Contribution to Substrate Specificity and Transport of Nonconserved Residues in Transmembrane Domain 12 of Human P-Glycoprotein

Abstract

P-glycoprotein (Pgp), the product of the MDR1 gene, confers multidrug resistance on cancer cells by ATP-dependent extrusion of anticancer drugs. Biochemical and genetic studies with Pgp have identified the putative transmembrane (TM) region 12 (residues 974-994) as a major region involved in drug interactions with amino acid residues conserved among Pgp family members shown to be essential for transport. To determine whether nonconserved residues might be involved in substrate specificity, 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 acid 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 function. However, when residues L975, V981, and F983 were replaced collectively, drug transport, drug-stimulated ATP hydrolysis, and photoaffinity labeling with the drug analogue, [125I]iodoarylazidoprazosin (IAAP), were abrogated, with little effect on [alpha-32P]-8-azido-ATP labeling and basal ATPase activity. Pairwise alanine substitutuions showed variable effects on function. Substitutions including L975A in combination with any one of the other two replacements had the least effect on Pgp function. The V981A and F983A double mutant showed the most effect on transport of fluorescent substrates. In contrast, alanine substitutions of all four nonconserved residues M986, V988, Q990, and V991 at the putative carboxy-terminal half of TM 12 showed no effect on drug transport except for a partial reduction in bodipy-verapamil extrusion. These results suggest that nonconserved residues in the putative amino-proximal half of TM 12 of Pgp play a more direct role in determining specificity of drug transport function than those in the putative carboxy-terminal half of TM 12.

Identification of a Nonconserved Amino Acid Residue in Multidrug Resistance Protein 1 Important for Determining Substrate Specificity

“…The topologically comparable transmembrane helix in P-gp, due to the five additional NH 2 -proximal TMs in MRP1/mrp1, is TM12, which has been demonstrated to play an important role in substrate specificity and drug binding. (35). Use of the thiol-reactive substrate, dibromobimane, together with cysteine-scanning mutagenesis has confirmed that hydrophobic residues Leu 975 , Val 982 , and Ala 985 in TM12 are important for the interaction of substrates with P-gp (36,37).…”

Functional polymorphisms of the human multidrug-resistance gene: Multiple sequence variations and correlation of one allele with P-glycoprotein expression and activity in vivo

“…Furthermore, the characterization of novel MDR-1 variants may be useful for the development of novel inhibitors that specifically modulate the activity of the individual types of MDR-1. The feasibility of using inhibitors of individual (artificially created) MDR-1 variants and their potential therapeutic application has recently been demonstrated in a model system (30,31). Mickley et al have recently described two genetic polymorphisms in MDR-1 at positions 2677 and 2995 in exons 21 and 24 (22).…”

“…On the other hand, experiments with artificially introduced MDR-1 mutations show clearly that PGP reacts quite sensitively to amino acid alterations. Mutations that change amino acids can alter the substrate spectrum of PGP, the effectiveness of transport, and also the sensitivity of PGP toward inhibition with specific inhibitory substances (2,(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). It is clear that some naturally occurring mutations and alleles-if they exist, and if we can discover themmay show similar effects.…”

Identification and Characterization of the Binding Sites of P-Glycoprotein for Multidrug Resistance-Related Drugs and Modulators

“…Considerable effort has been invested in characterizing drug resistance changes caused by Pglycoprotein mutations [52,53,[127][128][129][130][131][132][133][134][135][136][137][138][139][140][141][142][143][144][145]. P-glycoprotein mutations that have appeared in cultured cells selected for resistance to particular drugs [130][131][132], and mutations incorporated by site-directed mutagenesis have been evaluated [52,53,127,129,[134][135][136][137][138][139][140][141][142][143][144][145].…”