Received for publication August 11, 2006.
Revised November 16, 2006.
Accepted for publication November 20, 2006.

Modulating the Folding of P-glycoprotein and CFTR Truncation Mutants with Pharmacological Chaperones

Abstract

CFTR and P-glycoprotein (P-gp) are ABC transporters that have two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Defective folding of CFTR lacking phenylalanine 508 (F508) in NBD1 is the most common cause of cystic fibrosis. The F508 position appears to be universally important in ABC transporters because deletion of the equivalent residue (Y490) in P-gp also inhibits maturation of the protein. The pharmacological chaperone VRT-325 can repair the F508-type folding defects in P-gp or CFTR. VRT-325 may repair the folding defects by promoting dimerization of the two NBDs or by promoting folding of the TMDs. To distinguish between these two mechanisms, we tested the ability of VRT-325 to promote folding of truncation mutants lacking one or both NBDs. Sensitivity to glycosidases was used as an indirect indicator of folding. It was found that VRT-325 could promote maturation of truncation mutants lacking NBD2. Truncation mutants of CFTR or P-gp lacking both NBDs showed deficiencies in core-glycosylation that could be partially reversed by carrying out expression in the presence of VRT-325. The results show that dimerization of the two NBDs to form a 'nucleotide-sandwich' structure or NBD interactions with the TMDs are not essential for VRT-325 enhancement of folding. Instead, VRT-325 can promote folding of the TMDs alone. The ability of VRT-325 to promote core-glycosylation of the NBD-less truncation mutants suggests that one mechanism whereby the compound enhances folding is by promoting proper insertion of TM segments attached to the glycosylated loops so that they adopt an orientation favorable for glycosylation.

Key words: Ion transporters (SERCA, Na/K ATPase, CFTR), MDR/p-Glycoprotein, Mutagenesis/Chimeric approaches

This article has been cited by other articles:

				T. W. Loo, M. C. Bartlett, and D. M. Clarke Arginines in the First Transmembrane Segment Promote Maturation of a P-glycoprotein Processing Mutant by Hydrogen Bond Interactions with Tyrosines in Transmembrane Segment 11 J. Biol. Chem., September 5, 2008; 283(36): 24860 - 24870. [Abstract] [Full Text] [PDF]

				M. F. De Rosa, C. Ackerley, B. Wang, S. Ito, D. M. Clarke, and C. Lingwood Inhibition of Multidrug Resistance by AdamantylGb3, a Globotriaosylceramide Analog J. Biol. Chem., February 22, 2008; 283(8): 4501 - 4511. [Abstract] [Full Text] [PDF]

				Y. Wang, T. W. Loo, M. C. Bartlett, and D. M. Clarke Correctors Promote Maturation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-processing Mutants by Binding to the Protein J. Biol. Chem., November 16, 2007; 282(46): 33247 - 33251. [Abstract] [Full Text] [PDF]

				T. W. Loo, M. C. Bartlett, and D. M. Clarke Suppressor Mutations in the Transmembrane Segments of P-glycoprotein Promote Maturation of Processing Mutants and Disrupt a Subset of Drug-binding Sites J. Biol. Chem., November 2, 2007; 282(44): 32043 - 32052. [Abstract] [Full Text] [PDF]

				P. Gissen and E. R Maher Cargos and genes: insights into vesicular transport from inherited human disease J. Med. Genet., September 1, 2007; 44(9): 545 - 555. [Abstract] [Full Text] [PDF]