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Location of the Rhodamine-binding Site in the Human Multidrug Resistance P-glycoprotein

2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês

10.1074/jbc.m208433200

1083-351X

Tip W. Loo, David M. Clarke,

HIV/AIDS drug development and treatment

The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1–379 plus 681–1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2–12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding. The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1–379 plus 681–1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2–12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding. The human multidrug resistance P-glycoprotein (P-gp) 1The abbreviations used are: P-gp, P-glycoprotein(s); MTS, methanethiosulfonate; TM, transmembrane; HEK, human embryonic kidney is found in the plasma membrane and uses ATP to pump a wide variety of structurally diverse compounds out of the cell (reviewed in Refs. 1Hrycyna C.A. Semin. Cell Dev. Biol. 2001; 12: 247-256Crossref PubMed Scopus (52) Google Scholar and 2Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1365) Google Scholar). Expression of P-gp complicates treatment of AIDS and cancer because many of the therapeutic compounds are also substrates of P-gp (3Lee C.G. Gottesman M.M. Cardarelli C.O. Ramachandra M. Jeang K.T. Ambudkar S.V. Pastan I. Dey S. Biochemistry. 1998; 37: 3594-3601Crossref PubMed Scopus (464) Google Scholar, 4Krishna R. Mayer L.D. Eur. J. Pharmcol. Sci. 2000; 11: 265-283Crossref PubMed Scopus (1004) Google Scholar). P-gp also plays an important role in mediating the bioavailability of oral drugs because of its relatively high expression in the intestine, liver, kidney, and brain (5Thiebaut F. Tsuruo T. Hamada H. Gottesman M.M. Pastan I. Willingham M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7735-7738Crossref PubMed Scopus (2634) Google Scholar, 6Cordon-Cardo C. O'Brien J.P. Casals D. Rittman-Grauer L. Biedler J.L. Melamed M.R. Bertino J.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 695-698Crossref PubMed Scopus (1634) Google Scholar). P-gp is a member of the ATP-binding cassette family of transporters (7Higgins C.F. Annu. Rev. Cell Biol. 1992; 8: 67-113Crossref PubMed Scopus (3435) Google Scholar). Its 1280 amino acids are organized in two repeating units of 610 amino acids that are joined by a linker segment of 60 amino acids (8Chen C.J. Chin J.E. Ueda K. Clark D.P. Pastan I. Gottesman M.M. Roninson I.B. Cell. 1986; 47: 381-389Abstract Full Text PDF PubMed Scopus (1810) Google Scholar). Each repeat has six transmembrane (TM) segments and a hydrophilic domain containing an ATP-binding site (9Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 10Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The minimum functional unit is a monomer (11Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27488-27492Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), but both halves of the molecule do not have to be covalently linked for function (12Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7750-7755Abstract Full Text PDF PubMed Google Scholar, 13Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Both ATP-binding sites are required for activity (14Azzaria M. Schurr E. Gros P. Mol. Cell. Biol. 1989; 9: 5289-5297Crossref PubMed Scopus (290) Google Scholar, 15Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (141) Google Scholar, 16al-Shawi M.K. Urbatsch I.L. Senior A.E. J. Biol. Chem. 1994; 269: 8986-8992Abstract Full Text PDF PubMed Google Scholar, 17Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar) and likely function in an alternating mechanism (18Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (133) Google Scholar). An important goal in understanding the mechanism of drug transport is the identification of residues that line the drug-binding site. The drug-binding site(s) are within the TM domains of P-gp because a deletion mutant missing both nucleotide-binding domains could still interact with drug substrates (13Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). A useful method for identifying residues in the TM segments that contribute to drug binding is to use cysteine-scanning mutagenesis and reaction with thiol-reactive substrates. Such an approach is feasible with P-gp because a Cys-less mutant of P-gp is active (9Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar), and most single cysteine mutants retain activity (19Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). In previous studies we used the thiol-reactive substrates dibromobimane (20Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and MTS-verapamil (23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) to test the reactivity of the single cysteine mutants. These studies showed that residues in TMs 4–6 and 10–12 contributed to the drug-binding site. Some of these residues were common to the binding of both substrates. Although both dibromobimane and verapamil stimulate the ATPase activity of P-gp, it is not known whether both are transported by P-gp. Kinetic studies indicate that verapamil is a noncompetitive inhibitor of cytotoxic substrates transported by P-gp and may occupy separate site(s) from that of transported compounds (24Ayesh S. Shao Y.M. Stein W.D. Biochim. Biophys. Acta. 1996; 1316: 8-18Crossref PubMed Scopus (147) Google Scholar, 25Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (287) Google Scholar). It has been shown that all rhodamine compounds are transported by P-gp (26Eytan G.D. Regev R. Oren G. Hurwitz C.D. Assaraf Y.G. Eur. J. Biochem. 1997; 248: 104-112Crossref PubMed Scopus (107) Google Scholar). In this study, we used a thiol-reactive analog of rhodamine, MTS-rhodamine, to identify residues involved in its binding. The full-length human MDR1 cDNA was obtained by screening a human kidney cortex cDNA library (27Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar,28Bell G.I. Fong N.M. Stempien M.M. Wormsted M.A. Caput D., Ku, L.L. Urdea M.S. Rall L.B. Sanchez-Pescador R. Nucleic Acids Res. 1986; 14: 8427-8446Crossref PubMed Scopus (291) Google Scholar). The seven endogenous cysteines at positions 137, 431, 717, 956, 1074, 1125, and 1227 were replaced with alanine during construction of a Cys-less P-gp (9Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The Cys-less P-gp was functional. A (His)10 tag was attached at the COOH end of the molecule (Cys-less P-gp(His)10) to facilitate purification of the Cys-less P-gp by nickel-chelate chromatography (29Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Cysteine residues were then introduced into the predicted TM segments of Cys-less P-gp(His)10 as described previously (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The predicted boundaries of the TM segments are Met51 to Val71 (TM1), Tyr117 to Ala140(TM2), Lys189 to Thr209 (TM3), Lys213 to Ala233 (TM4), Thr294 to Tyr316 (TM5), Gln330 to Pro350(TM6), Val712 to Ile731 (TM7), Leu757 to Ala780 (TM8), Arg832 to Phe851 (TM9), Trp855 to Glu875(TM10), His936 to Phe957 (TM11), and Val974 to Phe994 (TM12). The integrity of the mutated cDNA was confirmed by sequencing the entire cDNA (30Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (57688) Google Scholar). The histidine-tagged P-gp mutants were expressed and purified as described previously (29Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Briefly, fifty 10-cm diameter culture plates of HEK 293 cells were transfected with the mutant cDNA, and the medium was replaced 24 h later with fresh medium containing 10 μm cyclosporin A. Cyclosporin A is a substrate of P-gp and is a powerful chemical chaperone for promoting maturation of P-gp (31Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 32Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). After another 24 h, the transfected cells were harvested and solubilized with 1% (w/v) n-dodecyl-β-d-maltoside, and the mutant P-gp was isolated by nickel-chelate chromatography (nickel-nitrilotriacetic acid columns; Qiagen, Inc., Mississauga, Canada). The P-gp(His)10 mutant proteins eluted from the nickel columns (in buffer containing 10 mm Tris-HCl, pH 7.5, 500 mm NaCl, 300 mm imidazole, pH 7.0, 0.1% (w/v)n-dodecyl-β-d-maltoside and 10% (v/v) glycerol) were mixed with an equal volume of 10 mg/ml sheep brain phosphatidylethanolamine (Type II-S; Sigma-Aldrich) that was washed and suspended in 10 mm Tris-HCl, pH 7.5, and 150 mmNaCl. The P-gp and lipid mixture was then sonicated for 45 s at 4 °C (bath-type probe, maximum setting; Branson Sonifier 450, Branson Ultrasonic, Danbury, CT). An aliquot of the sonicated P-gp and lipid mixture was assayed for drug-stimulated ATPase activity by the addition of an equal volume of buffer containing 100 mmTris-HCl, pH 7.5, 100 mm NaCl, 20 mmMgCl2, 10 mm ATP, and 2 mmverapamil. The samples were incubated for 30 min at 37 °C, and the amount of inorganic phosphate liberated was determined (33Chifflet S. Torriglia A. Chiesa R. Tolosa S. Anal. Biochem. 1988; 168: 1-4Crossref PubMed Scopus (426) Google Scholar). For inhibition with MTS-rhodamine (Toronto Research Chemicals, Toronto, Canada), the P-gp and lipid mixture was preincubated with 1 mm MTS-rhodamine for 15 min at 22 °C. Unreacted MTS-rhodamine was removed by gel filtration (Centri.Spin 20 columns, Princeton Separations, Inc., Adelphia, NJ). The columns were pre-equilibrated with nickel column elution buffer containing 5 mg/ml sheep brain phosphatidylethanolamine and 1 mm verapamil or 1 mm rhodamine B. The verapamil- or rhodamine-stimulated ATPase activity in the flow-through fraction was measured as described above. In the protection experiments, the P-gp and lipid samples were pretreated for 10 min at 22 °C with or without 3 mmrhodamine B. This is the saturating concentration of rhodamine B for stimulation of ATPase activity. We then added 0.3 mmMTS-rhodamine, and the samples were then incubated for 10 min at 22 °C to allow MTS-rhodamine to react with the mutant. Rhodamine B (3 mm) was then added to the sample without rhodamine B so that both samples now had the same amount of rhodamine B and MTS-rhodamine. The unreacted MTS-rhodamine was removed by gel filtration (Centri.Spin 20 columns). Rhodamine B-stimulated ATPase activity was then measured (final rhodamine B concentration was 1 mm). Drug-stimulated ATPase activity is a useful measure of drug interaction with P-gp. The ATPase activity of P-gp is increased 2–20-fold when it interacts with most substrates and modulators of P-gp (21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Furthermore, there is good correlation between substrate-stimulated ATPase activity and transport activity because the turnover numbers are quite similar (34Ambudkar S.V. Cardarelli C.O. Pashinsky I. Stein W.D. J. Biol. Chem. 1997; 272: 21160-21166Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Rhodamine dyes (Fig. 1) are transported by P-gp (26Eytan G.D. Regev R. Oren G. Hurwitz C.D. Assaraf Y.G. Eur. J. Biochem. 1997; 248: 104-112Crossref PubMed Scopus (107) Google Scholar) and also stimulate the ATPase activity of Cys-less P-gp. Fig. 2 shows rhodamine B stimulation of the ATPase activity of Cys-less P-gp. The ATPase activity was stimulated 10.5-fold with 1 mm rhodamine B, with half-maximal activation occurring at a concentration of 68 μm. Inhibition of ATPase activity was observed at 3 mm rhodamine B. Such inhibition of activity at high concentrations of substrate is characteristic of P-gp (21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 35Muller M. Bakos E. Welker E. Varadi A. Germann U.A. Gottesman M.M. Morse B.S. Roninson I.B. Sarkadi B. J. Biol. Chem. 1996; 271: 1877-1883Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar).Figure 2Effect of rhodamine B and MTS-rhodamine on Cys-less P-gp ATPase activity. Histidine-tagged Cys-less P-gp was expressed in HEK 293 cells and isolated by nickel-chelate chromatography. The isolated P-gp was mixed with lipid and sonicated, and ATPase activity was determined in the presence of various concentrations of rhodamine or MTS-rhodamine.View Large Image Figure ViewerDownload (PPT) Fig. 1 (lower panel) shows the structure of a thiol-reactive analog of rhodamine, MTS-rhodamine. An ethyl linker group attaches the methanethiosulfonate group to rhodamine. Alkylthiosulfonates react selectively with cysteines in a protein under relatively mild conditions, resulting in a disulfide attachment of the R group and release of a sulfinic acid byproduct (36Kenyon G.L. Bruice T.W. Methods Enzymol. 1977; 47: 407-430Crossref PubMed Scopus (178) Google Scholar, 37Bruice T.W. Kenyon G.L. J. Protein Chem. 1982; 1: 47-58Crossref Scopus (96) Google Scholar). Alkylthiosulfonate compounds react more rapidly with cysteines than other thiol-specific compounds such as maleimides or iodoacetamides (36Kenyon G.L. Bruice T.W. Methods Enzymol. 1977; 47: 407-430Crossref PubMed Scopus (178) Google Scholar). MTS-rhodamine appeared to be useful for identifying residues in P-gp that contribute to the binding site because of its ability to stimulate the ATPase activity of Cys-less P-gp (Fig. 2). Maximal stimulation of activity (6.2-fold) was at a concentration of 1 mm with half-maximal stimulation occurring at 110 μm. The slightly reduced affinity and stimulation observed with MTS-rhodamine could be due to the presence of the methanethiosulfonate group and/or the presence of the sulfate group rather than the carboxyl group present in rhodamine B. These characteristics were similar to those obtained with rhodamine B, indicating that they may occupy similar binding sites in P-gp. We tested whether rhodamine-like compounds interact with the TM domains with a "drug rescue" assay involving a P-gp deletion mutant that lacked the nucleotide-binding domains. Many P-gp mutations (processing mutations) cause it to be misfolded and trapped in the endoplasmic reticulum as a core-glycosylated intermediate (38Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 28683-28689Abstract Full Text PDF PubMed Google Scholar,39Loo T.W. Clarke D.M. FASEB J. 1999; 13: 1724-1732Crossref PubMed Scopus (81) Google Scholar). In a drug rescue assay, the misprocessed P-gp mutant is expressed in the presence of a drug substrate (chemical chaperone) that induces the misfolded protein to fold properly. The rescued P-gp bypasses the quality control system present in the endoplasmic reticulum (40Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21839-21844Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) and is further glycosylated in the Golgi and then trafficked to the cell surface in an active form. The drug substrate diffuses into the endoplasmic reticulum, where it may act as a "scaffold" for the partially folded P-gp to adopt a native conformation (32Loo T.W. Clarke D.M. J. Biol. Chem. 1998; 273: 14671-14674Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 41Loo T.W. Clarke D.M. Biochem. Cell Biol. 1999; 77: 11-23Crossref PubMed Scopus (88) Google Scholar). The P-gp deletion mutant (TMD1+TMD2; residues 1–379 plus 681–1025) is normally expressed as an immature core-glycosylated intermediate with an apparent mass of 80 kDa. Expression of the TMD1+TMD2 mutant in the presence of a drug substrate or modulator such as cyclosporin A, vinblastine, or capsaicin, however, results in a 100-kDa protein that is detected at the cell surface (13Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 24759-24765Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Accordingly, the TMD1+TMD2 mutant was expressed in HEK 293 cells in the presence of various concentrations of rhodamine B or 10 μm cyclosporin A and subjected to SDS-PAGE. Fig. 3 shows that the 100-kDa protein was not detected in the absence of drug substrate (No drug). By contrast, the 100-kDa product was readily detected when expression was in the presence of (10 μm) cyclosporin A or rhodamine B. The relative amount of the 100-kDa protein increased with increasing concentrations (16–125 μm) of rhodamine B. Higher concentrations (250–500 μm) of rhodamine B caused cell death. These results show that the TM domains alone are sufficient for binding rhodamine B. To identify residues in the TM segments that contribute to binding of rhodamine, we tested whether MTS-rhodamine inhibited the activity of single cysteine mutants. A total of 260 histidine-tagged single cysteine mutants were constructed. These mutations covered all of the residues predicted to be within the 12 TM segments of P-gp. When expressed in HEK 293 cells in the presence of cyclosporin A, the major product was mature 170-kDa P-gp on SDS-PAGE (data not shown). Eight mutants were not tested further because of low expression ((V52C(TM1), G54C(TM1), G62C(TM1), G122C(TM2), S344C(TM6), and G989C(TM12)) or low activity ((G722C(TM7) and G763C(TM8)). Six of these mutants involved substitution of a glycine. Replacement of glycine with the larger cysteine residue may have caused structural perturbations. Similar results were observed when glycine residues in the cytoplasmic loops connecting the TM segments were mutated (42Loo T.W. Clarke D.M. J. Biol. Chem. 1994; 269: 7243-7248Abstract Full Text PDF PubMed Google Scholar). The remaining 252 histidine-tagged mutants were expressed in HEK 293 cells in the presence of cyclosporin A. The mutant proteins were isolated by nickel-chelate chromatography, mixed with lipid, and reacted with 1 mm MTS-rhodamine for 15 min at 22 °C. Unreacted MTS-rhodamine and sulfinic acid byproducts were removed by passage through a gel filtration spin column. The verapamil-stimulated ATPase activity of the MTS-rhodamine-treated sample was then determined and compared with that of a mock treated sample. Verapamil was used to measure drug-stimulated ATPase activity because it is the most potent stimulator of the ATPase activity of Cys-less P-gp (about 13-fold) (20Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and all 252 single cysteine mutants retain verapamil-stimulated ATPase activity (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Mutants that showed at least a 25% decrease or a 50% increase in verapamil-stimulated ATPase activities after treatment with MTS-rhodamine are shown in Fig. 4. In TM1 (Fig. 4), MTS-rhodamine had only modest effects because most mutants retained at least 75% of their activity. The activity of M68C, was inhibited by 44%, whereas that of mutant L65C was increased (189%). Four mutants (Y118C, V125C, V133C, and C137) in TM2 were very sensitive to MTS-rhodamine because they were inhibited 94, 68, 80, and 93%, respectively. Mutant Q132C showed weaker inhibition (32%). In TM3, the activities of two mutants (K189C and Q195C) were inhibited 76 and 78%, respectively, by MTS-rhodamine. The activity of one mutant (S222C) in TM4 was strongly inhibited (98%) by MTS-rhodamine. This mutant appears to be particularly sensitive to modification by thiol-reactive reagents because dibromobimane (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) and MTS-verapamil (23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) also affect its activity. In TM5, however, there were significant differences in the inhibition pattern with MTS-rhodamine compared with that with dibromobimane (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar) or MTS-verapamil (23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The cysteines in TM5 showed little or no inhibition with dibromobimane or MTS-verapamil. In contrast, four mutants (N296C, G300C, Y310C, and F314C) were inhibited by >70% with MTS-rhodamine. The activities of two mutants (I340C and A342C) in TM6 were strongly inhibited (87 and 94%, respectively) by MTS-rhodamine. The activity of mutant F343C, however, was increased (347%) after treatment with MTS-rhodamine. In TMs 7–10, only a few residues were strongly affected by MTS-rhodamine. The activities of mutants A729C(TM7), F759C(TM8), G774C(TM8), A841C(TM9), and G872C(TM10) were inhibited 87, 80, 79, 78, and 76%, respectively, with MTS-rhodamine. The activities of 10 mutants in TMs 11 and 12 were quite sensitive (>50% inhibition) to inhibition by MTS-rhodamine. Mutants F942C(TM11), S943C(TM11), T945C(TM11), F957C(TM11), L975C(TM12), V981C(TM12), V982C(TM12), G984C(TM12), A985C(TM12), and S993C(TM12) were inhibited 67, 86, 79, 65, 78, 83, 87, 90, 94, and 51%, respectively, with MTS-rhodamine. The highly reactive nature of the methanethiosulfonate group allows the compound to react with any accessible cysteine residue in the molecule. To identify cysteines that are within the rhodamine-binding site, we tested whether rhodamine B could protect the reactive cysteine mutants from inhibition by MTS-rhodamine. The rationale is that rhodamine B would occupy the drug-binding site and prevent reaction of MTS-rhodamine with any reactive cysteine residue in the drug-binding site. The presence of a 10-fold excess of rhodamine B (3 mm) combined with the slightly higher apparent affinity of P-gp for rhodamine B (68 μm versus 110 μm for MTS-rhodamine) should protect the mutant from inactivation if the reactive cysteine is within or close to the drug-binding site. Twenty-eight mutants, Y118C(TM2), V125C(TM2), V133(TM2), C137C(TM2), K189C(TM3), Q195C(TM3), S222C(TM4), N296C(TM5), G300C(TM5), Y310C(TM5), F314C(TM5), I340C(TM6), A342C(TM6), A729C(TM7), F759C(TM8), G774C(TM8), A841C(TM9), G872C(TM10), F942C(TM11), S943C(TM11), T945C(TM11), F957C(TM11), L975C(TM12), V981C(TM12), V982C(TM12), G984C(TM12), A985C(TM12), and S993C(TM12) showed at least 50% inhibition with MTS-rhodamine and were subjected to protection assays with rhodamine B. The mutant proteins were preincubated with or without 3 mm rhodamine B for 10 min at 22 °C and then treated with 0.3 mm MTS-rhodamine for 10 min at 22 °C. The reaction mixtures was applied to a gel filtration column to remove the MTS-rhodamine and then assayed for rhodamine-stimulated ATPase activity and compared with that of mock treated samples. Rhodamine B protected five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12) from inhibition by MTS-rhodamine (Fig.5). The greatest protection was observed in mutant V981C(TM12). This mutant was inhibited by 80% by MTS-rhodamine but retained about 84% of its activity after treatment with rhodamine B. Lower levels of protection were observed with mutants I340C, A841C, L975C, and V982C (Fig. 5). No significant protection by rhodamine B was seen in the other 23 mutants (data not shown). Rhodamine compounds such as trimethylrosamine, rhodamines I, II, and II, rhodamine B, rhodamine G, and rhodamine 123 are transported by P-gp. These rhodamine dyes and MTS-rhodamine (this study) also stimulate ATPase activity and are considered to be substrates of P-gp (26Eytan G.D. Regev R. Oren G. Hurwitz C.D. Assaraf Y.G. Eur. J. Biochem. 1997; 248: 104-112Crossref PubMed Scopus (107) Google Scholar). Two mutants, L65C and F343C, showed increased activity after treatment with MTS-rhodamine. Because mutant L65C had only 51% of the verapamil-stimulated ATPase activity relative to that of the Cys-less parent (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), treatment with MTS treatment essentially restored the activity of the mutant to that of the Cys-less P-gp. Perhaps a bulky residue is required at position 56 for full activity. Mutant F343C showed about 3.5-fold increase in activity after treatment with MTS-rhodamine. Because mutant F343C had about 60% of the activity of Cys-less P-gp, reaction with MTS-rhodamine essentially causes a 2-fold increase in activity relative to the Cys-less parent. The presence of a bulky group a position 343 appears to enhance activity. We previously showed that the bulkiness of side chains in TMs 5 and 6 can have large effects on drug-stimulated ATPase activity (29Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 21449-21452Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 43Loo T.W. Clarke D.M. Methods Enzymol. 1998; 292: 480-492Crossref PubMed Scopus (20) Google Scholar). MTS-rhodamine inhibited the ATPase activities of 28 of the 252 single cysteine mutants by at least 50%. A cysteine mutant that was sensitive to inhibition was found in all other TM segments except in TM1. Three cysteine mutants (V52C, G54C, and G62C) in TM1 were not tested because of low expression, indicating that these residues must be important for structure and/or function. Therefore, it appears that all of the TMs are important for function because at least one position in each TM segment is sensitive to mutation or inhibition by MTS-rhodamine. The activity of mutant C137(TM2) was inhibited by 93% by MTS-rhodamine. This residue is interesting in that it is one of seven endogenous cysteines (other cysteines at positions 431, 717, 956, 1074, 1125, and 1227) found in wild-type P-gp. Previous studies on the inhibition of P-gp activity have shown that only the cysteines located in the cytoplasmic Walker A nucleotide-binding regions (Cys431 and Cys1074) are inhibited by thiol-reactive compounds (17Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 44Senior A.E. Gros P. Urbatsch I.L. Arch. Biochem. Biophys. 1998; 357: 121-125Crossref PubMed Scopus (11) Google Scholar, 45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (98) Google Scholar). Inhibition of the activity of the C137 mutant was quite specific because similar inhibition of activity was not observed when the mutant was treated with the thiol-reactive compounds such as biotin-maleimide (9Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 843-848Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar),N-ethylmaleimide (17Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar), disulfiram (45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (98) Google Scholar), dibromobimane (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar), or MTS-verapamil (23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). The ability to inhibit the activity of mutant C137 with MTS-rhodamine indicates that this residue could be a target for the development of novel inhibitory reagents that covalently modify P-gp. Rhodamine B significantly protected the activities of mutants I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12) from inhibition by MTS-rhodamine (Fig. 5). This indicates that these residues must be within or close to the rhodamine drug-binding site. The results from studies involving cysteine-scanning mutagenesis and reaction with structurally diverse thiol-reactive substrates (this study and Refs. 20Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 31945-31948Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 46Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, and 47Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 36877-36880Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), from photolabeling studies (48Bruggemann E.P. Germann U.A. Gottesman M.M. Pastan I. J. Biol. Chem. 1989; 264: 15483-15488Abstract Full Text PDF PubMed Google Scholar, 49Bruggemann E.P. Currier S.J. Gottesman M.M. Pastan I. J. Biol. Chem. 1992; 267: 21020-21026Abstract Full Text PDF PubMed Google Scholar, 50Demmer A. Thole H. Kubesch P. Brandt T. Raida M. Fislage R. Tummler B. J. Biol. Chem. 1997; 272: 20913-20919Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 51Demeule M. Laplante A. Murphy G.F. Wenger R.M. Beliveau R. Biochemistry. 1998; 37: 18110-18118Crossref PubMed Scopus (46) Google Scholar, 52Ecker G.F. Csaszar E. Kopp S. Plagens B. Holzer W. Ernst W. Chiba P. Mol Pharmacol. 2002; 61: 637-648Crossref PubMed Scopus (54) Google Scholar), and from mutational studies (27Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 3143-3149Abstract Full Text PDF PubMed Google Scholar, 53Gros P. Dhir R. Croop J. Talbot F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7289-7293Crossref PubMed Scopus (199) Google Scholar, 54Loo T.W. Clarke D.M. J. Biol. Chem. 1993; 268: 19965-19972Abstract Full Text PDF PubMed Google Scholar, 55Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (127) Google Scholar) point to the presence of a "common" drug-binding site. A model of such a common drug-binding site in P-gp is shown in Fig.6. The arrangement of the TM segments (Fig. 6) is also based on the results of disulfide cross-linking studies that show TMs 4–6 to be close to TMs 10–12 during the resting phase (19Loo T.W. Clarke D.M. J. Biol. Chem. 1996; 271: 27482-27487Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 46Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 31800-31805Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 56Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 20986-20989Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 57Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The MTS-rhodamine results (this study) show that TM9 must also be close to or within the binding site for rhodamine-type compounds and would be next to TM6 (Fig. 6). Song and Melera (58Song J. Melera P.W. Mol. Pharmacol. 2001; 60: 254-261Crossref PubMed Scopus (14) Google Scholar) showed in hamster P-gp that there is close interaction between TMs 6 and 9 during drug binding. Mutations in TM9 (I837L and N839I) or in TM6 (G388A and A339P) resulted in similar drug resistance profiles with four structurally different drugs (increased resistance to vincristine or actinomycin D but decreased resistance to colchicine or daunorubicin relative to wild-type P-gp). It is interesting that the equivalent residues in human P-gp (Ile840 and Asn842 in TM9 and Gly341 and Ala342in TM6) are adjacent to the cysteines (A841C(TM9) and I340C(TM6)) the activities of which are protected by rhodamine B from inhibition by MTS-rhodamine (Fig. 5). Some studies have suggested that P-gp has four different drug interaction sites that substrates occupy different sites during transport or that each substrate has a distinct binding site (25Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (287) Google Scholar, 59Dey S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10594-10599Crossref PubMed Scopus (367) Google Scholar,60Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (141) Google Scholar). These results are not incompatible with the model presented in Fig. 6. Although the model shows the presence of a common drug-binding site, it can be used to accommodate results that predict multiple drug-binding sites. We had proposed that substrates can create their own binding sites ("substrate-induced fit" hypothesis) by using a combination of residues from different TMs to form a particular drug-binding site (21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Forming the drug-binding site this way would depend on the TMs being quite mobile. The binding of a particular substrate would result in a more "rigid" P-gp. Evidence that the TM segments are quite mobile comes from disulfide cross-linking studies (57Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 5253-5256Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). When thermal motion is reduced (4 °C), only residues at the cytoplasmic side in TMs 4 and 5 were cross-linked with that in TM12. At higher temperatures (21 and 37 °C), these residues as well as residues at the cytoplasmic side in TM 6 were cross-linked with those in TMs 10 and 11. The substrate-induced fit hypothesis would also explain why P-gp binds substrates with different affinities. When a particular substrate induces a particular fit in P-gp, then the combined effects of contributing residues from each TM would determine the affinity. Also, some substrates may share the same residue(s) during binding. For example, the activities of mutants L339C(TM6) and A342(TM6) are protected from inhibition by dibromobimane (21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar) and MTS-verapamil (23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) but not by MTS-rhodamine, whereas that of mutant V982C(TM12) is protected from inhibition by dibromobimane and MTS-rhodamine but not by MTS-verapamil. An interesting feature of the cysteine mutants that are inhibited with thiol-reactive compounds is that most are located near the middle of each TM segment (Fig. 7). When the drug-binding site is opened as a fan, it appears that the reactive residues form a ring. The substrates may recognize various combinations of residues in this ring during binding. Future work with other structurally diverse thiol substrates will determine whether other residues contribute to this "ring of recognition." We thank Dr. Randal Kaufman (Boston, MA) for pMT21. We thank Claire Bartlett for assistance with tissue culture.