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Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface
Surtaj H Iram1, Susan P C Cole
1Division of Cancer Biology and Genetics, Queen's University Cancer Research Institute, Kingston, Ontario K7L 3N6, Canada.
Abstract:
Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter that effluxes drugs and organic anions across the plasma membrane. The 17 transmembrane helices of MRP1 are linked by extracellular and cytoplasmic loops (CLs), but their role in coupling the ATPase activity of MRP1 to the translocation of its substrates is poorly understood. Here we have examined the importance of CL5 by mutating eight conserved charged residues and the helix-disrupting Gly(511) in this region. Ala substitution of Lys(513), Lys(516), Glu(521), and Glu(535) markedly reduced MRP1 levels. Because three of these residues are predicted to lie at the interface of CL5 and the second nucleotide binding domain (NBD2), a critical role is indicated for this region in the plasma membrane expression of MRP1. Further support for this idea was obtained by mutating NBD2 amino acids His(1364) and Arg(1367) at the CL5 interface, which also resulted in reduced MRP1 levels. In contrast, mutation of Arg(501), Lys(503), Glu(507), Arg(532), and Gly(511) had no effect on MRP1 levels. Except for K503A, however, transport by these mutants was reduced by 50 to 75%, an effect largely attributable to reduced substrate binding and affinity. Studies with (32)P-labeled azido-ATP also indicated that whereas ATP binding by the G511I mutant was unchanged, vanadate-induced trapping of azido-ADP was reduced, indicating changes in the catalytic activity of MRP1. Together, these data demonstrate the multiple roles for CL5 in the membrane expression and function of MRP1.
Insights
Cytoplasmic loop 5 (CL5) of multidrug resistance protein 1 (MRP1) is crucial for its plasma membrane expression and function. Mutations in CL5 impact MRP1 levels and substrate transport, revealing its multifaceted roles.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Multidrug resistance protein 1 (MRP1) is an ATP-binding cassette transporter responsible for effluxing drugs and organic anions.
- The precise function of MRP1's cytoplasmic loops (CLs) in substrate translocation and ATPase activity remains largely unelucidated.
Purpose of the Study:
- To investigate the role of cytoplasmic loop 5 (CL5) in the membrane expression and functional activity of MRP1.
- To identify specific residues within CL5 critical for MRP1's structure and function.
Main Methods:
- Site-directed mutagenesis of conserved charged residues and helix-disrupting glycine in CL5 of MRP1.
- Assessment of MRP1 protein levels via Western blotting.
- Evaluation of MRP1 transport activity and substrate binding affinity.
- Analysis of ATP binding and catalytic activity using photoaffinity labeling with azido-ATP.
Main Results:
- Alanine substitution of Lys(513), Lys(516), Glu(521), and Glu(535) in CL5 significantly reduced MRP1 protein levels.
- Mutations at the CL5-NBD2 interface, including His(1364) and Arg(1367), also decreased MRP1 expression.
- Most CL5 mutants exhibited reduced transport activity (50-75%), linked to decreased substrate binding and affinity.
- The G511I mutant showed altered catalytic activity, despite unchanged ATP binding.
Conclusions:
- Cytoplasmic loop 5 plays a critical role in the proper plasma membrane expression of MRP1.
- CL5 is essential for coupling ATPase activity to substrate translocation, influencing both binding and catalysis.
- These findings highlight the multifaceted importance of CL5 in MRP1 function.
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