Domain exchangeability between the multidrug transporter (MDR1) and phosphatidylcholine flippase (MDR2)

Multidrug resistance (MDR) mediated by P-glycoprotein (MDR1) is clinically significant. Understanding how MDR1 substrate specificity is determined will help to overcome MDR to improve cancer treatment. One potential approach to achieve this goal is to study chimeras of MDR1 and its homolog MDR2 (also called MDR3), which has been identified as a phosphatidylcholine flippase. With an approach involving exchanging homologous segments of MDR1 and MDR2 and site-directed mutagenesis, we previously demonstrated MDR1 residues Q330, V331, and L332 in transmembrane domain 6 (TM6) are essential for multidrug transport activity; substituting these residues allows the N-terminal transmembrane region of MDR2 to support MDR1 activity. To further determine the exchangeability between MDR1 and MDR2, we constructed additional MDR1/MDR2 chimeras. We found that the N-terminal half of MDR1 and MDR2 was mostly exchangeable except for a few residues in TM6. However, this degree of exchangeability was not found in the C-terminal half of MDR1 and MDR2. In addition, with substitution of MDR1 residues 318-332 (TM6) and 937-994 (TM11-12), MDR2 had relatively normal affinity for MDR1 substrates, but it did not have multidrug transporter activity. These results suggest that the inability of MDR2 to transport most MDR1 drugs efficiently may be due to failure of those drugs to stimulate ATPase and activate transport as well as to decreased drug binding.