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 conf er multidrug resistance to tumor cells. In the absence of a high-resolution structure for this protein, several important and intriguing aspects of Pg p structure and function remain poorly understood. Fluorescence spectroscop y of endogenous or genetically engineered tryptophan residues represents a potentially powerful method to probe static and dynamic aspects of Pgp at h igh resolution. We have used site-directed mutagenesis to modify the wild-t ype (WT) mouse mdr3 Pgp for tryptophan fluorescence spectroscopy by replace ment 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 mu tant Trp-less mdr3 cDNA (mdr3F(1-11)) become multidrug-resistant and can ca rry out active transport of vinblastine, colchicine, and Calcein-AM. The md r3F(1-11) mutant has reduced activity compared with WT Mdr3, and shows a un ique pattern of drug resistance clearly distinct from WT and, as opposed to the latter, can neither confer FK-506 resistance nor functionally compleme nt ste6 in yeast. Studies with Pgp mutants containing either single or doub le tryptophan residues or with chimeric molecules constructed between wild- type Pgp and mdr3F(1-11) indicated that no single tryptophan residue was re sponsible for the reduced activity of the mdr3F(1-11) mutant. Likewise, all but one chimeric Pgp preserved the unique drug resistance profile of the m dr3F(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.