PSEUDOMONAS EXOTOXIN-MEDIATED SELECTION YIELDS CELLS WITH ALTERED EXPRESSION OF LOW-DENSITY-LIPOPROTEIN RECEPTOR-RELATED PROTEIN

The alpha(2)-macroglobulin (alpha(2)M) receptor/low-density lipoprotei n receptor-related protein (LRP) is important for the clearance of pro teases, protease-inhibitor complexes, and various ligands associated w ith lipid metabolism. While the regulation of receptor function is poo rly understood, the addition of high concentrations of the 39-kD recep tor-associated protein (RAP) to cells inhibits the binding and/or upta ke of many of these ligands. Previously, we (Kounnas, M. Z., J. Henkin , W. S. Argraves, D. K. Strickland. 1992. J. Biol. Chem. 267:12420-124 23) showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These f indings suggested that PE might use LRP to gain entry into toxin-sensi tive cells. Here we report on a strategy to select PE-resistant lines of Chinese hamster ovary cells that express altered amounts of LRP. An important part of this strategy is to screen PE-resistant clones for those that retain sensitivity to both diphtheria toxin and to a fusion protein composed of lethal factor (from anthrax toxin) fused to the a denosine diphosphate-ribosylating domain of PE. Two lines, with obviou s changes in their expression of LRP, were characterized in detail. Th e 14-2-1 line had significant amounts of LRP, but in contrast to wild- type cells, little or no receptor was displayed on the cell surface. I nstead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake o f chymotrypsin-alpha(2)M and was 10-fold resistant to PE compared with wild-type cells. A second line, 13-5-1, had no detectable LRP mRNA or protein, did not internalize alpha(2)M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to rece ptor-specific defects, since these mutant lines showed no resistance t o a PE chimeric toxin that was internalized via the transferrin recept or. The results of this investigation confirm that LRP mediates the in ternalization of PE.