Structural and functional characterization of the mouse multidrug resistance gene family

Abstract

The mouse multidrug resistance (mdr) gene family is composed of three closely related genes mdr1, mdr2 and mdr3. To clarify the role of these three genes in the emergence of multidrug resistance and to initiate a structure-function analysis of the corresponding proteins, we have isolated full length cDNA clones corresponding to their respective cellular RNA transcripts. Sequence analyses indicate that the three encoded polypeptides are highly similar sharing the same predicted structural features and a high degree of sequence homology (85% to 92%). The three genes are contiguous on a 625 kb chromosomal segment and appear to result from two consecutive gene duplication events. Hybridization studies with gene specific probes in independently derived multidrug resistant cell lines and transfection experiments with full length cDNA clones indicate that mdr1 and mdr3 but not mdr2 overexpression can induce multidrug resistance. In transfected cells, multidrug resistance is linked to a decreased drug accumulation and an concomitant increased ATP-dependent drug efflux. Mutational analysis indicates that both predicted ATP binding domains in mdr1 are absolutely essential for biological activity. The study of chimeric proteins constructed between biologically active mdr1 and inactive mdr2 indicate that both ATP binding domains of mdr2 are functional and suggest that transmembrane domains of mdr1 are essential for the drug resistance phenotype conferred by this protein. Finally, although mdr1 and mdr3 can confer multidrug resistance, drug survival characteristics of mdr1 and mdr3 transfectants indicate that both proteins have overlapping but distinct substrate specificities.