Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR) and P-glycoprotein (P-gp) are ATP-binding cassette (ABC) transporters that have two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). Defective folding of CFTR lacking phenylalanine 508 (ΔPhe508) in NBD1 is the most common cause of cystic fibrosis. The Phe508 position seems to be universally important in ABC transporters because deletion of the equivalent residue (Tyr490) in P-gp also inhibits maturation of the protein. The pharmacological chaperone VRT-325 can repair the ΔPhe508-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.
Footnotes
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This work was supported by a grant from the Canadian Institutes of Health Research and a grant from the National Cancer Institute of Canada through the Canadian Cancer Society. D.M.C. is the recipient of the Canada Research Chair in Membrane Biology.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.106.029926.
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ABBREVIATIONS: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; P-gp, P-glycoprotein; TM, transmembrane; TMD, transmembrane domain; NBD, nucleotide-binding domain; HEK, human embryonic kidney; DMSO, dimethyl sulfoxide; PAGE, polyacrylamide gel electrophoresis; E-64, N-(trans-epoxysuccinyl)-l-leucine 4-guanidinobutylamide; PNGase F, peptide N-glycosidase F; M14M, 3,6,9,12-tetraoxatetradecane-1,14-diyl bismethanethiosulfonate; VRT-532, 4-methyl-2-(5-phenyl-1H-pyrazol-3-yl)-phenol.
- Received August 11, 2006.
- Accepted November 20, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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