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J. Biol. Chem., Vol. 279, Issue 37, 38395-38401, September 10, 2004

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Processing Mutations Located throughout the Human Multidrug Resistance P-glycoprotein Disrupt Interactions between the Nucleotide Binding Domains^*

Tip W. Loo, M. Claire Bartlett, and David M. Clarke

From the Canadian Institutes of Health Research Group in Membrane Biology, Departments of Medicine and Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

The most common cause of cystic fibrosis is misfolding of the cystic fibrosis transmembrane conductance regulator (CFTR) protein because of deletion of residue Phe-508 (F508). P-glycoprotein (P-gp) is an ideal model protein for studying how mutations disrupt folding of ATP-binding cassette proteins such as CFTR because specific chemical chaperones can be used to correct folding defects. Interactions between the nucleotide binding domains (NBDs) are critical because ATP binds at the interface between the NBDs. Here, we used disulfide cross-linking between cysteines in the Walker A sites and the LSGGQ signature sequences to test whether processing mutations located throughout P-gp disrupted interactions between the NBDs. We found that mutations present in the cytoplasmic loops, transmembrane segments, and linker regions or deletion of Tyr-490 (equivalent to Phe-508 in CFTR) inhibited cross-linking between the NBDs. Deletion of Phe-508 in the P-gp/CFTR chimera also inhibited cross-linking between the NBDs. Cross-linking was restored, however, when the mutants were expressed in the presence of the chemical chaperone cyclosporin A. The "rescued" mutants exhibited drug-stimulated ATPase activity, and cross-linking between the NBDs was inhibited by vanadate trapping of nucleotide. These results together with our previous findings (Loo, T. W., Bartlett, M. C., and Clarke, D. M. (2002) J. Biol. Chem. 277, 27585–27588) indicate that processing mutations disrupt interactions among all four domains. It appears that cross-talk between the cytoplasmic and the transmembrane domains is required for establishment of proper domain-domain interactions that occur during folding of ATP-binding cassette protein transporters.

Received for publication, May 20, 2004 , and in revised form, July 6, 2004.

^* This work was supported in part by grants from the Canadian Cancer Society through the National Cancer Institute of Canada and the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of the Canada Research Chair in Membrane Biology. To whom correspondence should be addressed: Dept. of Medicine, University of Toronto, Rm. 7342, Medical Sciences Bldg., 1 King's College Circle, Toronto, Ontario M5S 1A8, Canada. Tel. and Fax: 416-978-1105; E-mail: david.clarke{at}utoronto.ca.

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This article has been cited by other articles:

				T. W. Loo, M. C. Bartlett, and D. M. Clarke Processing Mutations Disrupt Interactions between the Nucleotide Binding and Transmembrane Domains of P-glycoprotein and the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) J. Biol. Chem., October 17, 2008; 283(42): 28190 - 28197. [Abstract] [Full Text] [PDF]

				T. W. Loo, M. C. Bartlett, and D. M. Clarke The Dileucine Motif at the COOH Terminus of Human Multidrug Resistance P-glycoprotein Is Important for Folding but Not Activity J. Biol. Chem., January 28, 2005; 280(4): 2522 - 2528. [Abstract] [Full Text] [PDF]

				E. Y. Chen, M. C. Bartlett, T. W. Loo, and D. M. Clarke The {Delta}F508 Mutation Disrupts Packing of the Transmembrane Segments of the Cystic Fibrosis Transmembrane Conductance Regulator J. Biol. Chem., September 17, 2004; 279(38): 39620 - 39627. [Abstract] [Full Text] [PDF]