(Received for publication, May 24, 1995; and in revised form, August 1, 1995)
From the
The ATPase activity of P-glycoprotein is inactivated by N-ethylmaleimide (NEM), which is postulated to modify cysteine residues within either of the homology A consensus sequences for nucleotide binding (GNSGCGKS and GSSGCGKS, respectively) (Al-Shawi, M. K., Urbatsch, I. L., and Senior, A. E.(1994) J. Biol. Chem. 269, 8986-8992). To test this postulate as well as determine the contribution of either nucleotide-binding domain to function, a Cys-less mutant was constructed, and then a single cysteine residue was reintroduced back into each nucleotide-binding consensus sequence. We then tested the sensitivity of the ATPase activity of each mutant to covalent modification by NEM. It was found that covalent modification of a single cysteine residue within either nucleotide-binding consensus sequence (Cys-431 and Cys-1074, respectively) with NEM inhibited drug-stimulated ATPase activity of P-glycoprotein. The concentrations of NEM required for half-maximal inactivation of ATPase activity were 7 and 35 µM for mutants Cys-431 and Cys-1074, respectively. In both cases, inactivation of ATPase activity by NEM was prevented by ATP. These results suggest that both nucleotide-binding domains may need to bind ATP to couple drug binding to ATPase activity.
P-glycoprotein, also known as the multidrug-resistance protein, is a plasma membrane protein that is involved in the ATP-dependent efflux of a broad range of cytotoxic hydrophobic compounds from cells (reviewed by Endicott and Ling, 1989; Roninson, 1991; and Gottesman and Pastan, 1993). Many of the drugs used in cancer chemotherapy such as vinblastine, vincristine, taxol, doxorubicin, and daunomycin are substrates of P-glycoprotein. Therefore, this protein has significant clinical importance, since it may be one of several mechanisms responsible for the development of multidrug resistance to chemotherapeutic drugs during treatment of cancers.
The cDNAs coding for P-glycoprotein have been cloned from a variety of mammalian cells. They code for a protein of 1280 amino acids, which is organized into two homologous halves. Each homologous half is predicted to include a hydrophobic domain with six transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. The results from several studies support this domain organization of P-glycoprotein and provide insight into the structure and function of each domain. Studies on the topology of the full-length protein expressed in mammalian cells are consistent with a model of six transmembrane segments in each half of the molecule (Georges et al., 1993; Loo and Clarke, 1995; Kast et al., 1995). Genetic (Choi et al., 1988; Gros et al., 1991; Devine et al., 1992; Loo and Clarke, 1993a; 1993b; 1994a; 1994c) and biochemical studies (Bruggemann et al., 1989; 1992; Greenberger, 1993; Zhang et al., 1995) indicate that residues within the transmembrane domains contribute to drug binding. The hydrophilic domains containing the consensus nucleotide binding folds have been found to bind ATP (Azzaria et al., 1989; Baubichon-Cortay et al., 1994). It has been demonstrated that P-glycoprotein possesses high levels of drug-stimulatable ATPase activity (Ambudkar et al., 1992; Sarkadi et al., 1992; Sharom et al., 1993; Al-Shawi and Senior, 1993; Shapiro and Ling, 1994).
Al-Shawi et al.(1994) showed that the ATPase activity of
P-glycoprotein is inhibited by N-ethylmaleimide (NEM). ()Maximal inhibition occurred with labeling at two sites,
with equal distribution of the label between the NH
- 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; Walker et al.(1982)) in the two
nucleotide binding domains of Chinese hamster P-glycoprotein.
In this study, we used inactivation of ATPase activity by NEM to address the following questions: 1) Are the cysteines within the homology A regions (Cys-431 and Cys-1074, respectively) modified by NEM? 2) Is a mutant lacking these two cysteines still sensitive to inactivation by NEM? 3) If these two cysteines do react with NEM, then what is the effect of modifying only one of these residues? and 4) Does modification of the two cysteines outside the homology A regions (Cys-1125 or Cys-1227) affect ATPase activity?
Our approach was to utilize a Cys-less mutant of P-glycoprotein, which we have previously shown to retain the ability to confer drug resistance in transfected cells (Loo and Clarke, 1995). We then reintroduced a cysteine residue at its original position in the nucleotide-binding domains and assayed for ATPase activity after incubation with NEM. We found that inactivation of ATPase activity was indeed due to covalent modification of cysteine residues within the homology A sequences and that modification of either cysteine residue was sufficient to inactivate drug-stimulated ATPase activity of P-glycoprotein. These results suggest that both nucleotide-binding domains are capable of interacting with ATP and that both domains are essential for drug-stimulated ATPase activity.
Figure 1: Location of cysteines in the predicted structural model of P-glycoprotein. The model is that proposed by Juranka et al.(1989) and Gottesman and Pastan(1988). The positions of the cysteine residues are indicated by largecircles. TM1-TM12 correspond to the transmembrane helices. The positions of the glycosylated residues (Y) and sequences in the homology A regions (GNSGCGKS and GSSGCGKS) are shown.
To identify the cysteines
in the nucleotide-binding folds that were susceptible to covalent
modification by NEM, the cDNA coding for Cys-less P-glycoprotein-A52
was mutated to generate a series of mutants that contained a cysteine
at their original positions (residues 431, 1074, 1125, and 1227,
respectively) in the nucleotide-binding domains (Fig. 1). In a
previous study on the topology of P-glycoprotein, we found that Cys
residues located in the predicted transmembrane regions (Cys-137,
Cys-717, Cys-956) were not labeled with biotin maleimide. ()Therefore, inactivation of P-glycoprotein ATPase activity
by NEM (Al-Shawi and Senior, 1993) must involve the covalent
modification of one or more Cys residues in the nucleotide-binding
domains of P-glycoprotein. To identify the cysteine residues that were
sensitive to modification by NEM, it was necessary to measure the
ATPase activity of the mutant P-glycoproteins. Measurement of ATPase
activity requires a relatively large amount of P-glycoprotein, which is
difficult to achieve in mammalian cells. It has, however, been
demonstrated that expression of P-glycoprotein in insect cells yields
relatively large amounts of the transporter, which exhibits high levels
of drug-stimulated ATPase activity (Germann et al., 1990;
Sarkadi et al., 1992; Loo and Clarke. 1994b; Rao, 1995).
Accordingly, we subcloned the cDNA coding for each of these mutant
P-glycoproteins into a baculovirus expression vector for expression in
cultured Sf9 insect cells.
Recombinant baculoviruses containing the cDNA coding for the various Cys P-glycoprotein mutants were used to infect Sf9 insect cells. 5 days after infection, membranes were prepared from the infected cells. Fig. 2shows an immunoblot of membranes prepared from the infected cells. An immunoreactive protein of apparent mass of 140 kDa was the major product, and it was found to be present in similar amounts in cells expressing either wild-type P-glycoprotein-A52 or the various Cys mutants. This product has an apparent mass similar to that of the underglycosylated form of the mature P-glycoprotein and is consistently observed to be present when expressed in insect cells (Germann et al., 1990; Loo and Clarke, 1994b, Rao, 1995). In mammalian cells, P-glycoprotein is more extensively glycosylated and migrates on SDS-PAGE gels with an apparent mass of 170 kDa. The lack of cysteines did not appear to contribute significantly to instability of the protein since similar amounts of wild-type and mutant forms of the enzyme were expressed, with no significant difference in the amount of proteolytic degradation products.
Figure 2: Expression of wild-type and Cys mutants of P-glycoprotein (P-gp) in Sf9 insect cells. Membranes were prepared from cells infected with baculovirus (Control) or recombinant virus coding for wild-type P-glycoprotein or the Cys mutants. The membranes were subjected to SDS-PAGE, electrophoretically transferred to nitrocellulose, and probed with monoclonal antibody A52.
Figure 3:
Effect
of verapamil on the ATPase activity of membranes prepared from infected
cells. Membranes were prepared from Sf9 cells expressing wild-type
P-glycoprotein-A52 (), Cys-less P-glycoprotein (
), mutant
Cys-431 (
), mutant Cys-1074 (
), mutant Cys-1125 (
),
mutant Cys-1227 (+), or infected with wild-type virus (
). The
ATPase activity was measured in the presence of various concentrations
of verapamil as described under ``Experimental
Procedures.''
Figure 4:
Effect of NEM on verapamil-stimulated (panel A) and on the constitutive ATPase activity (panel
B) of membranes prepared from infected cells. Membranes were
prepared from Sf9 cells expressing wild-type P-glycoprotein-A52
(), Cys-less P-glycoprotein (
), mutant Cys-431 (
),
mutant Cys-1074 (
), mutant Cys-1125 (
), or mutant
Cys-1227 (
). NEM was added at various concentrations, and samples
were incubated for 10 min at room temperature. The reaction was stopped
by passage through 1-ml centrifuge columns (Penefsky, 1977) containing
Sephadex G-50 (Pharmacia). The ATPase activity was measured in the
presence of 25 µM verapamil (panelA) or
no added drug substrate (panelB) as described under
``Experimental Procedures.'' The results are expressed
relative that measured in the absence of NEM (100%) and have been
corrected to account for basal ATPase activity found in membranes from
Sf9 cells infected with control
baculovirus.
It is possible that NEM inhibition of drug-stimulated ATPase activity could be different from that of NEM inhibition of the constitutive ATPase activity of P-glycoprotein. Accordingly, we tested the effect of NEM on the ATPase activity of wild-type P-glycoprotein and the Cys mutants in the absence of verapamil. Fig. 4B shows that the pattern of inhibition of ATPase activity by NEM is similar to that observed in the presence of verapamil (Fig. 4B).
Figure 5: Labeling of Cys-less P-glycoprotein (P-gp) and mutants Cys-431 and Cys-1074 with biotin maleimide. Labeling with biotin-maleimide was carried out as described under ``Experimental Procedures.'' The concentration of biotin maleimide (µM) and position of P-glycoprotein-A52 are indicated.
The presence of MgATP has been shown to prevent inhibition of the ATPase activity of P-glycoprotein by NEM (Al-Shawi et al., 1994). Accordingly, we tested whether ATP could also prevent inhibition of the ATPase activity of the Cys mutants by NEM. Fig. 6shows the results of the ATP protection assays. The activities of wild-type and mutants Cys-431 and Cys-1074 P-glycoprotein were all protected from inactivation by NEM in the presence of MgATP. In all three cases, maximal protection was achieved in the presence of 2 mM ATP.
Figure 6:
Inhibition of NEM inactivation by ATP.
Membranes were prepared from Sf9 cells expressing Cys-less
P-glycoprotein (), mutant Cys-431 (
), or mutant Cys-1074
(
). ATP was added at various concentrations followed by
addition of either 10 µM NEM (Cys-less, Cys-1074) or 100
µM NEM (Cys-431), and samples were incubated for 10 min at
room temperature. The reaction was stopped by passage through 1-ml
centrifuge columns containing Sephadex G-50 (Penefsky, 1977). The
ATPase activity was measured in the presence of 25 µM verapamil as described under ``Experimental
Procedures.'' The results are expressed relative to that measured
in the absence of NEM (100%) and after correcting for basal ATPase
activity found in the membranes of Sf9 cells infected with control
baculovirus.
The ATPase activities of mutants Cys-1125 and Cys-1227 were relatively insensitive to NEM (Fig. 4). These results suggest that labeling of either of these cysteine residues by NEM either did not interfere with function or that they may be inaccessible to covalent modification. To distinguish between these two possibilities, membranes prepared from Sf9 cells expressing equivalent amounts of P-glycoprotein-A52 were labeled in the presence of 10 µM biotin maleimide. As shown in Fig. 7, mutants Cys-431 and Cys-1074 were again modified by biotin maleimide, while little or no labeling was observed with the Cys-less mutant or mutants Cys-1125 or Cys-1227. These results suggest that residues Cys-1125 and Cys-1227 are inaccessible to biotin maleimide and are likely to be hidden beneath the surface of the nucleotide-binding domain.
Figure 7: Labeling of NEM-insensitive P-glycoprotein Cys mutants with biotin maleimide. Labeling of membranes prepared from cells expressing the Cys mutants of P-glycoprotein with 10 µM biotin maleimide followed by immunoprecipitation with monoclonal antibody A52 and detection with horseradish peroxidase-conjugated streptavidin were carried out as described under ``Experimental Procedures.''
The results of this study show that inactivation of the ATPase activity of P-glycoprotein by NEM occurs through covalent modification of cysteine residues within the homology A regions of the nucleotide-binding domains. In this study, inactivation refers to the loss of ATPase activity. The relationship between ATPase activity and substrate transport has not yet been resolved. In some cases, there is a good correlation between the ability of a substrate to be transported by P-glycoprotein and its ability to stimulate ATPase activity. For example, Homolya et al.(1993) showed that transport of various fluorescent compounds by P-glycoprotein correlated with their ability to stimulate ATPase activity of P-glycoprotein. Recently, however, Sharom et al.(1995) reported that the level of stimulation of the ATPase activity of P-glycoprotein by hydrophobic peptides did not correlate with their affinity as transport substrates. In addition, it appears that the extent of ATPase stimulation by various drug substrates can be influenced by the host lipid environment (Doige et al., 1993; Urbatsch and Senior, 1995). Further studies will be needed to clarify these issues.
Inactivation of P-glycoprotein is due to the introduction of a bulky group into the consensus nucleotide-binding sequence rather than due to removal of a critical sulfhydryl group. This is in contrast to the results of Azzaria et al.(1989), who showed a conservative mutation of the lysine residues (to arginine) within the consensus nucleotide-binding sequences resulted in a protein that was unable to confer resistance to cytotoxic drugs in transfected cells. In a subsequent study, however, this mutant was found to be active when expressed in Escherichia coli (Bibi et al., 1993). There appears, however, to be some differences in the structure surrounding each nucleotide-binding fold, since higher levels of NEM were required to inactivate the ATPase activity of mutant Cys-431 relative to that of mutant Cys-1074. The two domains are not identical as they share approximately 43% amino acid identity (Choi et al., 1988), and this may influence the reactivity of Cys-431 and Cys-1074 to NEM.
The results of our study
also support the findings of Al-Shawi et al.(1994), who
reported that maximal inactivation occurred with the incorporation of
approximately two NEM per P-glycoprotein molecule, with an equal
distribution of radioactive NEM between the NH- and
COOH-terminal halves of P-glycoprotein. An interesting observation in
this study was that covalent modification of only one of the cysteine
residues in the homology A regions in either half of P-glycoprotein was
sufficient to inactivate the protein. Protection from inactivation by
NEM was afforded by ATP in each case. These results indicate that both
nucleotide-binding domains are capable of binding ATP and that both
domains may bind ATP for drug stimulation of ATPase activity to occur.
The reason for the presence of two ATP-binding regions is unknown.
Several possibilities exist (Gottesman et al., 1994; Senior et al., 1995). One of the sites may function as a regulator,
and the second may be catalytic in nature. Both sites may be needed
because they may participate in sequential steps during the transport
cycle. Indeed, in the cystic fibrosis transmembrane regulator, it was
recently shown that the NH-terminal nucleotide-binding
domain plays a catalytic role, while the COOH-terminal
nucleotide-binding domain functions as a regulator (Hwang et
al., 1994; Carson et al., 1995). In other members of the
ATP-binding cassette family of transporters, the presence of two intact
ATP-binding domains also seems to be necessary for transport. For
example, elimination of either one of the two domains of the Opp
oligopeptide transporter abolishes function (Hiles et al.,
1987). Similarly, mutations introduced into either one of the two
ATP-binding domains of STE6 inhibits function (Berkower and Michaelis,
1991). Some ATP-binding cassette transporters such as the histidine
permease have identical ATP-binding domains, suggesting that each
domain plays a similar role (Kerppola et al., 1991).
The finding that only the cysteines within the homology A regions of the nucleotide-binding domains are accessible to covalent modification makes them ideal targets for reporter molecules for studying conformational changes during transport. These studies are currently in progress.