title: Mammalian cell expression of an active site mutant of Pseudomonas exotoxin disrupts LRP1 maturation.
abstract: Low density lipoprotein receptor-related protein 1, (LRP1) is a large multifunctional receptor that binds more than 25 physiologic ligands. In addition, it functions as the surface receptor for several Rhinoviruses, HIV-tat and Pseudomonas exotoxin (PE). We report that the expression of PE within mammalian cells can serve as a probe of LRP1 maturation and functionality. To avoid cell killing, an enzymatically inactive form of the toxin (PEDelta553) was expressed. A permanent cell line (termed CY301) was established whereby PEDelta553 was expressed continually into the ER of CHO cells. CY301 cells were 100-fold resistant to exogenously added active PE but exhibited no cross-resistance to other toxins. Our studies indicate that PEDelta553 bound to immature LRP1 in the ER, prevented its maturation to the cell surface and thereby produced a toxin resistant phenotype. By confocal microscopy, cell-associated PEDelta553 was localized to the ER and co-localized with LRP1. Further characterization of CY301 cells indicated that RAP, the chaperone that aids in LRP1 folding, was released to the growth media. Thus the intracellular expression of PEDelta553 appears to be a valuable probe of LRP1 maturation and trafficking.
authors: Laboratory of Molecular Biology, CCR, National Cancer Institute, NIH, HHS, Bethesda, MD, USA.
authors: Diana V
authors: DV
authors: Pastrana
authors: Cheol H
authors: CH
authors: Yun
authors: Marian L
authors: ML
authors: McKee
authors: David J
authors: DJ
authors: Fitzgerald
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics*; D015870:Gene Expression; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015336:Molecular Probe Techniques; D050505:Mutant Proteins; D009154:Mutation; D011485:Protein Binding; D021381:Protein Transport; D011549:Pseudomonas / Q000737:chemistry*
chemical_list: D005098:Exotoxins; D008024:Ligands; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D050505:Mutant Proteins