CONTRIBUTION TO SUBSTRATE-SPECIFICITY AND TRANSPORT OF NONCONSERVED RESIDUES IN TRANSMEMBRANE DOMAIN-12 OF HUMAN P-GLYCOPROTEIN

P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer dr ugs. Biochemical and genetic studies with Pgp have identified the puta tive transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved amon g Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specific ity, seven amino acid residues were identified within TM 12 that were not strictly conserved among the MDR1 and MDR2 family of proteins from different mammalian species. We replaced all seven of these amino aci d residues with alanine, one at a time and in combinations, and used a vaccinia virus based transient expression system to analyze function. None of the single replacements caused any alteration in transport fu nction. However, when residues L975, V981, and F983 were replaced coll ectively, drug transport, drug-stimulated ATP hydrolysis, and photoaff inity labeling with the drug analogue, [I-125]iodoarylazidoprazosin (I AAP), were abrogated, with Little effect on [alpha-P-32]-8-azido-ATP l abeling and basal ATPase activity. Pairwise alanine substitutuions sho wed variable effects on function. Substitutions including L975A in com bination with any one of the other two replacements had the least effe ct on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine su bstitutions of all four nonconserved residues M986, V988, Q990, and V9 91 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extr usion. These results suggest that nonconserved residues in the putativ e amino-proximal half of TM 12 of Pgp play a more direct role in deter mining specificity of drug transport function than those in the putati ve carboxy-terminal half of TM 12.