Expression / Q000235:genetics; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured; D020668:rap GTP-Binding Proteins
D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C111480:RUNDC3A protein, human; C486394:Rap2ip protein, mouse; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D020668:rap GTP-Binding Proteins