Expression of the human multidrug transporter in insect cells by a recombinant baculovirus.
The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents. We expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action. MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter. Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection. The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope. The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells. Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by [3H]-azidopine, presumably at the same two sites as the native protein. Various drugs and reversing agents (e.g., daunomycin greater than verapamil greater than vinblastine approximately vincristine) compete with the [3H]azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart.
Germann
U A
UA
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Willingham
M C
MC
Pastan
I
I
Gottesman
M M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure; D004247:DNA / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008970:Molecular Weight; D009036:Moths; D010766:Phosphorylation; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D014162:Transfection
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D004247:DNA; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase