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.2000 Aug 1;19(15):3896-904.
doi: 10.1093/emboj/19.15.3896.

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression

Affiliations

CD147 is tightly associated with lactate transporters MCT1 and MCT4 and facilitates their cell surface expression

P Kirk et al. EMBO J..

Abstract

CD147 is a broadly expressed plasma membrane glycoprotein containing two immunoglobulin-like domains and a single charge-containing transmembrane domain. Here we use co-immunoprecipitation and chemical cross-linking to demonstrate that CD147 specifically interacts with MCT1 and MCT4, two members of the proton-linked monocarboxylate (lactate) transporter family that play a fundamental role in metabolism, but not with MCT2. Studies with a CD2-CD147 chimera implicate the transmembrane and cytoplasmic domains of CD147 in this interaction. In heart cells, CD147 and MCT1 co-localize, concentrating at the t-tubular and intercalated disk regions. In mammalian cell lines, expression is uniform but cross-linking with anti-CD147 antibodies caused MCT1, MCT4 and CD147, but not GLUT1 or MCT2, to redistribute together into 'caps'. In MCT-transfected cells, expressed protein accumulated in a perinuclear compartment, whereas co-transfection with CD147 enabled expression of active MCT1 or MCT4, but not MCT2, in the plasma membrane. We conclude that CD147 facilitates proper expression of MCT1 and MCT4 at the cell surface, where they remain tightly bound to each other. This association may also be important in determining their activity and location.

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Figures

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Fig. 1. Expression of MCT1, MCT2, MCT4, GP70 and CD147 in mammalian cell lines andXenopus oocytes. Crude membrane fractions were prepared from Y3, RBL and H4IIE cells andXenopus oocytes, proteins separated by SDS–PAGE and western blotting performed with antibodies against MCT1, MCT2, MCT4, GP70 and CD147 as indicated.
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Fig. 2. Immunoprecipitation of CD147 from Y3 and H4IIE cells co-precipitates MCT1 and MCT4 but not MCT2. CD147 was immunoprecipitated from Y3, RBL and H4IIE cells, proteins separated by SDS–PAGE and western blotted with antibodies against MCT1, MCT2 and MCT4 as shown. Control (OX-34) precipitate from Y3 cells did not cause co-precipitation of MCT1 (final lane) or MCT4 (data not shown).
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Fig. 3. Immunoprecipitation of CD147 from RBL cells co-precipitates MCT1 but not OX-101. CD147 was immunoprecipitated from RBL cells, and the precipitate and lysate separated by SDS–PAGE then western blotted for MCT1 [(A) left lane, lysate; right lane, immunoprecipitate) and the unrelated transmembrane protein OX-101 [(B) lanes 1 and 2, precipitate; lanes 3 and 4, lysate; lanes 2 and 4 were probed with secondary antibody only, to reveal non-specific bands].
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Fig. 4. DIDS cross-links MCT1 to CD147 but not CD2 in RBL cells. RBL cells transfected with CD2 were treated with DIDS or left untreated as indicated. Crude lysate and CD147 or CD2 immunoprecipitates were separated by SDS–PAGE and western blots performed with anti-MCT1 (A) and anti-CD147 (B) antibodies. Longer exposure did reveal the presence of MCT1 in the CD147 immunoprecipitate from untreated cells, but not in either of the CD2 immunoprecipitates (not shown).
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Fig. 5. The transmembrane and cytoplasmic regions of CD147 are sufficient to mediate the interaction with MCT1. The CD2–CD147 hybrid protein was immunoprecipitated from transfected RBL cells using anti-CD2 antibody, and western blotted for MCT1 (lane 1) and MCT2 (lane 2). MCT1 is clearly detected, contrasting with Figure 2, in which MCT1 is not detected in CD2 immunoprecipitate from CD2-transfected RBL cells. The bars represent 50 μm.
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Fig. 6. Co-localization of MCT1 and CD147 in isolated rat cardiac myocytes. Dual excitation confocal microscopy was performed on fixed isolated rat heart cells treated with antibodies against MCT1 (TRITC–secondary) and CD147 (FITC–secondary). The bars represent 50 μm.
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Fig. 7. Relocalization of CD147 by antibody treatment causes parallel relocalization of MCT1 and MCT4 but not GLUT1 or MCT2. In (A), Y3 cells were ‘capped’ (arrows) by CD147 primary and FITC–secondary antibody, fixed and treated with antibodies against MCT1, MCT4 (TRITC–secondary) or GLUT1 (CY3–secondary). Uncapped cells (top row) were treated as in Figure 6. Confocal fluorescence microscopy revealed the incorporation of MCT1 and MCT4 into the CD147 caps as confirmed by the orange/yellow in the overlay. (B) H4IIE cells, which contain MCT1 and MCT2 but not MCT4, were subjected to the same capping procedure as the Y3 cells although the capping (arrows) was less profound. The bars represent 20 µm.
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Fig. 7. Relocalization of CD147 by antibody treatment causes parallel relocalization of MCT1 and MCT4 but not GLUT1 or MCT2. In (A), Y3 cells were ‘capped’ (arrows) by CD147 primary and FITC–secondary antibody, fixed and treated with antibodies against MCT1, MCT4 (TRITC–secondary) or GLUT1 (CY3–secondary). Uncapped cells (top row) were treated as in Figure 6. Confocal fluorescence microscopy revealed the incorporation of MCT1 and MCT4 into the CD147 caps as confirmed by the orange/yellow in the overlay. (B) H4IIE cells, which contain MCT1 and MCT2 but not MCT4, were subjected to the same capping procedure as the Y3 cells although the capping (arrows) was less profound. The bars represent 20 µm.
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Fig. 8. Co-transfection with CD147 enables expression of MCT1 and MCT4, but not MCT2, at the plasma membrane. In (A), COS cells were transiently transfected with both CD147 and MCT1, MCT2 or MCT4 in the pCI-neo vector and the expression and location of CD147 (FITC–secondary) and MCT1, MCT2 or MCT4 (TRITC–secondary) were detected by dual excitation confocal microscopy. Within the field of cells shown for the MCT4–CD147 transfection, one cell overexpressed primarily MCT4 (M), another CD147 (O) and a third both proteins (OM). Only in the latter case were both proteins properly targeted to the plasma membrane and such cells frequently appeared more rounded. In (B), HeLa cells were transiently transfected with both CD147 and MCT1 or MCT4 in the pTRE vector. Appropriate levels of expression were achieved by addition of tetracycline (10 pg/ml). Dual excitation confocal microscopy was used to the reveal the expression and location of CD147, MCT1 and MCT4 as above. The bars represent 20 µm.
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Fig. 8. Co-transfection with CD147 enables expression of MCT1 and MCT4, but not MCT2, at the plasma membrane. In (A), COS cells were transiently transfected with both CD147 and MCT1, MCT2 or MCT4 in the pCI-neo vector and the expression and location of CD147 (FITC–secondary) and MCT1, MCT2 or MCT4 (TRITC–secondary) were detected by dual excitation confocal microscopy. Within the field of cells shown for the MCT4–CD147 transfection, one cell overexpressed primarily MCT4 (M), another CD147 (O) and a third both proteins (OM). Only in the latter case were both proteins properly targeted to the plasma membrane and such cells frequently appeared more rounded. In (B), HeLa cells were transiently transfected with both CD147 and MCT1 or MCT4 in the pTRE vector. Appropriate levels of expression were achieved by addition of tetracycline (10 pg/ml). Dual excitation confocal microscopy was used to the reveal the expression and location of CD147, MCT1 and MCT4 as above. The bars represent 20 µm.
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Fig. 9. Co-transfection of COS cells with CD147 and MCT1 leads to a large stimulation of lactate transport. In (A), COS cells that had been transiently transfected with both CD147 and MCT1 were loaded with BCECF and rates of transport by five individual cells determined from the rate of change of the 440/490 nm fluorescence ratio upon superfusion with the concentrations ofl-lactate or butyrate indicated. Butyrate was included to confirm that transfection did not change the plasma membrane pH gradient (Wanget al., 1994). The four cells labelled control showed similar rates of transport to non-transfected cells determined in a parallel experiment, and were therefore assumed to be untransfected. In (B), mean kinetic data are provided for four control cells and four transfected cells with elevated rates of transport similar to those shown in (A). Cells transiently transfected with MCT1 alone did not show rates of transport significantly different from non-transfected cells (data not shown and see the text for discussion).
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Fig. 10. Stable transfection of COS cells with MCT1 or MCT4 leads to a modest stimulation of lactate transport. Non-transfected COS cells or stable transfectants overexpressing either MCT1 or MCT4 were loaded with BCECF and rates of lactate transport by a field of cells determined from the rate of change of the 440/490 nm fluorescence ratio upon superfusion with 5 mMl-lactate as indicated. The expression of MCT1 and MCT4 in the transfected cells was visualized using immunofluorescence microscopy. The bars represent 20 µm.
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