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

Eur J Biochem. 1998 Mar 1;252(2):290-8. doi: 10.1046/j.1432-1327.1998.2520290.x.

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 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Brain / physiology
  • Carrier Proteins*
  • Cloning, Molecular
  • GTP-Binding Proteins / chemistry*
  • GTP-Binding Proteins / metabolism*
  • Gene Expression / genetics
  • Guanosine Triphosphate / analogs & derivatives
  • Guanosine Triphosphate / metabolism
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Mice
  • Molecular Sequence Data
  • Nerve Tissue Proteins / metabolism*
  • RNA, Messenger / analysis
  • Sequence Analysis, DNA
  • Transfection / genetics
  • Tumor Cells, Cultured
  • rap GTP-Binding Proteins

Substances

  • Carrier Proteins
  • Intracellular Signaling Peptides and Proteins
  • Nerve Tissue Proteins
  • RNA, Messenger
  • RUNDC3A protein, human
  • Rap2ip protein, mouse
  • Guanosine Triphosphate
  • GTP-Binding Proteins
  • rap GTP-Binding Proteins

Associated data

  • GENBANK/U73941
  • GENBANK/U93871
  • GENBANK/X94526