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.
Accelerated urokinase-receptor protein turnover triggered by interference with the addition of the glycolipid anchor
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Hector Avila, Heng Wang, Santosh Chauhan, Sean Hartig, Douglas D. Boyd; Accelerated urokinase-receptor protein turnover triggered by interference with the addition of the glycolipid anchor. Biochem J 1 March 2011; 434 (2): 233–242. doi: https://doi.org/10.1042/BJ20101573
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