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W2085202207 abstract "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 1The abbreviations used are: P-gp, P-glycoprotein(s); MTS, methanethiosulfonate; TM, transmembrane; HEK, human embryonic kidneyis 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 (51) Google Scholar and 2Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1330) 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 (457) Google Scholar, 4Krishna R. Mayer L.D. Eur. J. Pharmcol. Sci. 2000; 11: 265-283Crossref PubMed Scopus (964) 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 (2545) 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 (1583) 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 (3346) 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 (1709) 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 (261) Google Scholar, 10Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) 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 (61) 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 (133) 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 (270) Google Scholar, 15Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (137) 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 (174) Google Scholar) and likely function in an alternating mechanism (18Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) 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 (133) 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 (261) 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 (88) 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 (141) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) 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 (183) 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 (145) Google Scholar, 25Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (280) 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 (100) Google Scholar). In this study, we used a thiol-reactive analog of rhodamine, MTS-rhodamine, to identify residues involved in its binding.DISCUSSIONRhodamine 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 (100) 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 (131) 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 (189) 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 (174) Google Scholar, 44Senior A.E. Gros P. Urbatsch I.L. Arch. Biochem. Biophys. 1998; 357: 121-125Crossref PubMed Scopus (10) Google Scholar, 45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) 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 (261) Google Scholar),N-ethylmaleimide (17Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), disulfiram (45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) Google Scholar), dibromobimane (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) 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 (183) 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 (141) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (183) 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 (180) 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 (42) 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 (52) 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 (189) 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 (125) 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 (88) 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 (107) 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 (280) 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 (355) Google Scholar,60Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) 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 (118) 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 (118) 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 (183) 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.”Figure 7Location of the cysteine residues that interact with MTS-rhodamine, MTS-verapamil, or dibromobimane.The numbered cylinders represent TM segments 4–6 and 8–12 of P-gp. The TMs forming the drug-binding site are arranged as a fan. The residues that are protected from inhibition by MTS-rhodamine (this study) are in red, those protected by MTS-verapamil (23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar) are in black, and those protected by dibromobimane (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar) are inyellow.View Large Image Figure ViewerDownload (PPT) 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 1The abbreviations used are: P-gp, P-glycoprotein(s); MTS, methanethiosulfonate; TM, transmembrane; HEK, human embryonic kidneyis 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 (51) Google Scholar and 2Borst P. Elferink R.O. Annu. Rev. Biochem. 2002; 71: 537-592Crossref PubMed Scopus (1330) 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 (457) Google Scholar, 4Krishna R. Mayer L.D. Eur. J. Pharmcol. Sci. 2000; 11: 265-283Crossref PubMed Scopus (964) 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 (2545) 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 (1583) 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 (3346) 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 (1709) 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 (261) Google Scholar, 10Kast C. Canfield V. Levenson R. Gros P. J. Biol. Chem. 1996; 271: 9240-9248Abstract Full Text Full Text PDF PubMed Scopus (128) 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 (61) 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 (133) 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 (270) Google Scholar, 15Doige C.A., Yu, X. Sharom F.J. Biochim. Biophys. Acta. 1992; 1109: 149-160Crossref PubMed Scopus (137) 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 (174) Google Scholar) and likely function in an alternating mechanism (18Senior A.E. Gadsby D.C. Semin. Cancer Biol. 1997; 8: 143-150Crossref PubMed Scopus (129) 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 (133) 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 (261) 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 (88) 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 (141) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) 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 (183) 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 (145) Google Scholar, 25Shapiro A.B. Fox K. Lam P. Ling V. Eur. J. Biochem. 1999; 259: 841-850Crossref PubMed Scopus (280) 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 (100) Google Scholar). In this study, we used a thiol-reactive analog of rhodamine, MTS-rhodamine, to identify residues involved in its binding. DISCUSSIONRhodamine 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 (100) 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 (131) 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 (189) 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 (174) Google Scholar, 44Senior A.E. Gros P. Urbatsch I.L. Arch. Biochem. Biophys. 1998; 357: 121-125Crossref PubMed Scopus (10) Google Scholar, 45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) 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 (261) Google Scholar),N-ethylmaleimide (17Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), disulfiram (45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) Google Scholar), dibromobimane (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) 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 (183) 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 (141) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (183) 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 (180) 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 (42) 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 (52) 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 (189) 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 (125) 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 (88) 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 (107) 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 (280) 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 (355) Google Scholar,60Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) 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 (118) 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 (118) 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 (183) 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.” 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 (100) 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 (131) 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 (189) 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 (174) Google Scholar, 44Senior A.E. Gros P. Urbatsch I.L. Arch. Biochem. Biophys. 1998; 357: 121-125Crossref PubMed Scopus (10) Google Scholar, 45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) 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 (261) Google Scholar),N-ethylmaleimide (17Loo T.W. Clarke D.M. J. Biol. Chem. 1995; 270: 22957-22961Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), disulfiram (45Loo T.W. Clarke D.M. J. Natl. Cancer Inst. 2000; 92: 898-902Crossref PubMed Scopus (94) Google Scholar), dibromobimane (22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) 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 (183) 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 (141) Google Scholar, 21Loo T.W. Clarke D.M. J. Biol. Chem. 1999; 274: 35388-35392Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 22Loo T.W. Clarke D.M. J. Biol. Chem. 2000; 275: 39272-39278Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 23Loo T.W. Clarke D.M. J. Biol. Chem. 2001; 276: 14972-14979Abstract Full Text Full Text PDF PubMed Scopus (183) 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 (180) 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 (42) 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 (52) 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 (189) 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 (125) 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 (88) 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 (107) 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 (280) 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 (355) Google Scholar,60Pascaud C. Garrigos M. Orlowski S. Biochem. J. 1998; 333: 351-358Crossref PubMed Scopus (139) 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 (118) 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 (118) 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 (183) 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." @default.
W2085202207 created "2016-06-24" @default.
W2085202207 creator A5042609718 @default.
W2085202207 creator A5060459035 @default.
W2085202207 date "2002-11-01" @default.
W2085202207 modified "2023-10-14" @default.
W2085202207 title "Location of the Rhodamine-binding Site in the Human Multidrug Resistance P-glycoprotein" @default.
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