Analysis of Random Recombination between Human MDR1 and Mouse Mdr1a cDNA in a pHaMDR-Dihydrofolate Reductase Bicistronic Expression System

• Published in: Molecular pharmacology 1998-10, Vol.54 (4), p.623-630
• Main Authors: Shoshani, T, Zhang, S, Dey, S, Pastan, I, Gottesman, M M
• Format: Article
• Language: English
• Subjects: Animals; ATP-Binding Cassette, Sub-Family B, Member 1 - genetics; ATP-Binding Cassette, Sub-Family B, Member 1 - metabolism; Azides - metabolism; Biological Transport - physiology; DNA, Complementary - genetics; DNA, Complementary - metabolism; Genetic Vectors; Humans; Iodine Radioisotopes; KB Cells; Mice; Photoaffinity Labels - metabolism; Prazosin - analogs & derivatives; Prazosin - metabolism; Protein Structure, Tertiary; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombination, Genetic; Sensitivity and Specificity; Tetrahydrofolate Dehydrogenase - biosynthesis; Tetrahydrofolate Dehydrogenase - genetics; Transfection
• ISSN: 0026-895X; 1521-0111

Human P-glycoprotein (Pgp) confers multidrug resistance (MDR) to otherwise sensitive cells. The homologous mouse Pgps, which are encoded by mouse mdr 1a (also known as mdr 3) and mdr 1b (also known as mdr 1), confer different degrees of resistance to the same MDR drugs and inhibitors. To create recombinants for the study of sequences responsible for these differences in drug-resistance, chimeric cDNA libraries can be constructed by homologous recombination of pools of related sequences. This mutagenesis approach is called DNA shuffling. To select for chimeric Pgp with an altered resistance profile, DNA shuffling between the homologous but not identical drug interacting transmembrane domains 5 and 6 of human MDR 1 and mouse mdr 1a was used. The chimeric proteins were expressed in human KB-3-1 cells. One recombinant Pgp (clone 3-4) with a novel phenotype was analyzed in detail. Inhibitors of Pgp, including verapamil and cyclosporin A, were less effective in reversing resistance of the chimeric Pgp compared with wild-type Pgp, for certain drugs. However, [ 125 I]iodoarylazidoprazosin photoaffinity labeling of the chimeric Pgp and its binding competition with cyclosporin A, showed that cyclosporin A competed for the photoaffinity labeling. The chimeric Pgp cells stained less well with human-specific anti-Pgp mAb MRK16 than wild-type Pgp, despite having the described epitopes for MRK16. Staining with human-specific mAb UIC2 was increased when the chimeric protein was compared with wild-type Pgp. These results suggest an alteration in exposure of human Pgp specific epitopes in this chimeric Pgp, as well as a change in the interaction of reversing agents with the chimeric protein.