Role of proline 1150 in functional interactions between the membrane spanning domains and nucleotide binding domains of the MRP1 (ABCC1) transporter.
The ATP-binding cassette multidrug resistance protein 1 (MRP1) mediates ATP-dependent cellular efflux of drugs and organic anions. We previously described a mutant, MRP1-Pro1150Ala, which exhibits selectively increased estradiol glucuronide (E217betaG) and methotrexate transport as well as altered interactions with ATP. We have now further explored the functional importance of MRP1-Pro1150 at the interface of transmembrane helix 15 and cytoplasmic loop 7 (CL7) by replacing it with Gly, Ile, Leu and Val. All four mutants exhibited a phenotype similar to MRP1-Pro1150Ala with respect to organic anion transport and [gamma32P]8N3ATP photolabeling. They also displayed very low levels of substrate-independent vanadate-induced trapping of [alpha32P]8N3ADP. To better understand the relationship between the altered nucleotide interactions and transport activity of these mutants, [alpha32P]8N3ADP trapping experiments were performed under different conditions. Unlike leukotriene C4, E217betaG decreased [alpha32P]8N3ADP trapping by both wild-type and mutant MRP1. [alpha32P]8N3ADP trapping by MRP1-Pro1150Ala could be increased by using Ni2+ instead of Mg2+, and by decreasing temperature; however, the transport properties of the mutant remained unchanged. We conclude that the reduced [alpha32P]8N3ADP trapping associated with loss of Pro1150, or the presence of E217betaG, is due to enhanced ADP release following ATP hydrolysis rather than a reduction in ATP hydrolysis itself. We hypothesize that loss of Pro1150 alters the role of CL7 as a coupling helix that mediates signaling between the nucleotide binding domains and some substrate binding sites in the membrane spanning domains of MRP1.
Létourneau
Isabelle J
IJ
Department of Pharmacology & Toxicology, Division of Cancer Biology & Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario, Canada K7L 3N6.
Nakajima
Akio
A
Deeley
Roger G
RG
Cole
Susan P C
SP
D002460:Cell Line; D004958:Estradiol / Q000031:analogs & derivatives / Q000378:metabolism; D006801:Humans; D017997:Leukotriene C4 / Q000378:metabolism; D008727:Methotrexate / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D014162:Transfection; D022161:Transport Vesicles / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry
D004958:Estradiol; D017997:Leukotriene C4; D008727:Methotrexate; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011392:Proline; D014357:Trypsin; C025483:estradiol-17 beta-glucuronide; C421109:multidrug resistance-associated protein 1