Functional analysis of a tryptophan-less P-glycoprotein: a tool for tryptophan insertion and fluorescence spectroscopy.
P-glycoprotein (Pgp) functions as an ATP-dependent drug efflux pump to confer multidrug resistance to tumor cells. In the absence of a high-resolution structure for this protein, several important and intriguing aspects of Pgp structure and function remain poorly understood. Fluorescence spectroscopy of endogenous or genetically engineered tryptophan residues represents a potentially powerful method to probe static and dynamic aspects of Pgp at high resolution. We have used site-directed mutagenesis to modify the wild-type (WT) mouse mdr3 Pgp for tryptophan fluorescence spectroscopy by replacement of all 11 tryptophan residues individually with phenylalanine. None of the 11 tryptophans were found to be absolutely essential for Pgp activity, because Chinese hamster ovary cells transfected and overexpressing this mutant Trp-less mdr3 cDNA (mdr3F(1-11)) become multidrug-resistant and can carry out active transport of vinblastine, colchicine, and Calcein-AM. The mdr3F(1-11) mutant has reduced activity compared with WT Mdr3, and shows a unique pattern of drug resistance clearly distinct from WT and, as opposed to the latter, can neither confer FK-506 resistance nor functionally complement ste6 in yeast. Studies with Pgp mutants containing either single or double tryptophan residues or with chimeric molecules constructed between wild-type Pgp and mdr3F(1-11) indicated that no single tryptophan residue was responsible for the reduced activity of the mdr3F(1-11) mutant. Likewise, all but one chimeric Pgp preserved the unique drug resistance profile of the mdr3F(1-11) mutant. Altogether, we show that a Trp-less Pgp is functionally active and can be used as a molecular backbone for insertion of tryptophans in strategic locations to probe various aspects of Pgp function.
Kwan
T
T
Department of Biochemistry, McGill University, Montreal, Quebec, Canada.
Loughrey
H
H
Brault
M
M
Gruenheid
S
S
Urbatsch
I L
IL
Senior
A E
AE
Gros
P
P
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics / Q000378:metabolism; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D018432:Drug Resistance, Multiple / Q000502:physiology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; C095122:multidrug resistance protein 3; D000251:Adenosine Triphosphatases