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J. Biol. Chem., Vol. 279, Issue 13, 12325-12336, March 26, 2004

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Identification of Proline Residues in the Core Cytoplasmic and Transmembrane Regions of Multidrug Resistance Protein 1 (MRP1/ABCC1) Important for Transport Function, Substrate Specificity, and Nucleotide Interactions^*

Koji Koike, Gwenaëlle Conseil¶, Elaine M. Leslie¶||**, Roger G. Deeley, and Susan P. C. Cole||

From the Cancer Research Laboratories and the ^||Department of Pharmacology and Toxicology, Queen's University, Kingston, Ontario K7L 3N6, Canada

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette transporter that confers resistance to drugs and mediates the transport of organic anions. MRP1 has a core structure of two membrane spanning domains (MSDs) each followed by a nucleotide binding domain. This core structure is preceded by a third MSD with five transmembrane (TM) helices, whereas MSD2 and MSD3 each contain six TM helices. We investigated the consequences of Ala substitution of 18 Pro residues in both the non-membrane and TM regions of MSD2 and MSD3 on MRP1 expression and organic anion transport function. All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. In addition, five mutants containing substitutions of Pro residues in or proximal to the TM helices of MSD2 (TM6-Pro³⁴³, TM8-Pro⁴⁴⁸, TM10-Pro⁵⁵⁷, and TM11-Pro⁵⁹⁵) and MSD3 (TM14-Pro¹⁰⁸⁸) exhibited significantly reduced transport of five organic anion substrates. In contrast, mutation of Pro¹¹⁵⁰ in the cytoplasmic loop (CL7) linking TM15 to TM16 caused a substantial increase in 17-estradiol-17--(D-glucuronide) and methotrexate transport, whereas transport of other organic anions was reduced or unchanged. Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Our findings demonstrate the importance of TM6, TM8, TM10, TM11, and TM14 in MRP1 transport function and suggest that CL7 may play a differential role in coupling the activity of the nucleotide binding domains to the translocation of different substrates across the membrane.

Received for publication, October 17, 2003 , and in revised form, January 8, 2004.

^* This work was supported in part by Canadian Institutes of Health Research Grant MOP-10519. 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.

Present address: Dept. of Otorhinolaryngology, Kyushu University School of Medicine, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Japan.

^¶ Recipient of a Canadian Institutes of Health Research postdoctoral award.

^** Present address: NCI, NIEH, National Institutes of Health, 111 T.W. Alexander Dr., Bldg. 101, F018, Research Triangle Park, NC 27709.

Canada Research Chair in Cancer Biology. To whom correspondence should be addressed: Cancer Research Laboratories, Botterell Hall, Queen's University, Kingston, Ontario K7L 3N6, Canada. Tel.: 613-533-2636; Fax: 613-533-6830; E-mail: coles{at}post.queensu.ca.

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