ABCMdb

ABCC1 p.Gly511Ile

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Publications

PMID: 21177244 [PubMed] Iram SH et al: "Expression and function of human MRP1 (ABCC1) is dependent on amino acids in cytoplasmic loop 5 and its interface with nucleotide binding domain 2."

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. Login to comment
47 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. Login to comment
171 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. Login to comment
173 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). Login to comment
175 The reduced transport activity of the G511I mutant could be caused by a reduction in substrate affinity or transport capacity. Login to comment
176 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. Login to comment
183 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). Login to comment
184 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). Login to comment
185 These observations support the conclusion that decreased transport by the G511I mutant is caused by a reduction in substrate affinity. Login to comment
186 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. Login to comment
188 Labeling of the G511I mutant by [3 H]LTC4 was also reduced by 40% (Fig. 8B) (after correcting for differences in protein levels). Login to comment
189 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). Login to comment
190 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). Login to comment
192 For this reason, the interactions of the transport-deficient E507A and G511I mutants with ATP were examined. Login to comment
195 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). Login to comment
196 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. Login to comment
211 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. Login to comment
214 Protein expression, transport activity, and inhibition of [3 H]LTC4 and[3 H]E217betaG uptake by wild-type MRP1 and mutant G511I. Login to comment
215 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. Login to comment
220 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). Login to comment
236 [3 H]LTC4 photolabeling of wild-type and MRP1 mutants R501A, E507A, R532A, and G511I. Login to comment
240 Azido-[32 P]ATP labeling and vanadate-induced trapping of azido-[32 P]ADP by E507A and G511I mutant MRP1 proteins. Login to comment
263 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. Login to comment
264 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. Login to comment
267 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. Login to comment
272 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. Login to comment