ABCC1 p.Gly511Ile
Predicted by SNAP2: | A: D (80%), C: D (80%), D: D (91%), E: D (91%), F: D (91%), H: D (85%), I: D (91%), K: D (95%), L: D (91%), M: D (91%), N: D (75%), P: D (95%), Q: D (91%), R: D (95%), S: D (80%), T: D (85%), V: D (91%), W: D (95%), Y: D (95%), |
Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: 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, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[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.
Comments [show]
None has been submitted yet.
No. Sentence Comment
9 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.
X
ABCC1 p.Gly511Ile 21177244:9:83
status: NEW47 To generate point mutations in CL5, plasmid pBluescriptSK(ϩ)BamHI/SpHI-MRP1 containing a 1.9-kb fragment from pcDNA3.1(-)-MRP1k was used as the template with the following mutagenic primers (substituted nucleotides are underlined): R501A, 5Ј-GAG CAA AGA CAA TGC GAT CAA GCT GAT G-3Ј; K503A, 5Ј-GAC AAT CGG ATC GCG CTG ATG AAC G-3Ј; E507A, 5Ј-GCT GAT GAA CGC AAT TCT CAA TGG G-3Ј; G511I, 5Ј-CTC AAT ATT ATC AAA GTG CTA AAG CTT TAT GCC TGG GAG CTG GC-3Ј; K513A, 5Ј-CTC AAT GGG ATC GCA GTG CTA AAG C-3Ј; K513R, 5Ј-CTC AAT GGG ATC AGA GTG CTA AAG C-3Ј; K516A, 5Ј-GAT CAA AGT GCT AGC GCT TTA TGC CTG-3Ј; K516R, 5Ј-GAT CAA AGT GCT AAG ACT TTA TGC CTG-3Ј; E521A, 5Ј-CTT TAT GCC TGG GCG CTG GCA TTC-3Ј; E521D, 5Ј-CTT TAT GCC TGG GAC CTG GCA TTC-3Ј; R532A, 5Ј-GTG CTG GCC ATT GCG CAG GAG GAG CT-3Ј; E535A, 5Ј-CAT CAG GCA GGA GGC CTT GAA GGT GCT GAA G-3Ј; and E535D, 5Ј- CAG GCA GGA GGA TCT GAA GGT GCT GAA G-3Ј.
X
ABCC1 p.Gly511Ile 21177244:47:422
status: NEW171 Effect of Mutation G511I on MRP1 Expression and Function-Analyses of our homology model of MRP1 also suggested that substitution of the sterically unencumbered Gly511 with a bulky beta-branched amino acid like Ile could affect the geometry and/or mobility of CL5.
X
ABCC1 p.Gly511Ile 21177244:171:19
status: NEW173 As shown in Fig. 7A, substitution of Gly511 with Ile had no effect on MRP1 protein levels; however, transport of LTC4 and E217betaG by the G511I mutant was decreased by ϳ60 and 75%, respectively (Fig. 7, B and C).
X
ABCC1 p.Gly511Ile 21177244:173:37
status: NEWX
ABCC1 p.Gly511Ile 21177244:173:139
status: NEW175 The reduced transport activity of the G511I mutant could be caused by a reduction in substrate affinity or transport capacity.
X
ABCC1 p.Gly511Ile 21177244:175:38
status: NEW176 Because of the very low levels of E217betaG transport by G511I, the apparent affinity (Km) of this mutant for E217betaG could not be determined directly by kinetic analysis.
X
ABCC1 p.Gly511Ile 21177244:176:57
status: NEW183 As shown in Fig. 7D, E217betaG inhibited [3 H]LTC4 uptake by G511I and wild-type MRP1 with IC50 values that differed by 5-fold (40 versus 8 M, respectively).
X
ABCC1 p.Gly511Ile 21177244:183:61
status: NEW184 The ability of LTC4 to inhibit [3 H]E217betaG uptake by the G511I mutant relative to wild-type MRP1 was also reduced, as reflected by an ϳ3-fold difference in the IC50 values (Fig. 7E).
X
ABCC1 p.Gly511Ile 21177244:184:60
status: NEW185 These observations support the conclusion that decreased transport by the G511I mutant is caused by a reduction in substrate affinity.
X
ABCC1 p.Gly511Ile 21177244:185:74
status: NEW186 Photolabeling of MRP1 Mutants R501A, E507A, R532A, and G511I with [3 H]LTC4-To determine whether the decrease in LTC4 uptake by the R501A, E507A, R532A, and G511I mutants was associated with decreased binding of this substrate to MRP1, membrane vesicles enriched for wild-type or mutant MRP1 were photolabeled with [3 H]LTC4.
X
ABCC1 p.Gly511Ile 21177244:186:55
status: NEWX
ABCC1 p.Gly511Ile 21177244:186:157
status: NEW188 Labeling of the G511I mutant by [3 H]LTC4 was also reduced by 40% (Fig. 8B) (after correcting for differences in protein levels).
X
ABCC1 p.Gly511Ile 21177244:188:16
status: NEW189 These relative levels of photolabeling correlated well with the relative LTC4 transport activities of these mutants and, in the case of G511I, with the reduced affinity for this substrate suggested by the competition experiments (Fig. 7E).
X
ABCC1 p.Gly511Ile 21177244:189:136
status: NEW190 Photolabeling of Mutants E507A and G511I with 8-Azido- [␣-32 P]ATP and Orthovanadate- and BeF-induced Trapping of 8-Azido-[␣-32 P]ADP-The precise role of the CLs in coupling the ATPase activity of ABC transporters to the translocation of their substrates is not yet fully understood (12, 42).
X
ABCC1 p.Gly511Ile 21177244:190:35
status: NEW192 For this reason, the interactions of the transport-deficient E507A and G511I mutants with ATP were examined.
X
ABCC1 p.Gly511Ile 21177244:192:71
status: NEW195 Next, the catalytic activity of the E507A and G511I mutants was examined by measuring the amount of azido-[32 P]ADP trapped by orthovanadate under conditions permissive for ATP hydrolysis (26).
X
ABCC1 p.Gly511Ile 21177244:195:46
status: NEW196 As shown in Fig. 9B, [32 P]ADP trapping by the G511I mutant was reduced by 40%, whereas trapping by the E507A mutant was comparable to wild-type MRP1.
X
ABCC1 p.Gly511Ile 21177244:196:47
status: NEW211 LTC4 and E217betaG transport by the G511I mutant was also reduced, although their expression levels were comparable with wild-type MRP1. Together, these results illustrate the remarkable sensitivity of CL5 to mutation and thus establish its critical role in the expression and function of MRP1.
X
ABCC1 p.Gly511Ile 21177244:211:36
status: NEW214 Protein expression, transport activity, and inhibition of [3 H]LTC4 and[3 H]E217betaG uptake by wild-type MRP1 and mutant G511I.
X
ABCC1 p.Gly511Ile 21177244:214:122
status: NEW215 A, immunoblot of membrane vesicles (1 g of protein/lane) prepared from HEK293T cells transfected with wild-type MRP1 and mutant G511I cDNA expression vectors.
X
ABCC1 p.Gly511Ile 21177244:215:136
status: NEW220 D and E, membrane vesicles prepared from HEK293T cells transfected with wild-type (f, WT-MRP1) and mutant (Œ, G511I) MRP1 cDNA expression vectors were incubated for 1 min with either [3 H]LTC4 at 23 °C in the presence of E217betaG (0, 12.5, 25, and 50 M) (D) or [3 H]E217betaG at 37 °C in the presence of LTC4 (0, 300, 600, and 900 nM) (E).
X
ABCC1 p.Gly511Ile 21177244:220:116
status: NEW236 [3 H]LTC4 photolabeling of wild-type and MRP1 mutants R501A, E507A, R532A, and G511I.
X
ABCC1 p.Gly511Ile 21177244:236:79
status: NEW240 Azido-[32 P]ATP labeling and vanadate-induced trapping of azido-[32 P]ADP by E507A and G511I mutant MRP1 proteins.
X
ABCC1 p.Gly511Ile 21177244:240:87
status: NEW263 The fact that the G511I mutant was expressed at levels comparable with that of wild-type MRP1 suggests that even after the geometry of CL5 was altered, the integrity of its interface with NBD2 was not significantly altered.
X
ABCC1 p.Gly511Ile 21177244:263:18
status: NEW264 Nevertheless, the G511I mutant showed reduced transport activity, which was associated with an apparent decrease in substrate binding, again implicating a direct or indirect role for this region in substrate recognition by MRP1.
X
ABCC1 p.Gly511Ile 21177244:264:18
status: NEW267 Although binding of azido-[32 P]ATP by the mutants was not affected, vanadate-induced trapping of azido-[32 P]ADP was moderately reduced for G511I but not for E507A.
X
ABCC1 p.Gly511Ile 21177244:267:141
status: NEW272 Nevertheless, these authors reported that the LTC4 transport activity of the double mutant was reduced by Ͼ80%, a larger decrease than we observed for either of the single E507A and G511I mutants.
X
ABCC1 p.Gly511Ile 21177244:272:188
status: NEW