Domain organization of murine mdr1b P-glycoprotein: the cytoplasmic linker region is a potential dimerization domain.
P-glycoprotein is an integral membrane protein that functions as a cytotoxic drug-efflux pump. Studies suggest that the transporter exists in the membrane as a dimer and possibly higher order structures. We report the bacterial expression of the linker region (amino acids 621-688) of murine mdr1b P-glycoprotein and demonstrate that this region, which serves to link the two homologous halves of the transporter, has the potential to serve as a dimerization domain. The recombinant peptide (8742 daltons) eluted from a gel filtration column at a position corresponding to a dimer (i.e. 17,500 daltons). A dimer:monomer equilibrium, as a function of peptide concentration, was confirmed by large zone gel filtration chromatography. The dissociation constant (KD) for the dimer:monomer equilibrium was 350 nM. It was possible to dissociate the dimer by low pH (3.0) or high ionic strength (2.5 M NaCl). Dimerization was not affected by an alkaline pH of 10 or 5 mM EDTA. Studies with a truncated linker peptide indicated that the N-terminal 48 amino acids were sufficient for dimerization.
Juvvadi
S R
SR
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Glavy
J S
JS
Horwitz
S B
SB
Orr
G A
GA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D017931:DNA Primers; D019281:Dimerization; D004926:Escherichia coli; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017931:DNA Primers; D011994:Recombinant Proteins