Cloning and characterization of a novel GRP78-binding protein in the rat brain

J Biol Chem. 2003 Mar 21;278(12):10531-7. doi: 10.1074/jbc.M212083200. Epub 2003 Jan 3.

Abstract

The full-length cDNA clone of a novel GRP78-binding protein (GBP) was isolated from rat brain using PCR-selected cDNA subtraction. GBP was predominantly expressed in neuronal cells among various brain tissues. GBP mRNA was already detected in the E12 brain and then gradually increased to reach a peak within P0-2 weeks after birth. GBP expression in the brain decreased age-dependently to approximately 30% of the postnatal level at 12 months. GBP encoded 1021 amino acids and was predicted to have two transmembrane regions and glutamic acid- and proline-rich regions. Because the sequence of GBP offered few clues to the possible function, we performed a GST-tagged GBP pull-down assay in PC12 lysates and identified GRP78, one of the heat shock proteins, as a counterpart. Observation of COS7 cells expressing green fluorescent protein- or Myc-tagged GBP showed that GBP was localized in the endoplasmic reticulum-Golgi domain where BODIPY 558/568 (4,4-difluro-5-(2-thienyl)-4-bora-3alpha,4alpha-diaza-S-indacene)-labeled brefeldin A accumulated. To investigate a biological role for GBP, we established Neuro2a cells stably expressing Myc-tagged GBP. Overexpression of GBP did not affect cell growth or morphological features but attenuated the time-dependent decrease in cell viability caused by serum deprivation compared with control cells. After 48 h of serum starvation, Neuro2a cells overexpressing GBP were resistant to the cell death induced by serum withdrawal. These results suggest that GBP would have a relevant functional role in embryonic and postnatal development of the brain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Brain Chemistry
  • COS Cells
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Cell Death
  • Cloning, Molecular
  • Heat-Shock Proteins*
  • Immunohistochemistry
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • RNA, Messenger / analysis
  • Rats

Substances

  • Carrier Proteins
  • Heat-Shock Proteins
  • Molecular Chaperones
  • RNA, Messenger
  • Tmem132a protein, rat
  • molecular chaperone GRP78