Figure 5.

Functional interaction between rat MCT1 and CAIV in Xenopus oocytes requires direct binding of CAIV to Lys-73 in the Ig1 domain of rCD147. A, homology model of the Ig1 domain of rat CD147 (based on human CD147, PDB code 3B5H (37)). Glu-32 and Lys-73 are labeled in blue and red, respectively. B, original recordings of the change in intracellular H⁺ concentration during application of lactate and CO₂/HCO₃⁻ in Xenopus oocytes, expressing rMCT1 together with rCD147–WT (gray traces), rCD147–E32A (blue traces), and rCD147–K73A (red traces), respectively. Cells were either expressing rMCT1 + rCD147 alone (light traces) or rMCT1 + rCD147 and human CAIV (dark traces). C and D, rate of change in intracellular H⁺ concentration (Δ[H⁺]/Δt) during application of lactate (C) and 5% CO₂, 10 mm HCO₃⁻ (D) in Xenopus oocytes, expressing rMCT1 and rCD147–WT (gray dots), rCD147–E32A (blue dots), and rCD147–K73A (red dots), respectively. Cells were either expressing rMCT1 + rCD147 alone (light dots) or rMCT1 + rCD147 and CAIV (dark dots). The significance indicators above the dots with CAIV (dark dots) refer to the corresponding dots without CAIV (light dots of same color). E, representative Western blots of CAIV (left blot) and GST (right blot), respectively. CAIV was pulled down with GST (lane 1), a GST fusion protein of the Ig1 domain of rCD147–WT (lane 2), a GST fusion protein of Ig1 domain of rCD147–E32A (lane 3), and a GST fusion protein of Ig1 domain of rCD147–K73A (lane 4). Lysate of CAIV-expressing oocytes (lane 5) was added as positive control. F, relative intensity of the fluorescent signal of CAIV. For every blot, the signals for CAIV were normalized to the corresponding signals for GST–rCD147–WT. Each individual signal for CAIV was normalized to the intensity of the signal for GST in the same lane. The significance indicators above the dots refer to GST–rCD147–WT.