Comparison of the functional characteristics of the nucleotide binding domains of multidrug resistance protein 1.
Multidrug Resistance Protein 1 (MRP1) transports diverse organic anionic conjugates and confers resistance to cytotoxic xenobiotics. The protein contains two nucleotide binding domains (NBDs) with features characteristic of members of the ATP-binding cassette superfamily and exhibits basal ATPase activity that can be stimulated by certain substrates. It is not known whether the two NBDs of MRP1 are functionally equivalent. To investigate this question, we have used a baculovirus dual expression vector encoding both halves of MRP1 to reconstitute an active transporter and have compared the ability of each NBD to be photoaffinity-labeled with 8-azido-[(32)P]ATP and to trap 8-azido-[(32)P]ADP in the presence of orthovanadate. We found that NBD1 was preferentially labeled with 8-azido-[(32)P]ATP, while trapping of 8-azido-[(32)P]ADP occurred predominantly at NBD2. Although trapping at NBD2 was dependent on co-expression of both halves of MRP1, binding of 8-azido-ATP by NBD1 remained detectable when the NH(2)-proximal half of MRP1 was expressed alone and when NBD1 was expressed as a soluble polypeptide. Mutation of the conserved Walker A lysine 684 or creation of an insertion mutation between Walker A and B motifs eliminated binding by NBD1 and all detectable trapping of 8-azido-ADP at NBD2. Both mutations decreased leukotriene C(4) (LTC(4)) transport by approximately 70%. Mutation of the NBD2 Walker A lysine 1333 eliminated trapping of 8-azido-ADP by NBD2 but, in contrast to the mutations in NBD1, essentially eliminated LTC(4) transport activity without affecting labeling of NBD1 with 8-azido-[(32)P]ATP.
Gao
M
M
Cancer Research Laboratories, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Cui
H R
HR
Loe
D W
DW
Grant
C E
CE
Almquist
K C
KC
Cole
S P
SP
Deeley
R G
RG
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D016367:Baculoviridae / Q000378:metabolism; D002460:Cell Line; D015151:Immunoblotting; D007313:Insecta; D017997:Leukotriene C4 / Q000493:pharmacokinetics; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009711:Nucleotides / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D009711:Nucleotides; D017997:Leukotriene C4; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D008239:Lysine