title: Pseudomonas exotoxin-mediated selection yields cells with altered expression of low-density lipoprotein receptor-related protein.
abstract: The alpha 2-macroglobulin (alpha 2M) receptor/low-density lipoprotein receptor-related protein (LRP) is important for the clearance of proteases, protease-inhibitor complexes, and various ligands associated with lipid metabolism. While the regulation of receptor function is poorly understood, the addition of high concentrations of the 39-kD receptor-associated protein (RAP) to cells inhibits the binding and/or uptake of many of these ligands. Previously, we (Kounnas, M.Z., R.E. Morris, M.R. Thompson, D.J. FitzGerald, D.K. Strickland, and C.B. Saelinger. 1992. J. Biol. Chem. 267:12420-12423) [corrected] showed that Pseudomonas exotoxin (PE) could bind immobilized LRP. Also, the addition of RAP blocked toxin-mediated cell killing. These findings suggested that PE might use LRP to gain entry into toxin-sensitive 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 adenosine diphosphate-ribosylating domain of PE. Two lines, with obvious changes in their expression of LRP, were characterized in detail. The 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. Instead, receptor protein was found primarily within cells, much of it apparently in an unprocessed state. The 14-2-1 line showed no uptake of chymotrypsin-alpha 2M 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 2M-chymotrypsin, and exhibited a 100-fold resistance to PE. Resistance to PE appeared to be due to receptor-specific defects, since these mutant lines showed no resistance to a PE chimeric toxin that was internalized via the transferrin receptor. The results of this investigation confirm that LRP mediates the internalization of PE.
authors.affiliation: Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: D J
authors.initials: DJ
authors.lastname: FitzGerald
authors.forename: C M
authors.initials: CM
authors.lastname: Fryling
authors.forename: A
authors.initials: A
authors.lastname: Zdanovsky
authors.forename: C B
authors.initials: CB
authors.lastname: Saelinger
authors.forename: M
authors.initials: M
authors.lastname: Kounnas
authors.forename: J A
authors.initials: JA
authors.lastname: Winkles
authors.forename: D
authors.initials: D
authors.lastname: Strickland
authors.forename: S
authors.initials: S
authors.lastname: Leppla
mesh_terms: D036002:ADP Ribose Transferases; D000818:Animals; D001427:Bacterial Toxins; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005098:Exotoxins / Q000096:biosynthesis / Q000494:pharmacology* / Q000633:toxicity; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011550:Pseudomonas aeruginosa; D011971:Receptors, Immunologic / Q000032:analysis / Q000096:biosynthesis*; D011990:Receptors, Transferrin / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology / Q000633:toxicity; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D005098:Exotoxins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D011971:Receptors, Immunologic; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A