Abstract
P-glycoprotein (P-gp) is an integral membrane protein that causes multidrug resistance when overexpressed in tumor cells. Efforts to identify the position and polarity of its 12 putative transmembrane (TM) domains have so far failed to yield a consistent topological model. Recently, we have described a method for topology mapping based on the insertion of a small antigenic peptide epitope (YPYDVPDYA) in predicted intra- or extracellular loops of the protein. The tagged proteins are then functionally expressed in Chinese hamster ovary cells, and the polarity of the inserted tag with respect to plasma membrane is deduced by immunofluorescence in intact or permeabilized cells. We previously localized segments between TM1 and TM2, and TM5 and TM6 as extracellular and segments between TM2 and TM3 and downstream of TM6 as intracellular (Kast, C., Canfield, V., Levenson, R., and Gros, P. (1995) Biochemistry 34, 4402-4411). We have now inserted single epitope tags at positions 207, 235, 276, 741, 782, 797, 815, 849, 887, 961, and 1024; double epitope tags at positions 736, 849, and 961; and a triple epitope tag at position 849. Insertions of epitopes at positions 235, 736, 741, 849, 887, 961, and 1024 resulted in functional proteins, whereas insertions at positions 207, 276, 782, 797, and 815 abrogated the capacity of P-gp to confer multidrug resistance. The epitope tags inserted at positions 736, 849, and 961 were localized extracellularly, whereas tags at positions 235, 887, and 1024 mapped intracellularly. These results indicate that the intervening segments separated by TM4-TM5, TM10-TM11, and downstream of TM12 are cytoplasmic; segments delineated by TM7-TM8, TM9-TM10, and TM11-TM12 are extracellular. Our combined analysis of the amino- and carboxyl-terminal halves of P-gp supports a 12-TM domain topology with intracellular amino and carboxyl termini and ATP binding sites and an extracellular glycosylated loop (TM1-TM2) in agreement with hydropathy prediction. These results are clearly distinct from those obtained by the analysis of truncated P-gps in vitro and in heterologous expression systems.
Highlights
The abbreviations used areP-gp, P-glycoprotein; TM, transmemthe onset of multidrug resistance in cancer cells in vivo and in cultured cells in vitro (Gottesman and Pastan, 1993; Shustik et al, 1995)
¶ Supported by a senior scientist award from the Medical Research Council of Canada; International Research Scholar of the Howard Hughes Medical Institute
Two strategies have been used to map the topology of individual TM domains: (a) in vitro methods in which truncated P-gps are fused to an indicator molecule, and polarity is brane; MDR, multidrug resistance; EC, extracellular; IC, intracellular
Summary
P-gp, P-glycoprotein; TM, transmemthe onset of multidrug resistance in cancer cells in vivo and in cultured cells in vitro (Gottesman and Pastan, 1993; Shustik et al, 1995). Two strategies have been used to map the topology of individual TM domains: (a) in vitro methods in which truncated P-gps are fused to an indicator molecule, and polarity is brane; MDR, multidrug resistance; EC, extracellular; IC, intracellular. Deduced biochemically after insertion into microsomes or heterologous expression systems; and (b) in vivo methods in which discrete tags are inserted into key locations In this case, the full-length mutant cDNAs are expressed in mammalian cells, and the tags are mapped using epitope-specific antibodies or labeling reagents. Our findings contradict topological models deduced from the study of truncated P-gps fused to reporter genes and expressed in heterologous in vitro or in vivo systems (Zhang and Ling, 1991, 1993; Zhang et al, 1993; Bibi and Beja , 1994; Bejaand Bibi, 1995)
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