JOURNAL OF CELLULAR PHYSIOLOGY 148:479-484 19911

Modulation of ATP and Drug Binding by Monoclonal Antibodies Against P-Glycoprotein ELIAS GEORGES, ]IAN-TING ZHANG,

AND

VICTOR LING*

Ontario Cancer Institute, Department of Medical Biophysics, Division of Molecular and Structural Biology, Princess Margaret Hospital, Toronto, Ontario, Canada, M4X 1 K 9 The role of P-glycoprotein in mediating the drug-resistance phenotype in multidrug resistant cells is now well documented. It is thought to function as an energy-dependent drug-efflux pump of broad specificity. Structurally, P-glycoprotein is an internally duplicated molecule containing two large multi-spanning transmembrane domains and two cytoplasmicATP binding domains. In this report we demonstrate that monoclonal antibodies C219, C494, and C32 directed against short linear regions of the P-glycoprotein molecule inhibit ATP binding to P-glycoprotein in vitro. We also provide direct evidence that both predicted ATP-binding domains bind ATP and that there is co-operativity between the two sites. In addition, the capacity of P-glycoprotein to bind the calcium channel blocker, azidopine, is inhibited differentially by the antibodies. These observations are the first evidence linking specific perturbations of the P-glycoprotein molecule with ATP and drug binding. The successful treatment of cancer patients with chemotherapeutic drugs may be limited by the development of drug-resistant tumor cells. It has been postulated that a multidrug resistance (MDR) phenotype, first discovered in cell lines selected for resistance to drugs in vitro, may have clinical relevance (for reviews see Bradley et al., 1988; Gottesman and Pastan, 1988; Georges et al., 1990a).MDR is frequently associated with the over-expression of a 170 kDa membrane glycoprotein (P-glycoprotein) (Juliano and Ling, 1976). Transfection of P-glycoprotein full-length cDNA's was shown to confer the MDR phenotype to drug-sensitive cells (Gros et al., 1986a,b; Ueda et al., 1987). P-glycoprotein is a tandemly duplicated molecule; each half is predicted to encode a hydrophobic transmembrane domain thought to traverse the lipid bilayer six times and a large cytoplasmic domain with consensus sequences for nucleotide binding (Gerlach et al., 1986; Chen e t al., 1986; Gros et al., 1986a,b). It has been demonstrated that P-glycoprotein binds to photoactive analogues of ATP (Cornwell et al., 1987; Schurr et al., 1989), cytotoxic drugs (Safa et al., 1986; Safa e t al., 19891, and compounds which can reverse the MDR phenotype (chemosensitizers) (Georges et al., 1990a). ATPase activity has also been detected in immunopurified P-glycoprotein (Hamada and Tsuruo, 1988). Thus, i t has been postulated that P-glycoprotein functions as a n energy-dependent drug efflux pump of broad specificity to mediate the MDR phenotype. Structural features of P-glycoprotein, particularly the highly conserved ATP binding domain, are found in many membrane transport proteins in eukaryotes and prokaryotes. These proteins appear to be part of a superfamily of which P-glycoprotein is a member (Juranka et al., 1989). A major question concerning the function of Pglycoprotein is the role of the different predicted struc0 1991 WILEY-LISS, INC

tural domains in drug transport. Recently we have determined that three P-glycoprotein monoclonal antibodies bind to small specific continuous epitopes in P-glycoprotein (Georges et al., 1990b). In this study we determined if the binding of these antibodies to defined regions of the P-glycoprotein molecule affects its function.

MATERIALS AND METHODS Materials [3fH]-azidopine (75 mCiimmole) was purchased from Amersham Biochemical, Inc. (Mississauga, Ontario). Sequence grade staphylococcus aureus Glu-C endoprotease was purchased from Boehringer Mannheim (DorVal, Quebec). Trypsin from bovine pancreas (TPCK treated) was purchased from (Sigma, St. Louis, MO). All other chemicals used were of the highest grade available from commercial sources. Plasma membrane preparation Membrane enriched fractions were prepared essentially as described by Riordan and Ling (1979). Briefly cells from the multidrug resistant Chinese hamster ovary cell line B30 (Kartner et al., 1983) and the parental line AuxBl were suspended in hypotonic lysis buffer (10 mM KC1, 1.5 mM MgCl,, 10 mM Tris-HC1, pH 7.4) on ice for 10 min. Cells were lysed by passing them once through the Stansted Cell Disruptor at 250 psi in the presence of 2 mM phenylmethylsulfonyl fluoride (PMSF), and 2 pM leupeptin. The resulting cell lysate was centrifuged a t 4,OOOg in the Sorvall Received May 3, 1991.

*To whom reprint requestsicorrespondence should be addressed. Elias Georges is now a t the Institute of Parasitology of McGill University, Macdonald College, Ste-Anne-de Bellevue, Quebec, Canada.

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centrifuge using the SS34 rotor. The supernatant from this low speed spin was loaded onto a discontinuous sucrose gradient consisting of 16%, 31%,45%, and 60% sucrose. The plasma membrane enriched fraction at the 16131%interface of the sucrose gradient was used in the labelling experiments.

Photoaffinity cross-linking and immunoprecipitation Aliquots (10-25 pg) of plasma membrane fractions from drug sensitive (AuxB1) or resistant (B30) cells were suspended in 10 mM Tris-HC1 (pH 7.41, 250 mM sucrose in the presence of a protease inhibitor cocktail (2 mM PMSF, 2 pM leupeptin, 3 pgiml pepstatin). [3tH]-azidopine a t 0.2 pM final concentration was added in the dark to a reaction volume of 25-50 p1, and allowed to incubate a t room temperature for 1 hour prior to photo-cross-linking. Experiments with the radiolabeled ATP-analogue (8-a~ido-[a-~~P]-ATP) were undertaken with the plasma membranes as above and allowed to incubate on ice for 5 min before photo-crosslinking using a UV source (254 nm, 12 J, Stratalinker UV cross-linker, Stratagene, La Jolla, California) for 10 min on ice. Since our membrane vesicle preparations were likely to contain both inside-out and right-sideout vesicles, we optimized conditions for the P-glycoprotein ATP-binding domains to be accessible to ATP by using low concentrations of detergents such a s 1% Triton-X100 or 0.1% SDS. For immunoprecipitation, 50-100 pg aliquots of plasma membrane fractions were suspended in SDS buffer A (50 mM Tris-HC1 [pH 7.41, and 1%sodium dodecyl sulfate [SDSI) and then mixed with 9 volumes of Triton-X100 buffer B (50 mM TrisHCl I pH 7.4 1, 190 mM NaCI, and 1.25% Triton X-100). Protease inhibitors (2 mM PMSF, 2 pM leupeptin, and 3 pgiml pepstatin) were added to buffers A and B. The mixture was clarified by centrifugation in an eppendorf microfuge (15,000 rpm) for 2 min at 4°C. Ten micrograms of C219 monoclonal antibody was added to each aliquot and incubated overnight at 4°C. The resultant P-glycoprotein:C219 monoclonal antibody complex was precipitated by the addition of 50 p1 of Protein-A Sepharose (0.07mglml) (Pharmacia Inc., Quebec, Canada) to each aliquot and allowed to incubate for 3 h r a t 4°C. The Protein-A beads were washed 5~ with Tris wash buffer (50 mM Tris-HC1 [pH 7.41, 150 mM NaC1, 0.1% Triton-X100, 0.03%SDS, and 5 mglml of bovine serum albumin [BSAJ), and P-glycoprotein was eluted C219

from the beads in SDSlurea Fairbanks solubilization buffers (see SDS PAGE and Western blotting below), and analyzed directly without boiling.

Protease cleavage Plasma membrane fractions from B30 cells suspended in 10 mM Tris-HC1 (pH 7.4) containing 250 mM sucrose were incubated with Glu-C or trypsin in a reaction volume of 50-100 p1 a t a ratio of 1:50 wlw enzyme:substrate a t 37°C for 30 min. The digested samples were immunoprecipiated with C219 monoclonal antibody and fractionated on SDS-PAGE. SDS-PAGE and Western blotting Membrane protein fractions (10-25 pg) were resolved on SDS-PAGE using either the Fairbanks gel system with some modifications (Debenham et al., 1982) or the Laemrnli gel system (Laemmli, 1970). Briefly, proteins were dissolved in buffer I ( 2 % SDS, 50 mM dithiothreitol [DTTI, 1 mM ethylenediamine tetraacetic acid [EDTAI, and 10 mM Tris-HC1 [pH 8.01, and mixed with a n equal volume of buffer I1 (2x buffer I and 9 M urea). Samples were electrophoresed at constant power a t 5 W. Gel slabs containing the resolved membrane proteins were either fixed in 50% methanol, 10% acetic acid, and dried, or transferred to nitrocellulose membrane in Tris-glycine buffer in the presence of 20% methanol for Western blot analysis according to the procedure of Towbin et al. (1979). The dried nitrocellulose membrane or gel was covered with saran wrap, and exposed to Kodak x-ray film a t -70°C. Protein assay Plasma membrane proteins were measured using the Lowry protein assay (Lowry e t al., 1951). RESULTS We have previously determined the epitope sequences for three P-glycoprotein specific monoclonal antibodies C219, C494, and C32 (Georges e t al., 1990b). These epitopes comprise of short continuous sequences of about 6-13 amino acid residues and are located very close to the A and B consensus sequences of the ATP-binding domain (Fig. 1).Given the close proximity of these sequences to the predicted ATP-binding pocket in P-glycoprotein (Gerlach et al., 1986; Gros et al., 1986a,b; Chen et al., 19861, it was of interest to determine whether these monoclonal antibodies can

y4c:2

C219

I

COOH

"2

Fig. 1. Schematic drawing of the P-glycoprotein molecule. The epitope sequences of the three monoclonal antibodies (C219, C494, and C32) and their relative location on P-glycoprotein are shown. The small shaded boxes labelled 1-6 (or 1'-6') represent the predicted transmembrane domains in P-glycoprotein. The filled boxes, A and B (or A' and B'), show the relative location of the ATP binding domains.

ANTIBODIES AS SPECIFIC MODULATORS OF P-GLYCOPROTEIN

interfere with ATP binding. Membrane fractions from B30 drug-resistant cells were incubated with 8-azido[~x-~'P]-ATPand the photolabelling was initiated by UV light. The photoactive radiolabelled ATP was crosslinked to P-glycoprotein (Fig. 2 lanes 1, 4, 7, and 10). The binding specificity of 8-a~ido-[cw-~'PP]-ATP was demonstrated by competition with a n excess (100 FM) of cold ATP (Fig. 2, lane 13). When membrane fractions were incubated in the presence of increasing concentrations of P-glycoprotein-specific monoclonal antibodies (C219, C494, and C32) prior to UV cross-linking with the radiolabelled photoactive ATP analogue, a progressive decrease in the intensity of the radiolabelled P-glycoprotein band was seen in each instance (Fig. 2). However, no decrease in P-glycoprotein photolabelling was evident when a n irrelevant antibody was used even at higher concentrations (Fig. 2, lanes 1012).The two bands of approximate molecular weights of 24 kDa and 64 kDa are the light and heavy chains of immunoglobulin, respectively. The photolabelling of the immunoglobulin light and heavy chains is nonspecific and results from their presence at high amounts in the reaction mix. A previous study (Georges e t al., 1990b) had demonstrated that the epitopes for mAb C219 are found in both the C- and N-terminal halves of P-glycoprotein, while only the C-terminal half contained the C494 and C32 epitope sequences (see Fig. 1).It is interesting, therefore, that all three mAbs are effective in inhibit-

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ing ATP binding to P-glycoprotein. These results raise the possibility that both the N- and C-terminal halves of P-glycoprotein bind ATP and that perturbation of one ATP-binding domain (the C-terminal half) by a mAb such as C494 affects both halves. That is the ATP-binding domains of P-glycoprotein function in a cooperative manner. Alternatively, i t is possible that only the C-terminal half of P-glycoprotein binds ATP and the binding of mAbs inhibits ATP binding by more or less direct interference. To address these two alternatives, we worked out conditions to cleave P-glycoprotein into two halves. Membrane fractions from B30 cells were treated with protease (see Materials and Methods) and the digested P-glycoprotein was analyzed by Western blotting (Fig. 3a).The two bands migrating on SDS-PAGE with a n apparent molecular weights of 65 kDa and 100 kDa detected with C219 mAb represent the C-terminal and N-terminal halves of P-glycoprotein, respectively (Fig. 3, lane 2). However, only the C-terminal half was recognized by the monoclonal antibodies C494 and C32 (Fig. 3, lanes 4 and 6 respectively). These results confirm the previous epitope mapping studies (Georges et al., 1990b),and further suggest that a cleavage in the linker region of the native P-glycoprotein (see diagram in Fig. 3) generates two approximately equal halves of the P-glycoprotein molecule. The difference in their apparent molecular weights a s determined by SDSPAGE is due to the presence of carbohydrate moieties in the N-terminal half of P-glycoprotein (100 kDa band), which can account for as much as 30-50 kDa of its SDS-PAGE estimated size (Greenberger et al., 1988) 1 2 3 4 5 6 7 8 9 1 0 1 1 12 13 (Fig. 3, lane 2). The C-terminal half migrates as a 65 kDa band (Fig. 3, lanes 4, 6). Some residual full-length P-gP P-glycoprotein molecules, seen in Figure 3 (lanes 2, 4, 6), likely result from the presence of right-side-out vesicles with the proteolytic cleavage site protected on Hthe inside, since residual uncut P-glycoprotein remains after 2 h r of digestion. Using this cleavage assay, we determined if both Lhalves of P-glycoprotein (C- and N-terminal halves) can bind ATP. Sucrose purified B30 plasma membrane fractions were incubated with 8-azid04~~PI-ATP in the absence of detergents and then UV irradiated. The results in Figure 4 show a 170 kDa (P-glycoprotein) radiolabelled band in membrane fraction not treated with protease (lane l),while the treated plasma membrane (lane 2) revealed the presence of a 65 kDa and a 100 kDa fragment. This indicates that both halves of P-glycoprotein bind ATP. Immunoprecipitation using (us) o s g o s g o s g o g ; ; 0 C219 monoclonal antibody further confirmed the identity of the ATP-labelled 65 kDa and 100 kDa band (CmAb C219 c494 C32 'gG and N-terminal halves, respectively) of P-glycoprotein Fig. 2. ATP binding to P-glycoprotein in the absence or presence of (lanes 4). Similar results were obtained when memmonoclonal antibodies. B30 plasma membrane fractions were incu- brane fractions were photolabelled with the ATP-anabated with increasing concentrations (10 or 50 pg) of P-glycoprotein- logue after cleavage by protease (data not shown). specific monoclonal antibodies (C219, C494, and C32) or an irrelevant Taking together, these results indicate that the CIgG,, (50 to 100 pg) prior to the addition of the photoactive radiolaand N-terminal domains of P-glycoprotein bind ATP belled ATP analogue (8-azido-[~t~~P]-ATP). Lanes 1-3 of the autoradiogram show a radiolabelled P-glycoprotein band in the absence and and t h a t the perturbation by monoclonal antibodies of presence of 10 pg or 50 pg C219 mAb, respectively. Similarly lanes one domain inhibit the ATP-binding ability of both 4-6, 7-9 and 10-12 show P-glycoprotein photolabelling in the pres- domains. In addition, the fact that both domains can ence of the other antibodies. The binding of ATP analogue to Pglyeoprotein was abolished in the presence of excess cold ATP (lane bind ATP before and after proteolytic cleavage of 13). The radiolabelled protein bands migrating with an apparent P-glycoprotein suggest that the two cleaved halves molecular weight of 64 and 24 kDa represent mouse immunoglobulin remain functionally intact in the cell membrane, a t heavy and light chains, respectively (see results). least as far as ATP binding is concerned.

GEORGES ET AL.

482

1

2

3

4

P-9P

1

2

3

4

5

6

KD

N-Half P -9P 110-

N -Half

84-

c -Half

47-

C -Half

16-

C219

c494

C 32

Fig. 4. The binding of 8-azido-ATP to both the C- and N-terminal halves of P-glycoprotein. B30 plasma membrane fraction was crosslinked with azid~-[cy-~'P]-ATP and treated with trypsin (see Materials and Methods). Lanes 1and 2 show an autoradiogram of untreated and trypsin treated B30 membrane proteins resolved on a Laemmli SDS-PAGE. The ATP-analogue was predominantly cross-linked to P-glycoprotein in native B30 plasma membrane (lane 1). The trypsin treated fractions contained a 65 kDa and 100 kDa ATP cross-linked bands (lane 2). The identity of the two bands (65 kDa and 100 kDa) as the C- and N-terminal halves of P-glycoprotein was confirmed by immunoprecipitation with C219 monoclonal antibody (lane 4). Lane 3 shows an immunoprecipitation of trypsin treated plasma membrane with an irrelevant IgG.

1..

C219 ............................

..__________..__.___...... c32 ....i c219 Glu-C

Fig. 3. Proteolytic cleavage of P-glycoprotein into two halves. Enzymatic digestion of native P-glycoprotein yields two fragments with approximately equal polypeptide backbone that contain the epitope sequence of different monoclonal antibodies. Membrane fractions from drug-resistant B30 cells were digested with Glu-C or trypsin, and P-glycoprotein cleavage products were analyzed by Western blotting. Lanes 1,3,and 5 contain undigested B30 plasma membrane fractions probed with C219, C494, or C32 mAbs, respectively. A component a t about 170 kDa is detected by all three mAbs. Lanes 2, 4, and 6 represent digested plasma membrane similarly probed. Two fragments of 65 kDa, and 100 kDa are detected with C219 mAb (lane 2). In contrast, in lanes 4 and 6 , only the 65 kDa band is detected. The relative location of the monoclonal antibodies epitopes and the Glu-C cleavage site are indicated on the schematic diagram. The C- and N-terminal halves of P-glycoprotein which represent the two cleavage fragments (65 kDa and 100 kDa, respectively) detected by Western blotting are indicated by large boxes.

The role of P-glycoprotein a s a n energy-driven drug transport pump in MDR cells has been supported by studies demonstrating drug binding in vitro (Cornwell et al., 1986; Naito et al., 1988) and drug transport using membrane vesicles (Horio et al., 1988). Photoactive analogues of vinblastine (Safa et al., 1986) and calcium channel blocker, such as azidopine, were shown to cross-link to P-glycoprotein (Safa et al., 1987; Foxwell et al., 1989). This suggests that P-glycoprotein binds and transports drugs directly. However, it is not known if ATP binding by P-glycoprotein is required for drug binding and transport. As a n approach to this question, we used our panel of P-glycoprotein monoclonal antibodies to determine if they affected the binding of azidopine to P-glycoprotein. Plasma membrane fractions from B30 cells were pre-incubated with C219, C494, (232, or a n irrelevant antibod prior to the addition of the photoactive analogue [J+HI-azidopine. The results in Figure 5 show t h a t the binding of C219 to P-glycoprotein caused a significant decrease in azidopine binding (Fig. 5, lane 21, while C494 mAb was less effective than C219 (lane 31, and C32 mAb or an irrelevant antibody had no effect on the photolabelling

ANTIBODIES AS SPECIFIC MODULATORS OF P-GLYCOPROTEIN

1

2

3

4

5

Fig. 5. L3 ' HI-azidopine binding to P-glycoprotein in the presence of monoclonal antibodies. Plasma membrane fractions were separately pre-incubated with 50 pg of C219, C494, C32, or an irrelevant IgG prior to the addition of the photoactive [3+Hl-azidopine.Photolabelling with azidopine in the absence of antibody (lane 1) shows a 170 kDa band (P-glycoprotein)as the major radiolabelled band. Lanes 2-5 show the level of P-glycoprotein photolabelling with [3+H]-azidopine in the presence of C219, C494, C32, or an irrelevant antibody, respectively.

of P-glycoprotein (Fig. 5 , lanes 4, 5 ) . The addition of cold ATP (10 pM) to the above reaction mix did not alter the levels of azidopine cross-linking to P-glycoprotein (not shown). The fact that the amount of mAbs used in this experiment normally reduces ATP-binding to P-glycoprotein by a significant extent (Fig. 21, indicates that ATP-binding per se may not be a requirement for azidopine binding. The observed differential effects of the monoclonal antibodies on azidopine binding likely result from different perturbation of the conformation of the P-glycoprotein molecule.

DISCUSSION P-glycoprotein has been proposed to act as a n energydependent drug efflux pump. However, elucidation of its mechanism of action in intact cells is likely to be difficult due to the complexity of such a multi-component system. Ideally, reconstituted membrane vesicles containing purified P-glycoprotein and defined components are most suited for the study of P-glycoprotein transport properties. Alternatively, specific reagents which can perturb directly P-glycoprotein structure may provide a n approach towards delineating the functional properties of P-glycoprotein in a complex system. In this report, we demonstrate that monoclonal antibodies specific for P-glycoprotein can perturb P-

483

glycoprotein function. These monoclonal antibodies inhibit P-glycoprotein ATP binding. It may be speculated that the binding of a large molecule (such as a n immunoglobulin molecule) could directly block ATPbinding by steric hindrance limiting access to the ATP-binding pockets. Another possibility is that antibody binding may cause conformation changes in the ATP binding folds of P-glycoprotein reducing their binding affinity. Previous reports (Cornwell et al., 1987; Naito et al., 1988) have demonstrated specific photolabelling of P-glycoprotein by the ATP analogue 8-azido-ATP. However, in those studies, it was not known if one or both halves of the P-glycoprotein molecule are capable of binding ATP. In this report, we present the first direct evidence that ATP does in fact bind to both halves of P-glycoprotein. In addition, the finding that both C219 and C494 mAbs are equally effective in inhibiting ATP binding to P-glycoprotein was unexpected in view of the fact that C219 binds to both ATP binding domains while C494 recognizes only the Cterminal domain. This observation strongly suggests that the two ATP binding domains function co-operatively and that while these two domains are wellseparated in the linear sequence, they are likely to be spatially situated in close proximity in the native protein. Cytotoxic drugs and other lipophilic compounds are thought to interact directly with P-glycoprotein (Georges e t al., 1990a). The azidopine binding sites in P-glycoprotein have been mapped and are located in the transmembrane domains (manuscript in preparation), distant from the cytoplasmic ATP-binding domains in the linear sequence. Using the monoclonal antibodies as specific perturbants of P-glycoprotein, we have provided direct evidence that perturbation of the Pglycoprotein molecule affects drug binding. Remarkably, the binding of these monoclonal antibodies to P-glycoprotein appear to have differential effects on azidopine binding. C219 or C494 inhibits azidopine binding to P-glycoprotein, while C32 or a n irrelevant antibody does not. The fact that, under these conditions, ATP-binding by P-glycoprotein is inhibited by C32 provides evidence that azidopine binding (at least in vitro) is not dependent on a P-glycoprotein containing bound ATP. It is presently not clear how the binding of one ((3219 or C494) antibody near the ATP-binding folds affects a drug-binding site in Pglycoprotein, while another (C32) does not. We can only speculate that quite different conformational changes are imposed on P-glycoprotein as a result of binding to these monoclonal antibodies. Clearly, a better knowledge of the native 3-dimensional structure is needed to understand the role of the different structural domains in the transport function of this protein.

ACKNOWLEDGMENTS The authors will like to thank Drs. Grace Bradley and Marianne Poruchynsky for their critical reading of the manuscript. We also thank our colleagues at the Ontario Cancer Institute for helpful discussion. These studies were supported by the National Cancer Institute of Canada and by Public Health Service Grant CA 37130 from the National Institutes of Health USA.

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Jianting Zhang is a recipient of a National Cancer Institute of Canada post-doctoral fellowship.

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Horio, M., Gottesman, M.M., and Pastan, I. (1988) ATP-dependent transport of vinblastine in vesicles from human multidrug-resistant cells. Proc. Natl. Acad. Sci. U.S.A., 85r3580-3584. Juliano, R.L., and Ling, V. (1976) A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants. Biochim. Biophys. Acta., 455r152-162. Juranka, P.F., Zastawny, R.L., and Ling, V. (1989) P-glycoprotein: multidrug-resistance and a superfamily of membrane associated transport proteins. FASEB J . , 3r2583-2592. Kartner, N., Riordan, J.R., and Ling, V. (1983) Cell surface Pglycoprotein associated with multidrug resistance in mammalian cell lines. Science, 222,1285-1288. Laemmli, U.K. (1970) Cleavage of structural proteins during assembly of the head bacteriophage T,. Nature, 2271680485. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J . Biol. Chem., 193265-275. Naito, M., Hamada, H., and Tsuruo, T. (1988) ATP/Mg2’-dependent binding of vincristine to the plasma membrane of multidrugresistant K562 cells. J. Biol. Chem., 263t11887-11891. Riordan, J.R., and Ling, V. (1979) Purification of P-glycoprotein from plasma membrane vesicles of Chinese hamster ovary cell mutants with reduced colchicine permeability. J . Biol. Chem., 254t1270112705. Safa, A.R., Glover, C.J., Meyers, M.B., Biedler, J.L., and Felsted, R.L. (1986) Vinblastine photoaffinity labeling of a high molecular weight surface membrane glycoprotein specific for multidrug-resistant cells. J . Biol. Chem., 262:6137-6140. Safa, A.R., Glover, C.J., Sewell, J.L., Meyers, M.B., Biedler, J.L., and Felsted, R.L. (1987) Identification of the multidrug resistancerelated membrane glycoprotein as an acceptor for calcium channel blockers. J. Biol. Chem., 262r788P7888. Safa, A.R., Mehta, N.D., and Agresti, M. (1989)Photoaffinity labeling of P-glycoprotein in multidrug resistant cells with photoactive analogs of colchicine. Biocheni. Biophys. Res. Commun., 262:14021408. Schurr, E., Raymond, M., Bell, J.C., and Gros, P. (1989) Characterization of the multidrug resistance protein expressed in cell clones stably transfected with the mouse MDRl cDNA. Cancer Res., 49r2729-2733. Towbin, H., Staehelin, T., and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: orocedures and some aadications. Proc. Natl. Acad. Sci. U.S.A., >6:4350-4354. Ueda, K., Cardarelli, C., Gottesman, M.M., and Pastan, I. (1987) Exaression of a full-length cDNA for the human “MDR1” gene confers resistance to colckcine, doxorubicin, and vinblastine. Proc. Natl. Acad. Sci. U.S.A., 84t300P3008.