Molecular dissection of the human multidrug resistance P-glycoprotein

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REVIEW / SYNTHÈSE

Merck Frosst Award Lecture 1998 / La conference Merck Frosst 1998

Molecular dissection of the human multidrug resistance P-glycoprotein Tip W. Loo and David M. Clarke

Abstract: The human multidrug resistance P-glycoprotein is an ATP-dependent drug pump that extrudes a broad range of cytotoxic agents from the cell. Its physiological role may be to protect the body from endogenous and exogenous cytotoxic agents. The protein has clinical importance because it contributes to the phenomenon of multidrug resistance during chemotherapy. In this review, we discuss some of the results obtained by using molecular biology and protein chemistry techniques for studying this important and intriguing protein. Key words: P-glycoprotein, ABC transporters, drug transport, dibromobimane, mutagenesis, disulfide crosslinking, metal-chelate chromatography, ATPase activity. Résumé : La glycoprotéine P humaine de résistance multidrogue est une pompe ATP-dépendante qui expulse un large éventail de substances cytotoxiques de la cellule. Son rôle physiologique pourrait être de protéger l’organisme contre des substances cytotoxiques endogènes et exogènes. La protéine a une importance clinique car elle contribue au phénomène de résistance à plusieurs médicaments utilisés en chimiothérapie. Dans cette revue, nous discutons de résultats obtenus en appliquant des méthodes de biologie moléculaire et de chimie des protéines à l’étude de cette protéine importante et fascinante. Mots clés : glycoprotéine P, transporteurs ABC, transport de médicaments, dibromobimane, mutagenèse, liaison disulfure, chromatographie par chélation de métaux, activité ATPase. [Traduit par la Rédaction]

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Introduction The human multidrug resistance P-glycoprotein (P-gp) is clinically important because it contributes to the phenomenon of multidrug resistance during cancer and AIDS chemotherapy. P-gp is also interesting for biochemists because it uses the energy from ATP to transport a broad range of structurally unrelated cytotoxic compounds out of the cell. It also serves as a model protein for understanding the structures and mechanisms of other members of the ABC Received December 18, 1998. Accepted January 21, 1999. Abbreviations: dBBn, dibromobimane; NEM, N-ethylmaleimide; P-gp, P-glycoprotein; TM, transmembrane. T.W. Loo and D.M. Clarke.1 MRC Group in Membrane Biology, Department of Medicine and Department of Biochemistry, University of Toronto, ON M5S 1A8, Canada. 1

Author to whom all correspondence should be sent at the following address: Department of Medicine, University of Toronto, Room 7342, Medical Sciences Building, 1 King’s College Circle, Toronto, ON M5S 1A8, Canada (e-mail: [email protected]).

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(ATP-binding cassette) superfamily of proteins. The ABC family of proteins is the largest class of transport proteins. This minireview summarizes the clinical and physiological relevance of the protein and describes results from our laboratory that contribute to our understanding of the structure, mechanism and biosynthesis of this intriguing protein. More comprehensive reviews on P-gp have been published elsewhere (Chan et al. 1996; Gottesman et al. 1996; Sharom 1997). P-gp and multidrug resistance in cancer Chemotherapy is a major form of treatment for Hodgkin’s disease, testicular cancer, and many childhood cancers. Combination chemotherapy with cytotoxic agents that have different intracellular targets has been particularly effective. Unfortunately, the majority of cancers are either resistant to chemotherapy (renal, colon, etc.) or acquire resistance (such as in lymphoma, lung and breast cancers) during treatment (Lehnert 1996). This intrinsic or acquired ability of tumor cells to be resistant to multiple chemotherapeutic agents (multidrug resistance) is a major obstacle to successful cancer chemotherapy. P-gp, the product of the human MDR1 gene, was the first © 1999 NRC Canada


12 Fig. 1. P-gp and multidrug resistance. (A) Cytotoxic drugs such as vinblastine or colchicine rapidly diffuse into the lipid bilayer of a cell. The drugs enter the cytoplasm, bind to an intracellular target (microtubules in the case of colchicine or vinblastine) and cause cell death. Cells expressing P-gp, however, are protected because the protein extracts the drugs from the lipid bilayer and pumps them out in an ATP-dependent manner. (B) Schematic model of P-gp. It consists of four domains; two cytoplasmic ATP-binding domains, and two hydrophobic domains each containing six predicted TM segments. The protein is glycosylated at three sites in the first extracellular loop between TM1 and TM2.

important protein implicated in the phenomenon of multidrug resistance (see Fig. 1) (Juliano and Ling 1976). Mammalian cell lines overexpressing this protein were initially selected for resistance to a single cytotoxic drug, but were subsequently found to display cross-resistance to other structurally unrelated drugs (Biedler and Riehm 1970; Riordan and Ling 1985; Shen et al. 1986). Following this discovery, other proteins have been identified that may also confer multidrug resistance. These include the ABC multidrug resistance proteins, MRP1 and MRP2; as well as topoisomerase II and the lung resistance protein (Lehnert 1996). The clinical relevance of these other proteins in cancer multidrug resistance, however, is still under investigation. There is considerable evidence that many different tumours express P-gp (reviewed in Chan et al. 1996; Fisher et al. 1996; Goldstein 1996; Lehnert 1996). In renal (Fojo et al. 1987) or colon cancers (Weinstein et al. 1991), P-gp is constitutively expressed in relatively high amounts. In other cancers (lung, myeloma, breast, ovary, lymphoma, acute myeloid leukemia), the tumour cells frequently express P-gp only after exposure to chemotherapeutic drugs or during relapse (Chan et al. 1995). Several studies have shown P-gp expression to be predictive of poor response to chemotherapy and decreased overall survival (Chan et al. 1995; Fisher et al. 1996; Leighton and Goldstein 1995; Marie 1995). An interesting observation is that expression of P-gp is

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modulated when the tumour cells are stressed (e.g., heat, heavy metals) and by the recent observation that there is p53-dependent regulation of MDR1 expression (Thottassery et al. 1997). Inhibition of wild-type p53 resulted in increased expression of P-gp. Since deletion or mutation of the p53 (tumour suppressor gene) gene is also a frequent finding in human malignancies (Hollstein et al. 1991; Levine et al. 1991), it would be expected that tumours expressing the MDR1 gene would be highly resistant to cytotoxic drugs. The anticancer drugs most effectively extruded from tumor cells by P-gp are of natural origin. Examples include the anthracyclines (e.g., doxorubicin, daunorubicin, and mitoxantrone), vinca alkaloids (e.g., vincristine and vinblastine), epipodophyllotoxins (e.g., etoposide and teniposide), taxanes (e.g., taxol and taxotere), and actinomycin D (Sarkadi and Muller 1997). In cancer chemotherapy, the goal is to inhibit P-gp-mediated extrusion of these anticancer drugs to increase the effectiveness of treatment. Considerable efforts have been expended on finding chemosensitizers that will inhibit the function of P-gp and thereby reverse multidrug resistance. Initially, it was found that the calcium channel blocker verapamil greatly increased the sensitivity of multidrug resistant leukemia cells to cytotoxic agents in vivo and in vitro (Tsuruo et al. 1981, 1983). The most widely used compounds to inhibit P-gp function in initial clinical trials were verapamil and cyclosporin A. Unfortunately, the concentrations of these compounds required to inhibit P-gp lead to significant side effects. High verapamil levels cause cardiovascular toxicity and cyclosporin A enhances myeloid, renal, neural and hepatic toxicity. The use of short high-dose cyclosporin infusions at the same time of administration of vincristine, however, has been successful in the treatment of retinoblastoma (Chan et al. 1996). The side effects of verapamil and cyclosporin may be lessened with the development of second generation analogs such as R-verapamil and PSC 833. R-verapamil has less calcium channel inhibitory effect than the S-enantiomer of verapamil but with a similar ability to inhibit P-gp (Gruber et al. 1988). PSC 833 is a cyclosporin analog with virtually no immunosuppressive effect (Boesch et al. 1991). A desirable goal is to develop a more specific and effective modulator. The major drawback in modulator development has been the lack of structural information regarding specific binding sites or intramolecular arrangements during drug transport by P-gp. HIV-1 protease inhibitors and P-gp Shutting down P-gp during chemotherapy would also benefit treatment of other diseases, such as AIDS. Protease inhibitors are potent agents that are in vogue in the therapy of HIV-1 infection. Oral absorption and penetration of these inhibitors into the brain, however, are poor. It is now apparent that poor oral absorption and brain penetration of these FDA-approved protease inhibitors are due to the presence of P-gp (Kim et al. 1998; Lee and Gottesman 1998; Lee et al. 1998). Kim et al. (1998) recently showed that the HIV-1 protease inhibitors indinavir, nelfinavir, and saquinavir are all substrates of P-gp. The high levels of P-gp expressed in the blood-brain barrier may decrease the efficacy of these drug in the treatment of central nervous system infections in AIDS patients. Therefore, strategies to shut down P-gp © 1999 NRC Canada



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Fig. 2. Ball model of human P-gp.

could potentially benefit other diseases that involve a chemotherapy regimen. Physiological role of P-gp The major physiological role of P-gp is probably to protect the body from hydrophobic toxic agents. P-gp is found on the apical (or lumenal) surface of polarized epithelial cells of the small and large intestine, in the biliary canalicular membranes of hepatocytes, and on the apical surface of epithelial cells of the proximal cells of kidney (Baas and Borst 1988; Croop et al. 1989; Schinkel 1997; Thiebaut et al. 1987; Trezise et al. 1992; Van der Bliek et al. 1987). P-gp is also found in the blood–brain and blood–testis barrier (Cordon-Cardo et al. 1989) and probably serves to protect vital organs from naturally occurring xenotoxins ingested in food. Studies on P-gp knock-out mice show that the protein is not essential (Schinkel et al. 1994; Schuetz et al. 1996). The mice are viable and fertile and do not display obvious phenotypic abnormalities other than hypersensitivity to drugs. Therefore P-gp can provide protection by exclusion of these toxins in the intestine or blood–tissue barriers or by active excretion in the intestine, liver, or kidney. Evidence for such a protective role for P-gp in eliminating exogenous compounds was the recent demonstration that compounds found in the urine of rats and humans are substrates of P-gp. One such class of compounds excreted in human urine are the nonylphenol ethoxylates (Charuk et al. 1994, 1998;

Charuk and Reithmeier 1992). Perhaps the most important role of P-gp is to protect us from the numerous toxins present in our diet. Because of such a striking similarity in the secondary structure of P-gp and CFTR, it was thought that P-gp may also function as a chloride channel. Indeed, it was reported that P-gp was a volume-regulated chloride channel (Valverde et al. 1992), and that the chloride channel activity of P-gp could be separated from its drug transport activity (Gill et al. 1992). Several subsequent studies, however, have questioned this finding (Dong et al. 1994; Morin et al. 1995; Rasola et al. 1994; Tominaga et al. 1995) but some still believe that P-gp is a regulator of chloride channel activity (Valverde et al. 1996). Roepe (1995) have postulated an “altered partitioning model” for P-gp where altered sequestration of cytotoxic compounds by the cell occurs indirectly as a result of perturbation of the plasma membrane electrochemical potential by P-gp. This has been reviewed quite comprehensively (Roepe et al. 1996).

Structure–function analysis of the multidrug resistance P-gp Cloning of P-gp In parallel work, three groups cloned and sequenced the gene responsible for multidrug resistance by P-gp from ham© 1999 NRC Canada



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ster (Gerlach et al. 1986), mouse (Gros et al. 1986b), and human (Chen et al. 1986) cell lines. The 1280 amino acids of human MDR1 (Chen et al. 1986) are organized in two tandem repeats of 610 amino acids, joined by a linker region of 60 amino acids (Fig. 2). Each repeat consists of an NH2-terminal hydrophobic domain containing six potential TM sequences followed by a hydrophilic domain containing a nucleotide-binding site. The organization of the domains is characteristic of members of the ABC (ATP-binding cassette) superfamily of transporters (Higgins 1995). It was apparent that P-gp is a member of a multigene pgp or mdr family (Riordan and Ling 1985). The mdr/pgp gene family is composed of three members in rodents (hamster and mouse) and two members in humans (Ng et al. 1989). The close linkage of mdr/pgp genes on the chromosome implied that the gene family arose from one or more gene duplications (Bell et al. 1987; de Bruijn et al. 1986; Van der Bliek et al. 1988). A study on the intron–exon structure of human MDR1, however, suggests that P-gp arose by fusion of genes (Chen et al. 1990). Although all P-gp isoforms to date reveal the same overall structure, evidence suggests that they may have distinct functions. Expression vectors have been used to show that mdr1 of mouse or human is capable of conferring multidrug resistance when transfected and overexpressed in otherwise drug-sensitive, cultured cells (Gros et al. 1986a; Ueda et al. 1987). There is evidence that suggests that mouse mdr3 is associated with multidrug resistance, whereas human MDR3 and mouse mdr2 may not be associated with multiple drug resistance but act as phosphatidyl translocases (Ruetz and Gros 1994; Smit et al. 1993; Smith et al. 1994). The results indicate that mouse mdr1 and mdr3 have overlapping, but distinct, specificities (Raymond et al. 1990). The drug specificity of human P-gp differs from both mouse enzymes (Tang-Wai et al. 1995). Modification of the P-gp cDNA Understanding the structure of P-gp is central to our understanding of its mechanism. It is often quite difficult to obtain structural information on most polytopic membrane proteins because of technical difficulties in crystallizing membrane proteins. Our approach to obtaining structural and functional information about P-gp was to combine molecular biology with classical protein chemistry. This required several modifications of the MDR1 cDNA. The first modification was the construction of a functional epitope-tagged human P-gp. In initial studies, the mutants of human MDR1 were characterized by making stable cell lines in mouse NIH 3T3 cells. A difficulty in selecting for stable cell lines expressing the mutant P-gp was in distinguishing between clones expressing human MDR1 from those which could arise through induction of endogenous mouse mdr1 or mdr3. Accordingly, the human MDR1 cDNA (cloned from a human kidney cDNA library) (Loo and Clarke 1993b) was modified to encode for the epitope for monoclonal antibody A52 (derived from rabbit SERCA1 Ca2+-ATPase) at the COOH-terminal of the expressed protein. This modification did not alter its expression or drug transport activity (Loo and Clarke 1993b). This also allowed detection of very low levels of P-gp-A52 in Western Blots because the cells nor-


mally do not express SERCA1. In addition, the amount of P-gp-A52 expressed in whole cells could be quantitated by an ELISA assay by using purified SERCA1 as a standard. The SERCA1 Ca-ATPase is readily purified in large amounts from rabbit muscle. A second modification of the MDR1 cDNA was the addition of a polyhistidine tag at the COOH-terminal end of P-gp, and the development of a rapid expression, purification, and assay protocol for human P-gp (Loo and Clarke 1995d). This involved transient expression of the histidine-tagged P-gp in HEK 293 cells followed by purification by nickel-chelate chromatography and measurement of drug-stimulated ATPase activity. Expression, purification, and assay of ATPase activity can now be completed in 2–3 days, while previous methods using stable cell lines or expression in insect cells often took months. P-gp was the first ABC transporter and, to our knowledge, the first eukaryotic transporter that could be purified in an active state using this approach. The third modification was the construction of an active Cys-less P-gp (Loo and Clarke 1995b). Mutation of each of the seven endogenous cysteines to alanines yielded a mutant that was about 70% as efficient as wild-type protein in conferring resistance to various cytotoxic substrates. It is synthesized less efficiently than wild-type enzyme but this problem can be corrected by carrying out synthesis in the presence of non-toxic substrates such as cyclosporin A (Loo and Clarke 1997a). This was a very fortunate development because efforts to construct Cys-less versions of other eukaryotic transporters have usually failed because of problems with expression or protein stability. The Cys-less P-gp has been an invaluable tool in mapping the topology, studying the contribution of the two nucleotide-binding sites, determining packing of the transmembrane (TM) segments and in mapping the drug binding sites as described below. Topology of the TM domains of P-gp The TM domains appear to contain the drug-binding sites and likely form the translocation pathway through the membrane (Homolya et al. 1993; Raviv et al. 1990). The results from labeling studies with photolabeled analogs of drug substrates (Ambudkar et al. 1997; Bruggemann et al. 1989, 1992; Greenberger 1993; Greenberger et al. 1990; Morris et al. 1994), suggested that the labeled sites are closely associated with TM6 and TM12. These photolabeling studies suggest that there may be two distinct drug-labeling sites or that the two labeled segments are part of a single drug-labeling site. Therefore, knowledge about the topology of the TM segments is crucial for understanding how P-gp functions. To study the topology of P-gp, a Cys-less mutant of P-gp was constructed. Cysteine residues were re-introduced into the Cys-less P-gp containing an A52 epitope tag to create a series of single-Cys mutants that contained one cysteine in a predicted extracellular or cytoplasmic loop. The mutants were transiently expressed in HEK 293 cells and treated with membrane-permeant (biotin-maleimide) and -impermeant (stilbenedisulfonate maleimide) thiol-specific reagents. The rationale was that treatment with biotin maleimide would biotinylate any Cys residue. Biotinylation was monitored by immunoprecipitating © 1999 NRC Canada



Loo and Clarke

the labeled protein with A52 monoclonal antibody followed by blot analysis with streptavidin conjugated to a reporter molecule. Pre-treatment of the cells with membrane-impermeant stilbenedisulfonate maleimide was used to identify extracellular cysteines because they were unreactive when the cells were subsequently treated with biotin maleimide. The topology obtained was consistent with the predicted model of P-gp, which predicts six TM segments in each of the two homologous halves of the enzyme (Loo and Clarke 1995b). Subsequent mutational analyses (Loo and Clarke 1996c) and cross-linking experiments (Loo and Clarke 1996a) also suggest that each half of P-gp is symmetrically arranged in the membrane. Epitope insertion studies on the full-length protein have also confirmed the predicted topology (Kast et al. 1995, 1996). This topology, however, still remains controversial since studies using truncated molecules showed that some putative cytosolic or TM segments are located extracellularly (Skach et al. 1993; Zhang et al. 1996). It was not clear, however, whether these truncated molecules were functional. A resonance energy transfer study indicates that the nucleotide-binding domains are close (3.1–3.5 nm) to the membrane surface (Liu and Sharom 1998). Recently, a very low resolution structure (25 D; 1 D = 0.1 nm) for P-gp was described that suggested the TM segments surrounded a large aqueous pore (Rosenberg et al. 1997). The hydrophilic domains of P-gp The hydrophilic domains of P-gp containing the consensus nucleotide-binding folds bind ATP (Azzaria et al. 1989). It has been demonstrated that P-gp possesses high levels of drug-stimulatable ATPase activity (al-Shawi and Senior 1993; Ambudkar et al. 1992; Sarkadi et al. 1992; Shapiro and Ling 1994; Sharom et al. 1993). Al-Shawi et al. (1994) showed that the ATPase activity of P-gp is inhibited by N-ethylmaleimide (NEM). Maximal inhibition occurred with labeling at two sites, with equal distribution of the label between the NH2- and COOH-terminal halves of the molecule. ATP prevented inhibition by NEM. Therefore, it was predicted that the critical cysteines were located in the homology A nucleotide-binding consensus sequences (GNSGCGKS and GSSGCGKS, respectively) in the two nucleotide binding domains of Chinese hamster P-gp. To test the contribution of either nucleotide-binding domain to P-gp function, the Cys-less mutant was mutated to reintroduce a single cysteine back into each nucleotide-binding consensus sequence. The sensitivity of the ATPase activity of each mutant after covalent modification by NEM was then tested. It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys431 and Cys1074, respectively) with NEM inhibited drug-stimulated ATPase activity of P-gp. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide-binding domains are essential and that they probably operate in a cooperative manner. The need for both ATP-binding sites to be functional was also suggested by studies in which mutations introduced in either nucleotide-binding fold inactivated P-gp (Azzaria et al. 1989; Loo and Clarke 1995d).

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Mutational analysis to define amino acids important for drug transport Reconstitution studies with purified P-gp have shown that transport of hydrophobic drug substrates against a concentration gradient is coupled to ATP hydrolysis (Sharom et al. 1993). Also, evidence from photoaffinity labeling studies with drug analogues and from sequencing of P-gp from cells with altered drug resistance profiles indicate that the predicted TM domains play a critical role in recognition and transport of substrates. Therefore, one goal was to identify amino acids critical for binding of drug substrates and for transport of drugs out of the cell. Initially, the mutations were introduced into the MDR1 cDNA and then transfected into NIH 3T3 cells. The cells were then assayed for their ability to confer resistance to various cytotoxic drugs. Since there was little evidence to suggest which residues in the TM domains might be good candidates for drug binding, we used a systematic approach and tested the functional consequences of mutating each of the prolines (Loo and Clarke 1993b), phenylalanines (Loo and Clarke 1993a), or glycines (Loo and Clarke 1994a) located in TM regions. The potential role of the proline residues in the TM segments was investigated because of their unique structural and functional properties. The structural destabilization induced by prolines located in the middle of alpha-helices and the possibility of cis-trans isomerization of peptide bonds between prolines and their proceeding residues make prolines important for specific functions related to conformational changes in proteins (Brandl and Deber 1986). The putative TM segments of many membrane transport proteins contain a significantly higher number of proline residues than do the TM segments of nontransporting membrane proteins, suggesting some functional role of membrane-buried prolines in the transport reaction (Brandl and Deber 1986). In initial studies (Loo and Clarke 1993b), 13 proline residues in MDR1 were mutated individually (residues 32, 66, 233, 350, 373, 693, 694, 709, 726, 745, 807, 866, and 996) (see Fig. 2). Five residues are located in putative TM helices (TM1, TM4, TM6, TM7, and TM10); one is located in the extracellular loop connecting segments TM7 and TM8 and seven are located within intracellular loops. Each of the prolines was separately replaced with alanine since it is a small neutral amino acid. Drug transport characteristics were assessed by their ability to confer drug resistance after transfection into drug-sensitive NIH 3T3 cells. A dramatic shift in the drug resistance profile was observed for mutant P223A. Cells expressing the mutant were preferentially resistant to vinblastine with almost negligible resistance to colchicine. A similar, but less dramatic, shift in the drug-resistance profile was observed in cells expressing mutant P866A. The cells were preferentially resistant to vinblastine when compared to cells containing parental MDR1, but were relatively less resistant to colchicine, adriamycin, and actinomycin D. Therefore, Pro223 and Pro866 appear to play critical roles in the transport of colchicine by P-gp. No drug-resistant colonies were obtained when cells were transfected with mutant P709A. Immunoblots of cells ex© 1999 NRC Canada



16 Fig. 3. Structure of dibromobimane.

pressing this mutant revealed that the majority of the expressed protein product corresponded in molecular weight to an incompletely glycosylated form of the protein. Therefore, a mutation to Pro709, which is predicted to lie at or close to the intracellular end of TM7, affected the structural integrity of the protein. Mutations to the other 10 prolines yielded protein products that conferred drug resistance to transfected cells. Drug-resistant clones exhibited similar resistance profiles to cells expressing wild-type human MDR1. Each of the 31 phenylalanine residues in predicted TM regions was then systematically mutated to alanine (Loo and Clarke 1993a). Most of the mutants resembled wild-type enzyme except for F335A in TM6 and F978A in TM12. Both of these mutants exhibited drastically altered drug resistance profiles. It was interesting that these residues are found in identical positions when the homologous halves of the protein are aligned. The glycine residues in the cytoplasmic loop regions were also analyzed (Loo and Clarke 1994a). Glycine residues have the potential to play important roles in the transport reaction. The small side group of glycine makes this amino acid of unique importance in the mediation of inter-domain and protein–ligand contacts as well as in conferring the flexibility to the peptide backbone that is required for conformational changes. The lack of a side-chain on glycine permits a relatively wide range of flexibility. This property also allows bends to occur at glycine positions and may provide a hinge to impart flexibility to the polypeptide chain. Another reason for choosing glycine was because a spontaneous Gly to Val mutation, G185V, had been shown to confer an altered drug resistance profile when compared to wild-type enzyme (Choi et al. 1988). Therefore, the effects of introducing Gly to Val mutations at 20 other positions were tested (Loo and Clarke 1994a). Four of the mutations inhibited maturation while five other mutants exhibited large changes in the drug resistance profile conferred by P-gp. These results showed that the glycines in the cytoplasmic loops play important roles in structure and function of P-gp. Analysis of the mutations to prolines, phenylalanines, and glycines was informative, but time consuming. This was because of the need to establish stable cell lines for each mutant. In addition, one could never be certain that endogenous drug transporters were expressed when the cells were subjected to drug selection or that other cellular factors were influencing the drug resistance profiles. These potential problems required the development of a more reliable system for assessing the mutants. Accordingly, a rapid expression and purification method using P-gp containing 10

Biochem. Cell Biol. Vol. 77, 1999 Fig. 4. Distribution of dibromobimane-sensitive amino acids in TM6 and TM12. The results of cysteine-scanning mutagenesis and treatment of the Cys mutants with dBBn are shown. The black bars show the percentage of verapamil-stimulated ATPase activity remaining after treatment with dBBn. The TM segments are projected as helical wheels (3.6 amino acids per turn). The helices are oriented to reflect their predicted structure in a cell membrane; they start on the cytoplasmic side of the membrane and project into the membrane. Residues with an asterisk gave unstable proteins when the residue was mutated to Cys. Subsequent disulfide crosslinking experiments showed that the two mutation-sensitive faces of TM6 and TM12 are close to each other (Loo and Clarke 1997b).

tandem histidine residues at the COOH end of the molecule were developed (Loo and Clarke 1995d). The mutants could now be isolated by nickel chelate chromatography and assayed for drug-stimulated ATPase activity. These types of © 1999 NRC Canada



Loo and Clarke Fig. 5. Proposed model for the drug rescue of P-gp processing mutants. P-gp is first synthesized in the endoplasmic reticulum as a core-glycosylated intermediate. (A) The carbohydrates are modified in the Golgi and the protein is the delivered to the cell surface (Normal). (B) Processing mutants (Defective) are

17 recognized as defective and the core-glycosylated intermediate is rapidly degraded. (C) Synthesis of the processing mutant in the presence of drug substrates (Rescue), however, induces proper folding so that the mutant matures into a functional enzyme and is trafficked to the plasma membrane.

results are valid since it was recently shown that the turnover numbers for drug transport and for drug-stimulated ATP hydrolysis are comparable (Ambudkar et al. 1997). Therefore purification of histidine-tagged P-gp, together with alanine-scanning mutagenesis, could be used to examine the importance of most of the residues located within the predicted TM segments. There may be potential problems, however, in using alanine-scanning mutagenesis to study drug-protein interactions. One problem is that drug-protein interactions likely involve a large number of residues so that a single change may not have a measurable effect. If a change in substrate specificity is observed, it is then difficult to judge whether the change is due to local or global structural changes. Another problem is the difficulty in photolabeling P-gp with radioactive analogs of drug substrates. Usually, the concentration of radioactive analog required to achieve stoichiometric labeling of P-gp makes it economically unfeasible. To overcome these problems, a thiol-reactive compound, dibromobimane (dBBn), was identified that was a potent stimulator of the ATPase activity of Cys-less P-gp (Fig. 3). Cysteine-scanning mutagenesis was combined with dBBn modification of the mutant P-gp for studying the contribution of TM6 and TM12 residues towards coupling of drug binding and ATPase activity (Loo and Clarke 1997c). The rationale was that a thiol-reactive substrate would enter the drug-binding site of P-gp, covalently bind to a nearby cysteine residue, and inhibit drug-stimulated ATPase activity. Dibromobimane is a particularly useful compound because it is a potent stimulator of ATP hydrolysis and both its reactivity and ability to act as a substrate could be quenched with cysteine. The results suggested that the interface between TM6 and TM12 forms part of the potential drug-binding pocket in P-gp (Fig. 4). Indeed, recent cross-linking experiments show that these TM segments are close to each other and undergo conformational changes during the reaction cycle (Loo and Clarke 1996a; Loo and Clarke 1997b). Folding and maturation of P-gp P-gp is first synthesized in the endoplasmic reticulum as a core-glycosylated intermediate with a molecular mass of about 150 kDa. The carbohydrates are subsequently modified in the Golgi to yield a protein of about 170 kDa that is subsequently delivered to the cell surface (Fig. 5). During mutational studies, we found that about 10% of the point mutations affected processing of P-gp. These mutants are retained in the endoplasmic reticulum as core-glycosylated intermediates in association with the molecular chaperones calnexin (Loo and Clarke 1994b) and Hsc70 (Loo and Clarke 1995c) and are rapidly degraded. Recently, we found that the presence of drug substrates or modulators (Loo and Clarke 1997a) during biosynthesis prevented abnormal protein folding and trafficking (protein kinesis) of the misprocessed mutants. Remarkably, the presence of drug © 1999 NRC Canada



18 Fig. 6. Effect of drug substrates on interaction between the halves of a P-gp processing mutant expressed as separate polypeptides. The diagram shows the P-gp half-molecule polypeptides that would be recovered by nickel-chelate chromatography after coexpression of a histidine-tagged N-half protein with a processing mutation (X) and an epitope-tagged (A52) C-half polypeptide. Only the N-half polypeptide is recovered on a nickel column when coexpressed without drug substrate (No Drug). In the presence of drug substrate, the two halves appear to adopt an interactive structure and the C-half polypeptide copurifies with the N-half polypeptide (+ Drug Substrate).

substrates during biosynthesis rescued nearly all processing mutants including those with mutations in TM segments, intracellular or intracellular loops, the linker region and either nucleotide-binding domain. A possible explanation is that the hydrophobic drug substrates diffuse into the endoplasmic reticulum and induce correct folding by occupying the drug-binding site (Fig. 5). This observation has very important implications for the potential treatment of other diseases associated with defective protein kinesis, such as cystic fibrosis and Alzheimer’s disease. Interaction of large domains An important question was whether P-gp functions as a monomer or oligomer, because the drug-binding site formed by a monomer would be significantly different from that formed by an oligomer. Although P-gp has been shown to be present in the membranes as monomers and oligomers, it was not known if the monomers were functional (Poruchynsky and Ling 1994). The results from immunoprecipitation and nickel-chelate chromatography studies, however, showed that the minimum functional unit was a monomer (Loo and Clarke 1996b). Drug-stimulated ATPase activity requires the interaction of both halves of the molecule (Loo and Clarke 1994c). In addition, results obtained from mutational analyses (Loo and Clarke 1995d) and protein modification studies (Loo and Clarke 1995a) indicated that both nucleotide-binding sites

Biochem. Cell Biol. Vol. 77, 1999 Fig. 7. Drug substrates induce superfolding of the TM domains. The inset shows the four domain of P-gp. TMD1 contains TM segments 1 to 6 (residues 1–379), and TMD2 contains TM segments 7 to 12 (residues 681–1025). To determine the domains that are influenced by drug substrates during biosynthesis, each domain was expressed as a separate polypeptide (quarter-molecules), and then tested for sensitivity to digestion by trypsin. When TMD1 and TMD2 are expressed without drug substrates, TMD2 remained sensitive to trypsin (No Drug). Upon coexpression in the presence of drug substrates however, the two polypeptides interact and TMD2 adopts a trypsin-resistant conformation (+ Drug Substrate).

are critical for function. These results also suggested that P-gp activity must involve interaction of all four domains. This was confirmed by expressing each domain as a separate polypeptide and testing for associations using coimmunoprecipitation assays (Loo and Clarke 1995c). Each domain was also tested for interaction with the chaperones, calnexin and Hsc70. It was found that calnexin associated with the TM domains, whereas Hsc70 associated only with the cytoplasmic domains. It was possible that processing mutations interfere in folding of P-gp by preventing interactions between the various domains and that these deficiencies can be corrected by the presence of drug substrates. This was found to be the case for interactions between the homologous halves of P-gp expressed as separate polypeptides. Interactions between the half-molecules can be monitored by attaching a histidine tag to one half-molecule and an epitope tag to the other. After coexpression, the epitope tagged half-molecule will copurify with the histidine-tagged half-molecule using nickel-chelate chromatography. It was found that such interactions are inhibited by the presence of processing mutations. Interactions between half-molecules with processing mutations however, can be restored if the molecules are coexpressed in the presence of drug substrates (Fig. 6). These experiments suggest that the drug-binding site requires contributions from both halves of P-gp and that the presence of drug substrates during biosynthesis helps to pull the two halves together. The TM domain alone was recently shown to be sufficient for drug binding in experiments using quarter-molecule polypeptides. The N- and C-terminal hydrophobic domain quarter-molecule polypeptides, TMD1 (containing TM segments 1 to 6) and TMD2 (containing TM segments 7 to 12), are normally rapidly degraded when expressed in cells. When expressed together in the presence of a drug substrate, © 1999 NRC Canada



Loo and Clarke Fig. 8. Proposed working model for P-gp-mediated drug efflux. The hydrophobic substrate enters the lipid bilayer and interacts with residues in the TM domain that form the drug-binding domain (oval). Recent experiments with the thiol-reactive substrate dBBn suggests that TM6 and TM12 are close to the drug-binding domain. Upon ATP hydrolysis at alternating sites, there is a conformational change relayed to the TM domain such as TM6 and TM12 that reduces the affinity for the substrate and leads to drug efflux.

19

tide-binding domains required? Is ATP hydrolyzed at alternating sites? Are there multiple drug binding sites or a single site for accommodating all substrates? How are substrates extracted from the membrane? Is P-gp a flippase that moves substrates from one leaflet in the lipid bilayer to the other? Future studies will answer these questions and help us to understand the mechanism of P-gp mediated drug transport.

Acknowledgments This research was supported by a grant as part of a group grant from the Medical Research Council of Canada and by a grant from the Canadian Cystic Fibrosis Foundation. We thank Merck Frosst Canada for supporting the Merck Frosst Prize awarded by the Canadian Society for Biochemistry and Molecular & Cellular Biology. We thank all investigators for contributing to the understanding of P-glycoprotein.

References

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Model of the mechanism of drug transport The picture that has emerged is that drug transport by P-gp requires a co-ordinated effort between the two nucleotide-binding domains and the two TM domains. Inactivation of one nucleotide-binding domain by mutagenesis or chemical modification inhibits ATP hydrolysis at the other nucleotide-binding domain. This co-ordinated interaction between the two nucleotide-binding domains becomes uncoupled when they are expressed as separate half-molecule polypeptides. Each half-molecule will exhibit ATPase activity when expressed alone. Drug substrates however, do not stimulate the ATPase activity of half-molecule polypeptides. It is possible that binding of drug substrates by P-gp requires the presence of both TM domains. It appears that ligands for drug binding are contributed from residues in TM segments from both halves of the molecule. TM segments 6 and 12 seem to be particularly important since there is evidence that they are important for drug binding and undergo conformational changes during the reaction cycle. These TM segments seem to be ideally located for coupling drug efflux to ATP hydrolysis since each directly links a TM domain to a nucleotide-binding site. Accordingly, we have proposed a working model for drug transport (Fig. 8). There are many questions to be addressed about the structure and mechanism of P-gp. Why are two nucleo-

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Notes about the author

Notes sur l’auteur

This article is based on the Merck Frosst Award Lecture of the Canadian Society of Biochemistry and Molecular & Cellular Biology presented by Dr. David M. Clarke at the 41st Annual Meeting of the Canadian Federation of Biological Societies in Edmonton, in June 1998. Dr. Clarke was born in Windsor and raised in Sarnia, Ontario and obtained his Bachelor’s degree at the University of Windsor. He obtained his M.Sc. on “Post-Translational Modification of Peptides in the Brain” under the supervision of Dr. W.W.C. Chan at McMaster University. He completed his Ph.D. at the University of British Columbia in Vancouver under the supervision of Dr. P.O. Bragg, working on “Pyridine Nucleotide Transhydrogenase of E. coli : Nucleotide Sequence of the pnt Gene and Characterization of the Enzyme Complex.” Following his Ph.D., he did postdoctoral work at the Univeristy of British Columbia with Dr. Shirley Gillam on the “Cloning, Nucleotide Sequence and In vitro Expression

Cet article est basé sur la conférence d’acceptation du prix Merck Frosst de la Société canadienne de biochimie et de biologie moléculaire et cellulaire décerné à Dr David M. Clarke lors du 41e congrès annuel de la Fédération canadienne des sociétés de biologie tenu à Edmonton, Alberta, en juin 1998. Dr Clarke est né à Windsor et a grandi à Sarnia en Ontario. Il a obtenu un B.Sc. de l’Université de Windsor. Puis, il a étudié « Les modifications post-traductionnelles de peptides dans le cerveau » sous la direction de Dr W.W.C. Chan à l’Université McMaster à Hamilton, Ontario, et a obtenu un M.Sc. Par la suite, il a obtenu un Ph.D. de l’Université de la Colombie-Britannique à Vancouver après avoir déterminé « La séquence nucléotidique du gène pnt et caractérisé le complexe enzymatique de la transhydrogénase des nucléotides pyridiniques de E. coli » sous la direction de Dr P.O. Bragg. Après avoir obtenu un Ph.D., Dr Clarke a fait un stage post© 1999 NRC Canada



Loo and Clarke

of the Rubella Virus Subgenomic RNA.” He also did postdoctoral work at the University of Toronto with Dr. David H. MacLennan, on “The Mechanism of Ca2+ Transport by the SERCAI Ca2+- ATPase of Muscle.” After completing his postdoctoral studies he went to the Department of Medicine, University of Toronto in 1990 as Assitant Professor and is now an Associate Professor in the same department.

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doctoral avec Dr Shirley Gillam à l’Université de la Colombie-Britannique au cours duquel il a cloné, déterminé « La séquence nucléotidique et mesuré l’expression in vitro de l’ARN subgénomique du virus de la rubéole. » Il a également effectué un stage postdoctoral dans le laboratoire de Dr David H. MacLennan à l’Université de Toronto pour étudier « Le mécanisme de transport du Ca2+ par la Ca2+-ATPase SERCA1 du muscle. » En 1990, Dr Clarke a joint le département de médecine de l’Université de Toronto en tant que professeur adjoint. Il est maintenant professeur agrégé dans le même département.

© 1999 NRC Canada
