P-Glycoproteins (P-gps) are membrane glycoproteins encoded by the mdr gene family, and their overexpression is associated with multidrug resistance (MDR). Sequence analyses of mdr cDNAs predict a protein formed by two symmetrical halves, each composed of six transmembrane (TM) segments and one ATP-binding domain. To determine the topology of the N-terminal half of P-gp, a small antigenic peptide epitope (YPYDVPDYAIEGR) containing part of the hemagglutinin (HA) of influenza virus was inserted at six different positions of the Mdr3 protein (101, 161, 206, 244, 320, and 376). Functional integrity of the modified proteins was tested by measuring their capacity to confer MDR in Chinese hamster ovary cells. Intracellular and extracellular localization of the tag in the full-length protein was determined in intact or permeabilized cells by immunofluorescence using a mouse monoclonal antibody (12CA5) specific for the HA epitope. While insertions at positions 101, 161, 320, and 376 did not alter P-gp function, insertions at positions 206 and 244 abrogated the capacity of P-gp to confer drug resistance. The epitope tags inserted at positions 161 and 376 were found to be located intracellularly, whereas the tags at positions 101 and 320 were located on the extracellular side of the membrane. These results indicate that the intervening segments separating predicted TM1-TM2 and TM5-TM6 correspond to extracellular regions, while the segments linking TM2-TM3 and the one located downstream of TM6 correspond to intracellular regions. These results are consistent with a six TM domain model for the N-terminal half of P-gp with an extracellular glycosylated region (TM1-TM2) and an intracellular ATP-binding site (downstream TM6). Epitope insertion in segments linking TM3-TM4 and TM4-TM5 caused a loss of P-gp function, suggesting that the integrity of these sequences is essential either for drug transport or for proper maturation and accurate targeting of P-gp to the plasma membrane.
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