Multiple membrane-associated tryptophan residues contribute to the transport activity and substrate specificity of the human multidrug resistance protein, MRP1.
The multidrug resistance protein, MRP1, is a clinically important ATP-binding cassette transporter in which the three membrane-spanning domains (MSDs), which contain up to 17 transmembrane (TM) helices, and two nucleotide binding domains (NBDs) are configured MSD1-MSD2-NBD1-MSD3-NBD2. In tumor cells, MRP1 confers resistance to a broad spectrum of drugs, but in normal cells, it functions as a primary active transporter of organic anions such as leukotriene C(4) and 17beta-estradiol 17beta-(D-glucuronide). We have previously shown that mutation of TM17-Trp(1246) eliminates 17beta-estradiol 17beta-(D-glucuronide) transport and drug resistance conferred by MRP1 while leaving leukotriene C(4) transport intact. By mutating the 11 remaining Trp residues that are in predicted TM segments of MRP1, we have now determined that five of them are also major determinants of MRP1 function. Ala substitution of three of these residues, Trp(445) (TM8), Trp(553) (TM10), and Trp(1198) (TM16), eliminated or substantially reduced transport levels of five organic anion substrates of MRP1. In contrast, Ala substitutions of Trp(361) (TM7) and Trp(459) (TM9) caused a more moderate and substrate-selective reduction in MRP1 function. More conservative substitutions (Tyr and Phe) of the Trp(445), Trp(553), and Trp(1198) mutants resulted in substrate selective retention of transport in some cases (Trp(445) and Trp(1198)) but not others (Trp(553)). Our findings suggest that the bulky polar aromatic indole side chain of each of these five Trp residues contributes significantly to the transport activity and substrate specificity of MRP1.
Koike
Koji
K
Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Oleschuk
Curtis J
CJ
Haimeur
Anass
A
Olsen
Sharon L
SL
Deeley
Roger G
RG
Cole
Susan P C
SP
D000255:Adenosine Triphosphate / Q000378:metabolism; D000409:Alanine / Q000737:chemistry; D000465:Algorithms; D002412:Cations; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D017136:Ion Transport; D007477:Ions; D017997:Leukotriene C4 / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry*
D002412:Cations; D007477:Ions; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017997:Leukotriene C4; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000409:Alanine