Document Detail

Mammalian cell expression of an active site mutant of Pseudomonas exotoxin disrupts LRP1 maturation.
MedLine Citation:
PMID:  18351442     Owner:  NLM     Status:  MEDLINE    
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.
Diana V Pastrana; Cheol H Yun; Marian L McKee; David J Fitzgerald
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2008-03-20
Journal Detail:
Title:  Journal of biomedical science     Volume:  15     ISSN:  1423-0127     ISO Abbreviation:  J. Biomed. Sci.     Publication Date:  2008 Jul 
Date Detail:
Created Date:  2008-06-04     Completed Date:  2008-08-08     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9421567     Medline TA:  J Biomed Sci     Country:  Netherlands    
Other Details:
Languages:  eng     Pagination:  427-39     Citation Subset:  IM    
Laboratory of Molecular Biology, CCR, National Cancer Institute, NIH, HHS, Bethesda, MD, USA.
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MeSH Terms
Binding Sites / genetics
CHO Cells
Endoplasmic Reticulum / metabolism
Exotoxins / genetics*
Gene Expression
LDL-Receptor Related Protein 1 / antagonists & inhibitors*,  metabolism
Molecular Probe Techniques
Mutant Proteins
Protein Binding
Protein Transport
Pseudomonas / chemistry*
Reg. No./Substance:
0/Exotoxins; 0/LDL-Receptor Related Protein 1; 0/Ligands; 0/Mutant Proteins

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