Discrete Mutations Introduced in the Predicted Nucleotide-Binding Sites of the <i>mdr1</i> Gene Abolish Its Ability To Confer Multidrug Resistance

Azzaria, Marie; Schurr, Erwin; Gros, Philippe

doi:10.1128/mcb.9.12.5289-5297.1989

Cited by 12 publications

(2 citation statements)

References 36 publications

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“…In this regard, it is notable that the sequence homology between NBD1 and NBD2 in CFTR is much less 0-0 CPT-cAMP than for other proteins of similar overall structure with duplicate NBDs. Interestingly, the results discussed above contrast with those of similar studies on the NBDs of MDR, in which equivalent mutations in the two domains generally have the same effect on function (2).…”

Section: Discussioncontrasting

confidence: 74%

Maturation and Function of Cystic Fibrosis Transmembrane Conductance Regulator Variants Bearing Mutations in Putative Nucleotide-Binding Domains 1 and 2

Gregory¹,

Rich²,

Cheng³

et al. 1991

Molecular and Cellular Biology

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One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.

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Section: Discussioncontrasting

confidence: 74%

Maturation and Function of Cystic Fibrosis Transmembrane Conductance Regulator Variants Bearing Mutations in Putative Nucleotide-Binding Domains 1 and 2

Gregory¹,

Rich²,

Cheng³

et al. 1991

Molecular and Cellular Biology

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“…This switch from the inward- to the outward-facing orientation results in a redistribution of the drug-binding residues with more of these binding regions facing the extracellular side [ 79 ]. ATP plays an essential role in the stabilisation of this conformation and several studies have suggested an association between the ATPase activity of PGP and substrate efflux leading to drug resistance [ 87 , 88 , 89 , 90 , 91 ]. It has been reported that PGP can have lower drug affinity in the presence of ATP [ 92 , 93 , 94 , 95 , 96 ], while the use of the ATP analogue, adenylylimidodiphosphate, results in drug efflux [ 97 ].…”

Section: The Structure Of P-glycoproteinmentioning

confidence: 99%

P-glycoprotein: new insights into structure, physiological function, regulation and alterations in disease

Juvale

Hamid

Halim

et al. 2022

Heliyon

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Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site

Loo

Clarke

2002

Proc. Natl. Acad. Sci. U.S.A.

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The human multidrug resistance P-glycoprotein uses ATP to transport a wide variety of structurally unrelated cytotoxic compounds out of the cell. In this study, we used cysteine-scanning mutagenesis and cross-linking studies to identify residues that are exposed to the drug-binding site upon vanadate trapping. In the absence of nucleotides, C222(TM4) was cross-linked to C868(TM10) and C872(TM10); C306(TM5) was cross-linked to C868(TM10), C872(TM10), C945(TM11), C982(TM12), and C984(TM12); and C339(TM6) was cross-linked to C868(TM10), C872(TM10), C942(TM11), C982(TM12), and C985(TM12). These cysteines are in the middle of the predicted transmembrane (TM) segments and form the drug-binding site. Cross-linking between 332C(TM6) and cysteines introduced at the extracellular side of other TM segments was also done. In the absence of nucleotides, residues 332C and 856C on the extracellular side of TMs 6 and 10, respectively, were cross-linked with a 13-Å cross-linker (M8M, 3,6-dioxaoctane-1,8-diyl bismethanethiosulfonate). ATP plus vanadate inhibited cross-linking between 332C(TM6) and 856C(TM10) as well as those in the drug-binding site. Instead, vanadate trapping promoted cross-linking between 332C(TM6) and 976C(TM12) with a 10-Å cross-linker (M6M, 1,6-hexanediyl bismethanethiosulfonate). When ATP hydrolysis was allowed to proceed, then 332C(TM12) could form a disulfide bond with 975C(TM12). The cross-linking pattern of 332C(TM6) with residues in TM10 and TM12 indicates that the drug-binding site undergoes dynamic and relatively large conformational changes, and that different residues are exposed to the drug-binding site during the resting phase, upon vanadate trapping and at the completion of the catalytic cycle.T he human multidrug resistance P-glycoprotein (P-gp) is an ATP-dependent drug pump located at the plasma membrane that can transport a wide variety of structurally diverse compounds of different sizes (recently reviewed in ref. 1). P-gp is clinically important because overexpression of P-gp contributes to the phenomenon of multidrug resistance during treatment of AIDS and cancer. Many of these compounds in these treatment regimens are also substrates of P-gp (2-4). P-gp also plays an important role in mediating the bio-availability of oral drugs because of its relatively higher level of expression in the intestine, brain, liver, and kidney (5-10).P-gp consists of 1,280 aa that are organized in two repeating units of 610 aa that are joined by a linker region of about 60 aa (11). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (11-13).An important goal in understanding the mechanism of drug transport by P-gp is to determine how ATP hydrolysis is coupled to drug efflux. Both halves of P-gp can hydrolyze ATP (14, 15), but drug-stimulated ATPase activity (14) and conferring of drug resistance (16) requires interaction between the two halves of P-gp. Both halves of P-gp are required for activity because drug binding requires interaction between the NH 2 an...

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Discrete Mutations Introduced in the Predicted Nucleotide-Binding Sites of the mdr1 Gene Abolish Its Ability To Confer Multidrug Resistance

Cited by 12 publications

References 36 publications

Maturation and Function of Cystic Fibrosis Transmembrane Conductance Regulator Variants Bearing Mutations in Putative Nucleotide-Binding Domains 1 and 2

Maturation and Function of Cystic Fibrosis Transmembrane Conductance Regulator Variants Bearing Mutations in Putative Nucleotide-Binding Domains 1 and 2

P-glycoprotein: new insights into structure, physiological function, regulation and alterations in disease

Vanadate trapping of nucleotide at the ATP-binding sites of human multidrug resistance P-glycoprotein exposes different residues to the drug-binding site

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