Cloning and characterization of a novel GRP78-binding protein in the rat brain.
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.
Oh-hashi
Kentaro
K
Department of Anatomy and Neurobiology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Japan.
Naruse
Yoshihisa
Y
Amaya
Fumimasa
F
Shimosato
Goshun
G
Tanaka
Masaki
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001923:Brain Chemistry; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016923:Cell Death; D003001:Cloning, Molecular; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007150:Immunohistochemistry; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats
D002352:Carrier Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D012333:RNA, Messenger; C512354:Tmem132a protein, rat