ABCMdb

ABCC1 p.Asn19Gln

Predicted by SNAP2:	A: D (66%), C: D (66%), D: D (53%), E: D (53%), F: D (80%), G: D (66%), H: D (75%), I: D (75%), K: D (75%), L: D (75%), M: D (75%), P: D (75%), Q: D (63%), R: D (80%), S: N (66%), T: N (57%), V: D (71%), W: D (71%), Y: D (71%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D,

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[hide] The amino terminus of the human multidrug resistan... J Biol Chem. 2006 Oct 13;281(41):31152-63. Epub 2006 Aug 16. Chen Q, Yang Y, Li L, Zhang JT
The amino terminus of the human multidrug resistance transporter ABCC1 has a U-shaped folding with a gating function.
J Biol Chem. 2006 Oct 13;281(41):31152-63. Epub 2006 Aug 16., 2006-10-13 [PMID:16914551]

Abstract [show]
Multidrug resistance is a serious problem in successful cancer chemotherapy. Studies using model cell lines have demonstrated that overexpression of some members of the ATP-binding cassette (ABC) transporter superfamily, such as ABCC1, causes enhanced efflux and, thus, decreased accumulation of multiple anticancer drugs, which leads to increased cell survival. Unlike most other ABC transporters, ABCC1 has an additional membrane-spanning domain (MSD0) with a putative extracellular amino terminus of 32 amino acids. However, the function of MSD0 and the role of the extracellular amino terminus are largely unknown. In this study, we examined the structural folding and the function of the amino terminus. We found that it has a U-shaped folding with the bottom of the U-structure facing cytoplasm and both ends in extracellular space. We also found that this U-shaped amino terminus probably functions as a gate to regulate the drug transport activity of human ABCC1.

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No. Sentence Comment
196 It is also noteworthy that the mutants ABCC1FLAG-Q and ABCC1QQ , with the mutation of Asn19 to Gln, which is the last residue in the IU5C1 epitope, are still reactive to IU5C1 (Fig. 4, M and T). Login to comment

[hide] Membrane topology of the multidrug resistance prot... J Biol Chem. 1997 Sep 19;272(38):23623-30. Hipfner DR, Almquist KC, Leslie EM, Gerlach JH, Grant CE, Deeley RG, Cole SP
Membrane topology of the multidrug resistance protein (MRP). A study of glycosylation-site mutants reveals an extracytosolic NH2 terminus.
J Biol Chem. 1997 Sep 19;272(38):23623-30., [PMID:9295302]

Abstract [show]
Multidrug resistance protein, MRP, is a 190-kDa integral membrane phosphoglycoprotein that belongs to the ATP-binding cassette superfamily of transport proteins and is capable of conferring resistance to multiple chemotherapeutic agents. Previous studies have indicated that MRP consists of two membrane spanning domains (MSD) each followed by a nucleotide binding domain, plus an additional extremely hydrophobic NH2-terminal MSD. Computer-assisted hydropathy analyses and multiple sequence alignments suggest several topological models for MRP. To aid in determining the topology most likely to be correct, we have identified which of the 14 N-glycosylation sequons in this protein are utilized. Limited proteolysis of MRP-enriched membranes and deglycosylation of intact MRP and its tryptic fragments with PNGase F was carried out followed by immunoblotting with antibodies known to react with specific regions of MRP. The results obtained indicated that the sequon at Asn354 in the middle MSD is not utilized and suggested approximate sites of N-glycosylation. Subsequent site-directed mutagenesis studies established that Asn19 and Asn23 in the NH2-terminal MSD and Asn1006 in the COOH-terminal MSD are the only sites in MRP that are modified with N-linked oligosaccharides. N-Glycosylation of Asn19 and Asn23 provides the first direct experimental evidence that MRP has an extracytosolic NH2 terminus. This finding, together with those of previous studies, strongly suggests that the NH2-terminal MSD of MRP contains an odd number of transmembrane helices. These results may have important implications for the further understanding of the interaction of drugs with MRP.

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66 The N19Q, N1006Q, and N1156Q mutations were generated using the TransformerTM site-directed mutagenesis kit (CLONTECH Laboratories, Inc., Palo Alto, CA) based on the method developed by Deng and Nickoloff (35). Login to comment
67 The templates for mutagenesis were prepared by cloning the BamHI fragment as above (for N19Q) and the XmaI fragment (MRP nucleotides 2337-4322) (for N1006Q and N1156Q) from pcDNAI-MRP1 into pGEM®-3Z (Promega, Madison, WI). Login to comment
68 Mutagenesis was then performed according to the manufacturer`s instructions using the ScaI/ StuI and SspI/EcoRV selection primers (for N19Q and N1006Q/N1156Q mutations, respectively), and the following sense mutagenic oligonucleotide primers: 5Ј-C TGG GAC TGG CAG GTC ACG TGG-3Ј (N19Q), 5Ј-C CCC ATC GTC CAG GGG ACT CAG G-3Ј (N1006Q), and 5Ј-C TAT TCC CAT TTC CAG GAG ACC TTG C-3Ј (N1156Q). Login to comment
69 The N19Q/N23Q double mutant was also generated by this method using the N19Q mutagenic primer with the N23Q BamHI fragment in pGEM¾1;-3Z as a template. Login to comment
72 The N19Q/N23Q/N1006Q triple mutant was prepared by cloning the BamHI fragment containing the N19Q/N23Q mutation into pcDNAI-MRP1/N1006Q. Login to comment
164 Introduction of the individual N19Q, N23Q, and N71Q mutations in MSD1 did not result in a detectable alteration of the electrophoretic mobility of the intact proteins (Fig. 4A, lanes 3-5). Login to comment
165 However, after digestion with trypsin, the N-2 fragments of wild-type MRP and MRP-(N71Q) co-migrated at 43-60 kDa, whereas the N-2 fragments of MRP-(N19Q) and MRP-(N23Q) migrated slightly faster at approximately 38-50 kDa (Fig. 4B, lanes 2-5). Login to comment
166 The MRP-(N19Q) and MRP-(N23Q) N-2 fragments are glycosylated because they have lower electrophoretic mobility than the deglycosylated 25-kDa N-2 fragment of wild-type MRP (compare with Fig. 4B, lane 1). Login to comment
168 To confirm that Asn19 and Asn23 are the only glycosylated sites in MSD1, we also produced an N19Q/N23Q MRP double mutant. Login to comment
169 MRP-(N19Q/ N23Q) migrated faster than wild-type MRP and the N19Q, N23Q, and N71Q single mutants (Fig. 4A, compare lane 6 with lanes 2-5). Login to comment
178 A, crude membrane proteins from Cos-1 cells expressing wild-type MRP (WT) treated with (ϩ) or without (-) PNGase F, the single glycosylation site mutant MRP constructs N19Q, N23Q, N71Q, N1006Q, and N1156Q, the double mutant N19Q/N23Q, and the triple mutant N19Q/N23Q/N1006Q were separated by SDS-polyacrylamide gel electrophoresis on 5% gels and immunoblotted with mAb QCRL-1. Login to comment
70 The N19Q/N23Q double mutant was also generated by this method using the N19Q mutagenic primer with the N23Q BamHI fragment in pGEMt-3Z as a template. Login to comment