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J. Biol. Chem., Vol. 280, Issue 29, 27213-27221, July 22, 2005

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Basigin (CD147) Is the Target for Organomercurial Inhibition of Monocarboxylate Transporter Isoforms 1 and 4

THE ANCILLARY PROTEIN FOR THE INSENSITIVE MCT2 IS EMBIGIN (gp70)^*

Marieangela C. Wilson, David Meredith, Jocelyn E. Manning Fox, Christine Manoharan¶, Andrew J. Davies, and Andrew P. Halestrap||

From the Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol BS8 1TD, United Kingdom

Translocation of monocarboxylate transporters MCT1 and MCT4 to the plasma membrane requires CD147 (basigin) with which they remain tightly associated. However, the importance of CD147 for MCT activity is unclear. MCT1 and MCT4 are both inhibited by the cell-impermeant organomercurial reagent p-chloromercuribenzene sulfonate (pCMBS). Here we demonstrate by site-directed mutagenesis that removal of all accessible cysteine residues on MCT4 does not prevent this inhibition. pCMBS treatment of cells abolished co-immunoprecipitation of MCT1 and MCT4 with CD147 and enhanced labeling of CD147 with a biotinylated-thiol reagent. This suggested that CD147 might be the target of pCMBS, and further evidence for this was obtained by treatment of cells with the bifunctional organomercurial reagent fluorescein dimercury acetate that caused oligomerization of CD147. Site-directed mutagenesis of CD147 implicated the disulfide bridge in the Ig-like C2 domain of CD147 as the target of pCMBS attack. MCT2, which is pCMBS-insensitive, was found to co-immunoprecipitate with gp70 rather than CD147. The interaction between gp70 and MCT2 was confirmed using fluorescence resonance energy transfer between the cyan fluorescent protein- and yellow fluorescent protein-tagged MCT2 and gp70. pCMBS strongly inhibited lactate transport into rabbit erythrocytes, where MCT1 interacts with CD147, but not into rat erythrocytes where it interacts with gp70. These data imply that inhibition of MCT1 and MCT4 activity by pCMBS is mediated through its binding to CD147, whereas MCT2, which associates with gp70, is insensitive to pCMBS. We conclude that ancillary proteins are required to maintain the catalytic activity of MCTs as well as for their translocation to the plasma membrane.

Received for publication, October 21, 2004 , and in revised form, May 9, 2005.

^* This work was supported in part by the Royal Society, the Wellcome Trust, and The British Heart Foundation and by an Infrastructure Award and Joint Research Equipment Initiative Grant from the Medical Research Council to establish the School of Medical Sciences Cell Imaging Facility. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

Current address: Dept. of Human Anatomy & Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, UK.

Funded by a Research Studentship from the Medical Research Council. Current address: Dept. of Pharmacology, 9-58 Medical Sciences Bldg., Edmonton, Alberta T6G 2H7, Canada.

^¶ Funded by a Research Studentship from the Biotechnology and Biological Sciences Research Council.

^|| To whom correspondence should be addressed. Tel.: 44-117-928-8592; Fax: 44-117-928-8274; E-mail: a.halestrap{at}bristol.ac.uk.

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