title: Accelerated urokinase-receptor protein turnover triggered by interference with the addition of the glycolipid anchor.
abstract: u-PAR (urokinase-type plasminogen activator receptor), anchored to the cell surface via a glycolipid moiety, drives tumour progression. We previously reported that colon cancer cells (RKO clone 2 FS2), attenuated for in vivo tumorigenicity, are diminished >15-fold for u-PAR display when compared with their tumorigenic isogenic counterparts (RKO clone 2), this disparity not reflecting altered transcription/mRNA stability. FACS, confocal microscopy and Western blotting using a fused u-PAR-EGFP (enhanced green fluorescent protein) cDNA revealed a >14-fold differential in the u-PAR-EGFP signal between the isogenic cells, ruling out alternate splicing as a mechanism. Although metabolic labelling indicated similar synthesis rates, pulse-chase revealed accelerated u-PAR-EGFP turnover in the RKO clone 2 FS2 cells. Expression in RKO clone 2 cells of a u-PAR-EGFP protein unable to accept the glycolipid moiety yielded diminished protein amounts, thus mirroring the low endogenous protein levels evident with RKO clone 2 FS2 cells. Transcript levels for the phosphatidylglycan anchor biosynthesis class B gene required for glycolipid synthesis were reduced by 65% in RKO clone 2 FS2 cells, and forced overexpression in these cells partially restored endogenous u-PAR. Thus attenuated u-PAR levels probably reflects accelerated turnover triggered by inefficient addition of the glycolipid moiety.
authors.affiliation: Department of Cancer Biology, MD Anderson Cancer Center, Houston, TX 77030, USA.
authors.forename: Hector
authors.initials: H
authors.lastname: Avila
authors.forename: Heng
authors.initials: H
authors.lastname: Wang
authors.forename: Santosh
authors.initials: S
authors.lastname: Chauhan
authors.forename: Sean
authors.initials: S
authors.lastname: Hartig
authors.forename: Douglas D
authors.initials: DD
authors.lastname: Boyd
mesh_terms: D017398:Alternative Splicing; D003110:Colonic Neoplasms / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015398:Signal Transduction; D014162:Transfection; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D006017:Glycolipids; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014568:Urokinase-Type Plasminogen Activator