Membrane Topology of the Multidrug Resistance Protein (MRP)
A STUDY OF GLYCOSYLATION-SITE MUTANTS REVEALS AN EXTRACYTOSOLIC NH2 TERMINUS*
- David R. Hipfner‡§,
- Kurt C. Almquist‡§,
- Elaine M. Leslie¶‖,
- James H. Gerlach**,
- Caroline E. Grant**,
- Roger G. Deeley‡**‡ and
- Susan P. C. Cole‡¶§§
- From the Departments of ‡Pathology and¶Pharmacology & Toxicology, and the **Cancer Research Laboratories, Queen’s University, Kingston, Ontario, Canada K7L 3N6
Abstract
Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents. Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD. Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP. To aid in determining the topology most likely to be correct, we have identified which of the 14N-glycosylation sequons in this protein are utilized. Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP. The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation. Subsequent site-directed mutagenesis studies established that Asn19and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides.N-Glycosylation of Asn19 and Asn23provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus. This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices. These results may have important implications for the further understanding of the interaction of drugs with MRP.
Footnotes
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↵* This work was supported by Grant MT-10519 from the Medical Research Council of Canada.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ Supported by a Medical Research Council of Canada studentship.
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↵‖ Supported by an Ontario Graduate Scholarship.
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↵‡ Stauffer Research Professor of Queen’s University.
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↵§§ Senior Scientist of the Ontario Cancer Foundation. To whom correspondence and reprint requests should be addressed: Cancer Research Laboratories, Rm. 328, Botterell Hall, Queen’s University, Kingston, Ontario, Canada K7L 3N6. Tel.: 613-545-6507; Fax: 613-545-6830.
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↵1 The abbreviations used are: MRP, multidrug resistance protein; ABC, ATP-binding cassette; MSD, membrane spanning domain; NBD, nucleotide binding domain; CFTR, cystic fibrosis transmembrane conductance regulator; SUR, sulfonylurea receptor; MOAT, multispecific organic anion transporter; mAb, monoclonal antibody; PCR, polymerase chain reaction; PNGase F, protein N-glycosidase F.
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↵2 R. G. Deeley and S. P. C. Cole, unpublished results
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- Received May 19, 1997.
- Revision received July 14, 1997.
