The P-glycoprotein (ABCB1) linker domain encodes high-affinity binding sequences to alpha- and beta-tubulins.
P-Glycoprotein (or ABCB1) has been shown to cause multidrug resistance in tumor cell lines selected with lipophilic anticancer drugs. ABCB1 encodes a duplicated molecule with two hydrophobic and hydrophilic domains linked by a highly charged region of approximately 90 amino acids, the "linker domain" with as yet unknown function(s). In this report, we demonstrate a role for this domain in binding to other cellular proteins. Using overlapping hexapeptides that encode the entire amino acid sequence of the linker domain of human ABCB1, we show a direct and specific binding between sequences in the linker domain and several intracellular proteins. Three different polypeptide sequences [617EKGIYFKLVTM627 (LDS617-627), 657SRSSLIRKRSTRRSVRGSQA676 (LDS657-676), and 693PVSFWRIMKLNLT705 (LDS693-705)] in the linker domain interacted tightly with several proteins with apparent molecular masses of approximately 80, 57, and 30 kDa. Interestingly, only the 57 kDa protein (or P57) interacted with all three different sequences of the linker domain. Purification and partial N-terminal amino acid sequencing of P57 showed that it encodes the N-terminal amino acids of alpha- and beta-tubulins. The identity of the P57 interacting protein as tubulins was further confirmed by Western blotting using monoclonal antibodies to alpha- and beta-tubulin. Taken together, the results of this study provide the first evidence for ABCB1 protein interaction mediated by sequences in the linker domain. These findings are likely to provide further insight into the functions of ABCB1 in normal and drug resistant tumor cells.
Georges
Elias
E
Institute of Parasitology, McGill University, Quebec H9X 1C0, Canada. elias.georges@mcgill.ca
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002457:Cell Extracts / Q000737:chemistry; D045744:Cell Line, Tumor; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000737:chemistry; D006801:Humans; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D013462:Sulfur Radioisotopes / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; C493964:CDKN1C protein, human; D002457:Cell Extracts; D050761:Cyclin-Dependent Kinase Inhibitor p57; D009842:Oligopeptides; D013462:Sulfur Radioisotopes; D014404:Tubulin; D008715:Methionine