ABCC1 p.His1049Ala
| Predicted by SNAP2: | A: D (75%), C: D (75%), D: D (91%), E: D (85%), F: D (71%), G: D (85%), I: D (85%), K: D (85%), L: D (80%), M: D (80%), N: D (80%), P: D (91%), Q: D (80%), R: D (80%), S: D (80%), T: D (75%), V: D (80%), W: D (91%), Y: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] Expression and function of human MRP1 (ABCC1) is d... J Biol Chem. 2011 Mar 4;286(9):7202-13. Epub 2010 Dec 20. Iram SH, Cole SP
Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2.
J Biol Chem. 2011 Mar 4;286(9):7202-13. Epub 2010 Dec 20., 2011-03-04 [PMID:21177244]
Abstract [show]
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.
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No. Sentence Comment
49 To create the CL6 mutation H1049A, a 2-kb XmaI fragment (containing nucleotides 2337-4322) in pGEM-3Z (Promega, Madison, WI) was used as the template with the mutagenic primer 5Ј-CCG CTG TCT GGC CGT GGA CCT GC-3Ј.
X
ABCC1 p.His1049Ala 21177244:49:27
status: NEW158 To test this hypothesis, mutants H1049A, H1364A, and R1367A were generated, and the levels of the mutant proteins in HEK293T cells determined as described above.
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ABCC1 p.His1049Ala 21177244:158:33
status: NEW160 The levels of the NBD2 mutant H1364A and the CL6 mutant H1049A were also reduced by Ͼ50%.
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ABCC1 p.His1049Ala 21177244:160:56
status: NEW178 Homology models and levels of MRP1 mutants H1049A, H1364A, and R1367A.
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ABCC1 p.His1049Ala 21177244:178:43
status: NEW233 If such interdomain interactions exist and are important for the proper folding and assembly of MRP1, it was reasonable to expect that, as we observed for the Ala substitution of Glu535 , replacing His1049 with Ala would also affect the levels of MRP1.
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ABCC1 p.His1049Ala 21177244:233:198
status: NEW234 Indeed, this was the case, although the reduction in E535A protein levels was significantly greater (Ͼ90%) than the reduction observed for H1049A (60%).
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ABCC1 p.His1049Ala 21177244:234:145
status: NEW