©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Evidence That Transmembrane 12 and the Loop between Transmembrane 11 and 12 Form Part of the Drug-binding Domain in P-glycoprotein Encoded by MDR1 (*)

(Received for publication, September 2, 1994; and in revised form, December 15, 1994)

Xiaoping Zhang (§) Karen I. Collins Lee M. Greenberger (¶)

From the Oncology and Immunology Research Section, Lederle Laboratories, American Cyanamid Company, Pearl River, New York 10965

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Human MDR1 encodes an ATP-binding cassette transporter, P-glycoprotein, that mediates multiple drug resistance (MDR) to antitumor agents. It has been previously shown that photoaffinity drug-labeling sites reside within, or near, the last transmembrane loop of each cassette within P-glycoprotein (transmembrane domains (TM) 5-6 and 11-12). A genetic approach was used to determine if the drug-labeling site in the second cassette contains functionally important amino acids. Since human MDR3 is 77% identical to MDR1 but does not mediate MDR, the region from TM10 to the C terminus of MDR1 was replaced with the corresponding sequences from MDR3. The resultant chimeric protein was expressed but not functional. By using progressively smaller replacements, we show that replacements limited to TM12 markedly impaired resistance to actinomycin D, vincristine, and doxorubicin, but not to colchicine. The phenotype was associated with an impaired ability to photoaffinity label the chimeric P-glycoprotein with [I]iodoaryl azidoprazosin. In contrast, replacement of the loop between TM11 and 12 appears to create a more efficient drug pump for actinomycin D, colchicine, and doxorubicin, but not vincristine. These results suggest that, similar to voltage-gated ion channels, amino acids within and immediately N-terminal to the last transmembrane domain of P-glycoprotein compose part of the drug-binding pocket and are in close proximity to photoaffinity drug-labeling domains.


INTRODUCTION

P-glycoprotein is a plasma membrane protein which is believed to utilize ATP to export, and thereby mediate multiple drug resistance (MDR) (^1)to, widely used anticancer drugs including anthracyclines, taxanes, Vinca alkaloids, mitoxantrone, and etoposide(1, 2, 3) . It is one of many ATP-binding cassette (ABC) proteins which contain six putative transmembrane (TM) alpha-helices and an ATP-binding motif(4) . As with many ABC transporters, P-glycoprotein contains and requires two cassettes to form a functional unit(1, 4) . Other ABC transporters permit the movement of a variety of small molecules through the membrane including Cl (the cystic fibrosis transmembrane conductance regulator), peptides associated with antigen processing (TAP proteins), and multiple hydrophobic anticancer drugs (the multidrug resistance-associated protein)(4, 5) . Indeed, P-glycoprotein itself may also be a volume-regulated Cl channel(6) , an efflux pump for ATP(7) , and a pH regulator(8) . On a broader level, many membrane proteins contain one or more cassettes, which are composed of six putative transmembrane alpha-helices, and participate in the transport of a variety of small molecules including ions (e.g. voltage-gated ion channels), sugars, peptides, and neurotransmitters(9, 10) .

P-glycoprotein overexpression has been associated with a poor outcome in the treatment of some cancers(11) . In addition, a variety of small molecules, such as verapamil, dihydropyridines, forskolin, and cyclosporine A, bind to P-glycoprotein and inhibit its function(12) . Therefore, the clinical utility of non-toxic derivatives of these and other molecules as chemosensitizing agents has been studied intensively (12) . However, the molecular basis of interaction of anticancer drugs and inhibitors with P-glycoprotein are poorly understood.

We have attempted to define drug-binding domains in P-glycoprotein, and thereby to understand how multiple agents are exported by, or inhibit, P-glycoprotein. Based on photoaffinity labeling studies, probes derived from anticancer drugs or chemosensitizing agents are preferentially competed by vinblastine > doxorubicin > colchicine(13, 14) . Azidopine (a photoactive Ca channel blocker), and photoaffinity analogs of forskolin and prazosin bind to small common domains (15, 16) that are present in each cassette of P-glycoprotein(17, 18) . The photoaffinity labeling domains map to TM5-6 (forskolin analog;(15) ) and within, or immediately C-terminal to, TM6 and TM12 (prazosin analog;(19) ). Similarly, TM6 and the loop between TM5-6 (known as the H5, P, or SS1-SS2 region) of cassettes which compose voltage-gated Ca, Na, and K channels form part of the ion pore and contain receptor sites for inhibitors and photoaffinity labeled drugs(9, 20, 21, 22) . This may help explain why some agents, such as verapamil and azidopine, inhibit both P-glycoprotein and the L-type Ca channel.

Here we examine if the photoaffinity drug-labeling domains of P-glycoprotein contain amino acid residues that mediate resistance to anticancer drugs. To do this, we have made and evaluated the function of chimeric P-glycoproteins encoded by human MDR1 and MDR3. This method is logical since, in man, P-glycoprotein is encoded by two highly related genes, MDR1 and MDR3, which share 77% amino acid identity and similar putative topology(23, 24) . (MDR3 is also known as MDR2(25) ). However, only MDR1, or its mouse equivalent, mdr1 and mdr3 (also known as mdr1a and mdr1b(26) ), mediates MDR in transfected cells(27, 28, 29, 30, 31) . The human MDR3 homologue in the mouse, mdr2, appears to be a translocase for phospholipids (32, 33) . Functional differences between MDR1 and MDR3 may be mediated by regions of greatest amino acid diversity between these genes. Such regions are located in the transmembrane domains as well as the so-called linker region connecting each cassette. Consistent with this, chimeric P-glycoproteins composed of human MDR1 and MDR3 (34) or mouse mdr1 and mdr2(35) , were defective or had altered ability to mediate MDR if a large span of transmembrane domains or the first intracytoplasmic loop (between TM2 and 3) from MDR3 replaced the corresponding region in MDR1, respectively. However, the nucleotide binding regions in mdr2 (which is highly conserved between all MDR genes) were functional in the mdr1 environment(35) .

In this report, we focused on the second cassette in P-glycoprotein. In particular, sequences from TM10 to the C terminus, or subdivisions within this region, from human MDR3 replaced the corresponding region in human MDR1. We find that TM12 and the loop between TM11 and TM12 of P-glycoprotein is intimately involved in substrate specificity and alters the ability to photoaffinity label P-glycoprotein.


MATERIALS AND METHODS

Cells

The human melanoma cell line, SK-Mel-21(36) , was obtained from Dr. M. Eisinger (Lederle Laboratories, Pearl River, NY). A clone of SK-Mel-21, designated SK2, was used for transfection experiments. Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 5000 units/liter of penicillin, and 5 mg/liter streptomycin. Two sets of cell lines that overexpressed endogenous human MDR1 were used as controls: a bisantrene-resistant line designated S1-B1-20 (derived from the colon carcinoma cell line LS174T)(37) , and drug-resistant cell lines known as KB-85 and KB-V1 (gifts from Drs. Pastan and Gottesman, National Cancer Institute, Bethesda, MD)(38) .

Vector Construction

To construct the vector containing MDR genes, the pcDNA1 vector (Invitrogen, San Diego, CA) was modified to create expression vector pcDNA1tkpASP (obtained from Dr. Tom Jones, Lederle Laboratories, Pearl River, NY). This allowed the placement of the first intron of the human cytomegalovirus (HCMV) major IE gene (39) downstream of the HCMV IE enhancer/promoter, but just 5` of the multiple cloning site of pcDNA1tkpASP. Genes cloned in pcDNA1tkpASP are expressed at a much higher level than those using pcDNA1. (^2)For MDR1 insertions, a unique SacII site was introduced into the multiple cloning site of pcDNAItkpASP between SacI and BamHI by site-directed mutagenesis(40) . Human MDR1 cDNA was cut at its flanking SacII and XhoI sites from pHaMDR 1/A (obtained from Drs. Gottesman and Pastan(41) ) and ligated into the SacII/XhoI-cut pcDNA1tkpASP backbone, resulting in pCMVHMDR 1. Site-directed mutagenesis was used to return the G185V mutation in pHaMDR 1A to the wild-type codon (see (42) ).

To facilitate the construction of chimeric MDR 1/3 genes, pCMVHMDR1 underwent several rounds of site-directed mutagenesis to introduce and eliminate several restriction enzyme sites (Fig. 1). This resulted in pCMVHMDR1C, D, E, and F. In all cases, no changes in amino acid identity occurred. Compared to pCMVHMDR1, pCMVHMDR1C had 1) a HindII site introduced at the bases encoding amino acid residues 856 and 857 of MDR1, and 2) two PstI sites eliminated; one at the multiple cloning site and the other in the beta-lactamase gene. Compared to pCMVHMDR1C, pCMVHMDR1D had a KpnI site eliminated at the multiple cloning site. Compared to pCMVHMDR1D, pCMVHMDR1E had a PstI site introduced at bases that correspond to amino acid residue 954 of MDR1. Compared to pCMVHMDR1E, pCMVHMDR1F had a PstI site eliminated at bases encoding amino acid residue 1125 in MDR1.


Figure 1: Amino acid sequence alignment from residue 858 to the C terminus of human P-glycoprotein encoded by MDR1 with the homologous region (from residue 857 to the C terminus) of human MDR3 gene product. Dashes represent amino acid identity between the gene products. Brackets above sequence indicates predicted TM domains or the Walker motifs A and B of the nucleotide binding fold. Vertical lines inserted in the sequence indicate the positions of restriction enzymes sites used to construct chimeric P-glycoprotein genes. Chimeric construct K was made by replacing MDR1 with MDR3 in the region spanning the KpnI to the second PstI site. Hatched bars indicate the position and sequence of mutagenesis primers used with the K construct to create chimeric construct R, Q, and P. These primers were also used in combination to create chimeric constructs M, N, and O.



Plasmid pHepG2-16 contains a human MDR3 cDNA fragment (43) and was obtained from the American Type Culture Collection (Rockville, MD). A 21-base pair insert in the MDR3 cDNA fragment in pHepG2-16 was eliminated by site-directed mutagenesis before being used for construction of chimeric genes. Chimeric expression vectors pMDR1/3 I, H, G, J, K, and L were constructed by ligation of inserts from pHepG2-16 and corresponding backbones from pCMVHMDR1C, D, or E. Prior to ligation, construction of pMDR1/3 G and I required the introduction of an XhoI site, by the polymerase chain reaction, at the 3` non-translated region of the MDR3 cDNA fragment in pHepG2-16. Construction of pMDR1/3 K, required the introduction of a KpnI site in MDR3 cDNA fragment in pHepG2-16 prior to ligation. This necessitated the conversion of two consecutive codons at residues 924 and 925 from MDR3 identity to MDR1 identity (see Fig. 1). As a result, codon 926 in pMDR1/3 K and its derivatives, pMDR1/3 R, Q, P, M, O, and N, was valine instead of glycine. Chimeric expression vectors pMDR1/3 R, Q, P, M, O, and N were constructed from pMDR1/3 K by site-directed mutagenesis with the mutagenesis primers shown in Fig. 1. Under this condition, preservation of Leu from MDR3 was allowed in constructs pMDR1/3 R, Q, P, M, N, and O (Fig. 1). No attempt was made to return this base pair change to MDR1 identity, since point mutation of MDR1 at this residue (I1003L) did not alter the drug resistance phenotype compared to wild-type MDR1 (data not shown). All constructs were verified by DNA sequencing.

Transfection of Mammalian Cells

DNA transfection of SK2 melanoma cells was performed by calcium phosphate precipitation according to published methods (44) using a kit from Life Technologies, Inc. DNAs used for transfection were prepared with Midiprep kit from Qiagen (Studio City, CA). Ten µg of plasmid DNA plus 15 µg of carrier DNA were transfected into SK2 cells plated at 0.5 million cells/100-mm culture dish. Twenty-four h after transfection, the transfection medium was replaced with tissue culture medium. At 48 h post-transfection, the plate of cells was passaged at a 1:10 dilution. At 72 h post-transfection, two plates were treated with 3 nM of vincristine, and another two plates were treated with 15 nM of colchicine. (These values were determined empirically and allowed selection of SK2-transfected cells that express exogenous P-glycoprotein, but not endogenous P-glycoprotein in mock transfected cells). The plates were replaced with fresh drug-containing media every 4 days thereafter until resistant colonies were cloned at day 12 post-exposure to the drugs. Under these conditions, plates transfected with a reporter plasmid, pSVbetaGAL (obtained from Promega, Madison, WI), did not produce any resistant colonies. Staining pSVbetaGAL-transfected plates indicated that the transfection efficiency was usually about 100 cells/µg of plasmid DNA.

Analysis of P-glycoprotein Expression

For estimation of P-glycoprotein cell surface levels by fluorescence analysis, cells were washed, treated with trypsin (Life Technologies, Inc.) for 1 min, and harvested from the flask. Five-hundred-thousand cells/100 µl were resuspended in phosphate-buffered saline containing 5% fetal bovine serum (solution A) with or without 2 µg of monoclonal antibody 4E3.16 (45) for 1 h at 4 °C. Following repeated washing in solution A, cells were incubated in a 1:20 dilution of fluorescein-conjugated goat anti-rabbit Fab(2) (EY Laboratories, Burlingame, CA) for 1 h at 4 °C. Cells were washed, resuspended in 0.5 ml of solution A, and analyzed using fluorescence detection. Analysis was done on a FACScan 440 (Becton Dickinson, CA) using a 488 nm emission wavelength and a 517 nm excitation filter.

The level of P-glycoprotein was also estimated by immunoblot analysis using crude membranes. To prepare crude membranes from cell lines, 10^7 cells were washed in phosphate-buffered saline and then lysed in 1 ml of 10 mM Tris, pH 8.0, 10 mM NaCl, 1 mM MgCl(2) in the presence of a protease inhibitor mixture (100 units/ml aprotinin, 30 µM leupeptin, 1 µg/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride) for 20 min at 4 °C. All subsequent steps were conducted at 4 °C. After homogenization with a Tenbrock mortar and pestle (30 strokes), the suspension was centrifuged for 10 min at 450 times g. The supernatant was centrifuged at 100,000 times g for 1 h. Subsequently, the pellet was resuspended in 10 mM Tris, pH 7.4, in the presence of 100 units/ml aprotinin. Protein was determined by the Lowry method(46) . Proteins within crude membranes were resolved on gels and transferred to nitrocellulose. Blots were probed by a peptide-directed antibody (1:500) that specifically recognized the sequence after the Walker B-motif of the second cassette in P-glycoprotein (obtained from Oncogene Sciences Inc, Uniondale, NY). Bound antibody was detected by using 1:2000 donkey anti rabbit Ig whole antibody linked to horseradish peroxidase (Amersham Corp.), followed by ECL detection (Amersham) using XAR-5 film (less than 1 min). P-glycoprotein levels were quantitated using an image scanner (Molecular Devices, Menlo Park, CA).

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for Detection of Vector Expression

RT-PCR was carried out with GeneAmp RNA PCR kit from Perkin Elmer Cetus following the manufacturer's procedure. Total cellular RNA (prepared using RNAzol, Biotecx, Houston, TX) was used as template for RT-PCR. To detect endogenous mRNA for P-glycoprotein, the published primer pair 1A/1AP (47) was used. To detect mRNAs in stably transfected cells (i.e. mRNAs expressed from exogenously introduced expression vectors), primer pair 1A/135B was used. Primer 135B has the following sequence: TTTTTTTTTTTTTTGCGTTTTATTCTGTCTTTTTAT. PCR parameters for 1A/1AP pairs were: 94 °C for 1 min, followed by 50 cycles of 94 °C for 30 s, 55 °C for 40 s, and 72 °C for 40 s, ending with a 5-min extension at 72 °C. PCR parameters for 1A/135B were modified from those for 1A/1AP in the cycle number (45 versus 50) and annealing temperature (57 versus 55 °C). The two RT-PCR products were verified by their sizes on agarose gel and DNA sequencing.

Characterization of Resistance Profiles

The amount of drug required to kill 50% of the cells (ED) after 3 days continuous exposure was determined in 96-well plates using the sulforhodamine B assay as described previously(37) . To compare the resistance between cell lines transfected with exogenous genes, it was necessary to minimize the effect of differences in P-glycoprotein expression level between cells expressing distinct chimeric molecules. To do so, the ED of a transfected cell line (X) is normalized against the ED of a cell line transfected with a wild-type (wt) MDR1 gene (pCMVHMDR1F) by their P-glycoprotein expression levels according to the following equation: Normalized ED(X) = ED(X) times [FL(wt) - FL(SK2)]/[FL(X) - FL(SK2)] where FL is the mean channel number of events detected by flow cytometry using the procedure described above for cell surface P-glycoprotein expression. (FL(SK2) is an estimate of the amount of fluorescent signal detected in non-transfected cells and is negligible.) Then, the normalized relative resistance compared to cells transfected with the wild-type gene was determined by the following equation: [normalized ED(X)/ED(wt)] times 100. A similar standardization of resistance based on relative P-glycoprotein expression using immunoblotting was used in a study comparing 32 transfected cell lines(48) .

Photoaffinity Labeling of P-glycoprotein

One-hundred µg of crude membrane was incubated in 6 nM [I]iodoaryl azidoprazosin (2200 Ci/mmol; DuPont NEN) as described previously(16) . After 1 h of incubation at 25 °C, material was subjected to ultraviolet irradiation (lamp no. R-52G; UVP, San Gabriel, CA) for 5 min at 4 °C. Then, samples were solubilized in Laemmli sample buffer, resolved on SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose. Radioactive bands were identified by autoradiography (approximately 6 h of exposure). The amount of radiolabel incorporated into the band that migrated in the position of P-glycoprotein was determined by densitometric scanning as described above. Alternatively, radiolabel incorporated into P-glycoprotein was estimated by excising the region known to contain P-glycoprotein and counted in a gamma counter. Either measurement produced the same relative results. The relative amount of radiolabel bound to P-glycoprotein was determined by following equation: % relative radiolabel (x) = ([cpm (x) - cpm (SK2)]/[cpm (WT) - cpm (SK2)]) times 100, where x is the transfected cell line, and cpm (WT) and cpm (SK2) correspond to the amount of radiolabel found in each cell line in the position of P-glycoprotein, respectively. (SK2 cells do not contain P-glycoprotein, but the amount of radiolabel in the expected position of P-glycoprotein was used to subtract the amount of nonspecific binding.) Where densitometric analysis was done, volume integration replaced the cpm determination. These results were compared with the relative amount of P-glycoprotein that was assessed by immunoblot analysis using densitometric scanning described above and identical quantitative analysis as just described.


RESULTS

The experimental strategy initially required expression of wild-type MDR1 in drug-sensitive cells. Therefore, a clonal cell line derived from human melanoma cells was transfected with MDR1. Multiple cell colonies were established and evaluated. One clone, SK2-1F8, was established as a standard with which future constructs were compared. Relative to SK2, 1F8 cells were resistant to colchicine, vincristine, actinomycin D, and doxorubicin, but not to the non-MDR drug, cisplatin (Table 1).



P-glycoprotein expressed in SK2-1F8 cells was detected by fluorescent staining using an antibody, 4E3.16, specific for a cell surface epitope in P-glycoprotein (Fig. 2A) as well as by immunoblot analysis (see below). No P-glycoprotein was found in SK2 cells by either method. Since the human MDR1 gene was used to transfect human cells, we were concerned that the selection procedures used to clone such transfected cells might permit the elevation of endogenous MDR1. While this seemed unlikely since no colonies arose in mock transfected cells, RT-PCR analysis was used to test this possibility (Fig. 2B). In this case, RT-PCR exploited the differences in the 3`untranslated region between the endogenous and exogenous MDR mRNAs. The primer pair specific for endogenous MDR1 was able to detect a strong signal in KB-85 cells. These cells are known to have low level expression of P-glycoprotein and low level resistance(38) . No signal was detected in drug-sensitive KB cells or SK2-1F8 cells. A very weak signal was detected with SK2 cells. In contrast, the primer pair specific for exogenous MDR1 was able to detect exogenous MDR mRNA only in SK2-1F8 cells. Sequence analysis of the resultant PCR products verified that the products came from the expected genes (data not shown).


Figure 2: P-glycoprotein expression in transfected cells. Panel A, expression of P-glycoprotein on the surface of non-transfected cells (SK2) and cells transfected with MDR1 (SK2-1F8). Cells were incubated in 4E3.16 as described and analyzed by fluorescence analysis. Histogram displays the number of cells that bound the 4E3.16 antibody as indirectly monitored by fluorescent intensity. Panel B, RT-PCR analysis of endogenous and exogenous MDR gene expression. Primer pairs specific for the 3`-untranslated region of MDR1 (endogenous gene; primer pair 1A/1AP) or the expression vector (exogenous gene; primer pair 1A/135B) were used to amplify reversed-transcribed mRNA from the indicated cell line. Control cell lines were KB and its drug-selected counterpart, KB-85. PCR product was resolved on a 1% agarose gel. Standard markers are from top to bottom: 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234-220, and 154 base pairs.



A series of MDR3 replacements within the MDR1 environment was done (Fig. 3). After transfection of SK2 cells and subsequent selection, individual colonies were expanded and then evaluated for resistance to actinomycin D, colchicine, vincristine, and doxorubicin using 3-day survival assays. P-glycoprotein levels varied from 0.4 to 1.5 times the amount found in cells transfected with MDR1. Therefore, the amount of resistance was normalized based on P-glycoprotein levels. The data were most striking for actinomycin D resistance. The largest replacement, which spanned Thr to the C terminus (Leu), contained 72 amino acid differences ( Fig. 1and 3) and is designated construct pMDR1/3 I (or I). In this case, resistance to actinomycin D (as well as the other three drugs) was markedly impaired. The I region was subdivided into the H and G regions which contained 59 and 13 amino acid differences compared to MDR1, respectively. The H construct encoded a P-glycoprotein that was defective for actinomycin D resistance. By sequential subdivision of the H and K regions, the defect in actinomycin D resistance was localized to TM12 which contained 6 different amino acids compared to MDR1 (construct P; see Fig. 1and Fig. 3). In contrast, replacement of the TM 11 region from MDR3 (construct R), which has only six differences compared to MDR1, had no effect on actinomycin D resistance. Surprisingly, the chimera that contained the loop between TM11 and 12 from MDR3 (construct Q) was associated with a 2.7-fold elevation in actinomycin D resistance above that found in cells transfected with MDR1. As shown below in immunoblot analysis, in cells transfected with the Q construct, P-glycoprotein was expressed at approximately 0.36-fold of the wild-type levels. Since the amount of actinomycin D required to kill 50% of 1F8 and Q3 cells was similar (21 ± 2.9 versus 22.6 ± 3.3; mean ± S.E.; n = 5 or 7, respectively), P-glycoprotein encoded by construct Q behaved as a more efficient drug efflux pump compared to wild-type MDR1. Similar results were obtained with another P and Q clonal cell line (data not shown).


Figure 3: Effects of chimeric P-glycoproteins on actinomycin D resistance in transfected cells. Bottom left, proposed orientation of human MDR1 based on hydropathy plot (see Refs. 63, 64 for modifications of this model). Upper left, map of replacement from TM10 to the C terminus of MDR1 with the corresponding region from MDR3 is shown. Top line shows the C-terminal one-third of P-glycoprotein encoded by MDR1. Three filled rectangles represent TM10, 11, and 12. Two ovals indicate the Walker A and B consensus sites within the nucleotide binding fold (NBF). Hatched bars indicate replacements from MDR3. Selected restriction sites (dashed lines) in the nucleotide sequence were either present in MDR1 or created by site-directed mutagenesis and were used to allow replacement with the corresponding region from MDR3. Replacement regions from MDR3 are coded by letters indicated to the right. The number after letter indicates clone number. Checkered bar at the bottom of the replacement map indicates the proposed photoaffinity drug-binding site for iodoaryl azidoprazosin identified by immunological mapping(19) . Upper right, drug resistance profile of cells transfected with MDR1/3 constructs compared to MDR1. The amount of actinomycin D resistance was determined as described in Table 1. This value was adjusted to account for difference in P-glycoprotein expression and then compared with the resistance found in cells transfected with wild-type MDR1 (SK2-1F8 cells, abbreviated as 1F8). Resistance in non-transfected cells (SK2) is shown for comparison. Values are mean ± S.E., n = 3-6 independent experiments except for construct I where n = 2. Significant differences compared to 1F8 cells were determined by two-tailed Student's t test (* p < 0.05,** p < 0.01,*** p < 0.005).



A detailed drug resistance profile of chimeric proteins with mutations in the TM11-TM 12 region was compiled (Fig. 4). The most striking observation was that replacement of TM 12 from MDR3 (construct P) impaired resistance to vincristine and doxorubicin. However, colchicine resistance was normal. Therefore, the TM12 region of MDR3 is capable of mediating resistance to at least one P-glycoprotein substrate. The loop between TM11 and 12 mediated supernormal levels of resistance for colchicine and doxorubicin, but no enhancement of vincristine resistance was observed.


Figure 4: Resistance profile of P-glycoproteins with replacement in the TM11-12 region. Resistance to colchicine, doxorubicin, and vincristine were evaluated in chimeric P-glycoproteins that contain a replacement of TM11, the loop between TM11-12, and TM12 from MDR3. Methodology is described in the legend to Fig. 3. Comparisons were made with cells transfected with full-length MDR1 (WT). The resistance profile to actinomycin D is repeated from Fig. 3for comparison.



If the loop between TM11-12 and TM12 alone has a positive and negative effect on actinomycin D resistance, respectively, these regions may interact to form a drug binding domain in P-glycoprotein. Therefore, we determined if the effects of TM12 and the loop between TM11 and TM12 were additive. The TM11 region from MDR3, since it had no effect on resistance, was used a control. To do this, constructs M, N, and O were transfected into SK2 cells (Fig. 3, lower portion of the graph). Expression of construct M, which encodes TM11 and TM12 from MDR3, resulted in a defect for actinomycin D resistance. Construct O, which encodes TM11 plus the loop between TM11 and TM12 from MDR3, mediated a 2.9-fold increase in resistance to actinomycin D compared to 1F8 cells. However, construct N, which encodes TM12 plus the loop between TM11 and TM12 from MDR3, mediated normal levels of resistance to actinomycin D compared to 1F8 cells. Therefore, the neutral, positive and negative effects on drug resistance from TM11, the loop between TM11 and TM12, and TM12 regions, respectively, were additive. Less prominent, but consistent effects were seen with doxorubicin, colchicine, or vincristine (data not shown).

Since a major photoaffinity drug labeling site in P-glycoprotein has been localized to the TM11-12 region (see Fig. 3), the photoaffinity drug binding capacity of chimeric P-glycoproteins was investigated (Fig. 5). Characterization was limited to the TM11-12 region. To properly interpret the results, both the photoaffinity drug binding capacity and the amount of P-glycoprotein need to be compared. Therefore, after photoaffinity labeling with [I]iodoaryl azidoprazosin, material was resolved on gels and transferred to nitrocellulose. Blots were quantitated for both immunoreactivity and photolabeling of P-glycoprotein. S1 cells and their drug-resistant counterpart designated S1-B1-20, which highly overexpress MDR1 P-glycoprotein, were used as controls(37) . It was found that all cell lines that expressed chimeric genes also expressed P-glycoprotein which could be photoaffinity labeled (Fig. 5A). The binding was specific since labeling of the species that migrated at the position of P-glycoprotein was competed by 20 µM vinblastine (Fig. 5B). In the chimera that contained TM11 from MDR3, which was expressed in the R1 cell line, both the amount of photoaffinity labeling and immunoreactive P-glycoprotein were increased 80% above the level found in wild-type MDR1 (Fig. 5C). However, replacement of TM12 from MDR3 markedly inhibited photolabeling since the level of P-glycoprotein in the P2 cell line was 15% higher than wild-type P-glycoprotein, yet photoaffinity labeling was actually reduced 75% compared to wild-type P-glycoprotein. In chimeras that contained the TM11-12 loop from MDR3, expressed in the Q3 cell line, the P-glycoprotein level was reduced, but photoaffinity labeling of P-glycoprotein was reduced even more than expected. Similar results occurred with the Q2 cell line (data not shown).


Figure 5: Photoaffinity labeling of P-glycoprotein. Panel A, crude membranes from the indicated cell lines were photoaffinity labeled with [I]iodoaryl azidoprazosin. After gel electrophoresis and transfer to nitrocellulose, the resultant labeled proteins were detected by autoradiography (6 h of exposure). The blot was subsequently reacted with antibody for the detection of P-glycoprotein and detected by the ECL method using a 15-s exposure. Panel B, photoaffinity labeling as in panel A in the presence (+) or absence(-) of 20 µM vinblastine. Panel C, quantitative analysis of photoaffinity labeled P-glycoprotein encoded by MDR1/3 chimeras. The relative amount of P-glycoprotein (as determined by densitometic scans of immunoblots) or photoaffinity labeled P-glycoprotein was determined and compared. Values (mean ± S.E.; n = 3 independent experiments) were standardized to cells that expressed wild-type MDR1 (1F8).




DISCUSSION

Previously, we and other others have shown that TM11-12 of P-glycoprotein, and the corresponding region of the first cassette, TM5-6, contain photoaffinity labeling sites which are likely to form part of the drug binding domain in P-glycoproteins that mediate MDR (human MDR1, and mouse mdr1 and mdr3)(15, 17, 19) . Because of the technical limitations with the immunological mapping studies of drug binding sites, and in particular, the multiple conformations that photoaffinity labeled molecules can assume(49) , the drug labeling site may or may not contain residues that form the actual drug binding domain in P-glycoprotein. Therefore, a genetic approach was used to further this analysis. The genetic approach used here demonstrates that mutations in TM12 and the loop between TM11 and TM12 of MDR1 can alter the specificity of P-glycoprotein for anticancer drugs. These data verify and refine the current model. However, we still cannot rule out the possibility that these regions indirectly participate in drug efflux (e.g. by inducing conformational changes at a distant site within P-glycoprotein).

Additional evidence argues that TM5-6 and TM11-12 form part of the drug-binding domain in P-glycoprotein (although TM4, TM10, and the cytoplasmic loop between TM2 and TM3 also play functional roles(34, 50) ). This includes point mutations in TM6 and TM12 of human MDR 1 (F335A and F978A)(48) , in TM11 of mouse mdr genes (S941F of mdr1 or S943F of mdr3)(51) , and two consecutive mutations (G338A and A339P) in TM6 of hamster pgp1(50) , which cause complex perturbations in the drug resistance phenotype mediated by P-glycoprotein. Beyond this, point mutations in the fifth and sixth transmembrane domains of cassettes within other ABC transporters (cystic fibrosis transmembrane conductance regulator, plasmodium falciparum MDR, and the Drosophilia melanogaster eye color genes) either alter specificity or are associated with a loss in function of these proteins (53, 54, 55) . Future analysis of double mutants in TM5-6 and TM11-12 of P-glycoprotein is likely to further define the interaction between these regions.

Since the ``mutations'' were created in a cassette-like fashion using replacement regions from MDR3, the data may also help explain why MDR3 does not mediate drug resistance(30) . Human MDR3 may not be able to transport certain antitumor drugs because of the 6 amino acid residues within TM12 that differ from those in MDR1. Consistent with this, the mouse homolog of MDR3, designated mdr2, participates in the translocation of phosphotidylcholine in the membrane while one of the mouse genes that mediates MDR (mdr3) is incapable of this function(31, 33) . The amino acid residues in mouse mdr2 and human MDR3 are perfectly conserved within TM12. Whether or not residues in this region are critical for lipid translocation needs to be investigated.

The mutations in TM12 and the loop between TM11-12 are associated with fundamentally different phenotypes. Compared to cells transfected with wild-type P-glycoprotein, replacement in TM12 was associated with markedly impaired resistance to actinomycin D, vincristine, and doxorubicin, but not colchicine as well as reduced photoaffinity labeling of P-glycoprotein (construct P). Similar to this, a point mutation within TM12 of MDR1 eliminated resistance to all of these drugs(48) , and mouse mdr2 can be expressed at high amounts but had considerably reduced binding to the same photoaffinity probe used here (31) . In contrast, alteration of the loop between TM11-12 was associated with approximately a 2.5-fold reduction in P-glycoprotein expression but a 2-3-fold elevation in resistance to actinomycin D, doxorubicin, and colchicine. Therefore, the latter mutant protein behaves as a more efficient pump for certain drugs, although the molecular basis for this observation is unknown. A similar observation has been made when the two mouse genes that mediate MDR were compared (56) . These two regions are likely to interact with each other, since positive and negative effects on resistance were additive. Additivity between point mutations within other proteins is common(57) .

The molecular alterations induced by these mutations in P-glycoprotein probably alter the contact points between amino acids and substrates (or inhibitors) for the protein. This could influence initial drug binding, translocation of bound drug through the plasma membrane, release of drug, as well as energy coupling between ATP hydrolysis and the site of energy utilization. Consistent with this, mutations in TM12 within MDR1 (shown here) or TM11 from mouse mdr1 or mdr3 impair the ability of P-glycoprotein to mediate MDR to some drugs and bind photoaffinity analogs(58) . In contrast, the G185V point mutation (59) and the TM11-12 replacement (shown here) are associated with an increase in drug resistance to certain drugs and a decrease in photoaffinity labeling of P-glycoprotein. The mechanistic implications of changes in photoaffinity labeling of P-glycoprotein are not clear and will need further kinetic analysis with reversible ligand-binding and/or transport methods. This is true since photoaffinity labeling is not reversible, the fate of photoactivated ligands can be altered by changes in the local lipid environment, and covalent linkage of the photolabel to P-glycoprotein is known to be an inefficient process.

Limitations of the Chimeric Approach

An alternative explanation for impaired P-glycoprotein function in some chimeric constructs is that mutations in TM11-12 may make P-glycoprotein unstable and lead to increased protein turnover and/or improper plasma membrane targeting. The latter phenomenon is found within a point mutation (albeit within the ATP binding region) of a P-glycoprotein homologue, the cystic fibrosis transmembrane regulator(60) . However, this possibility seems unlikely in our chimeric proteins since the amount of P-glycoprotein detected by surface labeling matches the amount found by immunoblotting in crude membrane preparations, and no aberrant molecular weight species (due to degradation or alterations in biosynthesis) were observed by immunoblot analysis. A second possibility is that the TM11-12 region encoded by MDR3 is not efficiently anchored in the plasma membrane. Consistent with this, the TM11-12 region from mouse homologue of human MDR3 does not efficiently insert into the membrane when this region is expressed by itself in an in vitro translation/translocation system(61) . In this assay system, non-conserved residues in the loop between TM11-12 are critical for proper insertion. Therefore, this may explain why alterations in the loop between TM11 and TM12, which occur in the Q-type P-glycoprotein, would not be expressed at normal levels. However, this does not explain the loss of activity in the P-type P-glycoprotein, since the changes in TM12 are conserved and suggest that the insertion into the membrane between wild-type and P constructs would be identical. Future studies of the membrane orientation of the loop between TM11 and TM12 and TM12 itself, when the entire protein is expressed, appear to be critical for understanding the interaction of selected substrates with P-glycoprotein.

The chimeric approach itself has certain unique limitations. It would not indicate if conserved residues between MDR1 and MDR3, such as Phe (in TM12 of MDR1) or the homolog of Ser (in TM11 of mdr1), which have been previously implicated, participate in drug resistance. Additionally, because of additivity, it is possible to miss regions of importance. Therefore, point mutation of divergent amino acids within TM12 or the loop between TM11-12 of MDR1 and MDR3 may or may not result in the same phenotype as the chimeric molecules.

Comparison with Voltage-gated Ion Channels

On a broader level, topological similarities have been noted between voltage-gated ion channels, which contain 1 (K channels) or 4 (Na and Ca channels) cassettes (I-IV) composed of six transmembrane domains (S1-S6) and P-glycoprotein(9, 10) . The pore in voltage-gated ion channels is likely to consist in part of the loop between the last two transmembrane domains (H5, P, or SS1-SS2 domains) (see (9) ), the S6 domain(20, 21) , since point mutations within these regions alter ion specificity and/or channel gating. Ca and K channel antagonists bind in close proximity to, or within, the P region and the S6 domains of these molecules(21, 22) . It now appears that homologous regions in P-glycoprotein interact with a variety of drugs. Therefore, a general rule may be that the TM5-6 region within cassettes composed of six transmembrane domains forms part of a three-dimensional pore in related types of pumps and ion channels(9) . This leads us to speculate that voltage-gated ion channels and ABC transporters may operate using related mechanisms. Subtle variations have evolved within these regions and can alter substrate specificity. It may also help explain why P-glycoprotein can behave as a drug efflux pump and a Cl channel(6, 62) . If this hypothesis is correct, this information could be used to guide the understanding of a wide variety of related proteins. Ultimately, a more complete understanding of drug-binding pores in such proteins will require structural resolution in three dimensions.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: New England Medical Center, Division of Geographic Medicine and Infectious Disease, Boston, MA 02111.

To whom correspondence should be addressed. Tel.: 914-732-3487; Fax: 914-732-5695.

(^1)
The abbreviations used are: MDR, multiple drug resistance; ABC, ATP-binding cassette; RT-PCR, reverse transcriptase-polymerase chain reaction; TM, transmembrane domain; WT, wild-type; cpm, counts/min.

(^2)
T. Jones, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. Sridhar Rabindran for critical review of this manuscript.


REFERENCES

  1. Gottesman, M. M., and Pastan, I. (1993) Annu. Rev. Biochem. 62, 385-427 [CrossRef][Medline] [Order article via Infotrieve]
  2. Ruetz, S., Raymond, M., and Gros, P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11588-11592 [Abstract]
  3. Ruetz, S., and Gros, P. (1994) J. Biol. Chem. 269, 12277-12284 [Abstract/Free Full Text]
  4. Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113 [CrossRef]
  5. Cole, S. P. C., Bhardwaj, G., Gerlach, J. H., Mackie, J. E., Grant, C. E., Almquist, K. C., Stewart, A. J., Kurz, E. U., Duncan, A. M. V., and Deeley, R. G. (1993) Science 258, 1650-1654
  6. Valverde, M. A., Diaz, M., Sepulveda, F. V., Gill, D. R., Hyde, S. C., and Higgins, C. F. (1992) Nature 355, 830-833 [CrossRef][Medline] [Order article via Infotrieve]
  7. Abraham, E. H., Prat, A. G., Gerweck, L., Seneveratne, T., Arceci, R. J., Kramer, R., Guidotti, G., and Cantiello, H. F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 312-316 [Abstract]
  8. Roepe, P. (1992) Biochemistry 31, 12555-12564 [Medline] [Order article via Infotrieve]
  9. Jan, L. Y., and Jan, Y. N. (1992) Cell 69, 715-718 [Medline] [Order article via Infotrieve]
  10. Greenberger, L. M., and Ishikawa, Y. (1994) Trends Cardiovas. Med. 4, 193-198
  11. Arceci, R. J. (1993) Blood 81, 2215-2222 [Medline] [Order article via Infotrieve]
  12. Yang, C., Greenberger, L. M., and Horwitz, S. B. (1991) in Synergism and Antagonism in Chemotherapy (Chou, T.-C., and Rideout, D. C., eds) pp. 311-338, Academic Press, New York
  13. Beck, W. T., and Qian, X. (1992) Biochem. Pharmacol. 43, 89-93 [Medline] [Order article via Infotrieve]
  14. Greenberger, L. M., Cohen, D., and Horwitz, S. B. (1994) in Anticancer Drug Resistance (Ozols, R. F., and Goldstein, L. J., eds) pp. 69-106, Norwell, Kluwer Academic Publishers, MA
  15. Morris, D. I., Greenberger, L. M., Bruggemann, E. P., Cardarelli, C., Gottesman, M. M., Pastan, I., and Seamon, K. B. (1994) Mol. Pharmacol. 46, 329-337 [Abstract]
  16. Greenberger, L. M., Yang, C.-P. H., Gindin, E., and Horwitz, S. B. (1990) J. Biol. Chem. 265, 4394-4401 [Abstract/Free Full Text]
  17. Bruggemann, E. P., Currier, S. J., Gottesman, M. M., and Pastan, I. (1992) J. Biol. Chem. 267, 21020-21026 [Abstract/Free Full Text]
  18. Greenberger, L. M., Lisanti, C. J., Silva, J. T., Horwitz, S. B. (1991) J. Biol. Chem. 266, 20744-20751 [Abstract/Free Full Text]
  19. Greenberger, L. M. (1993) J. Biol. Chem. 268, 11417-11425 [Abstract/Free Full Text]
  20. Ragsdale, D. S., McPhee, J. C., Scheuer, T., and Catterall, W. A. (1994) Science 265, 1724-1728 [Medline] [Order article via Infotrieve]
  21. Lopez, G. A., Jan, Y. N., and Jan, L. Y. (1994) Nature 367, 179-182 [CrossRef][Medline] [Order article via Infotrieve]
  22. Catterall, W. A., and Striessnig, J. (1992) Trends. Pharmacol. Sci. 13, 256-262 [CrossRef][Medline] [Order article via Infotrieve]
  23. Van der Bliek, A. M., Kooiman, P. M., and Borst, P. (1988) Gene (Amst.) 71, 401-411 [CrossRef][Medline] [Order article via Infotrieve]
  24. Chen, C., Chin, J. E., Ueda, K., Clark, D. P., Pastan, I., Gottesman, M. M., and Roninson, I. B. (1986) Cell 47, 381-389 [Medline] [Order article via Infotrieve]
  25. Roninson, I. B., Chin, J. E., Choi, K., Gros, P., Housman, D. E., Fojo, A., Shen, D., Gottesman, M. M., and Pastan, I. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4538-4542 [Abstract]
  26. Hsu, S., Lothstein, L., and Horwitz, S. B. (1989) J. Biol. Chem. 264, 12053-12062 [Abstract/Free Full Text]
  27. Devault, A., and Gros, P. (1990) Mol. Cell. Biol. 10, 1652-1663 [Medline] [Order article via Infotrieve]
  28. Ueda, K., Cardarelli, C., Gottesman, M. M., and Pastan, I. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 3004-3008 [Abstract]
  29. Lincke, C. R., van der Bliek, A. M., Schuurhuis, G. J., van der Velde-Koerts, T., Smit, J. J. M., and Borst, P. (1990) Cancer Res. 50, 1779-1785 [Abstract]
  30. Schinkel, A., Roelofs, M. E. M., and Borst, P. (1991) Cancer Res 51, 2628-2635 [Abstract]
  31. Bushman, E., and Gros, P. (1994) Cancer Res. 54, 4892-4898 [Abstract]
  32. Smit, J. J. M., Schinkel, A. H., Oude Elferink, R. P. J., Groen, A. K., Wagenaar, E., van Deemter, L., Mol, C. A. A. M., Ottenhoff, R., van der Lugt, N. M. T., van Roon, M. A., van der Valk, M. A., Offerhaus, G. J. A., Berns, A. J. M., and Borst, P. (1993) Cell 75, 451-462 [Medline] [Order article via Infotrieve]
  33. Ruetz, S., and Gros, P. (1994) Cell 77, 1071-1081 [Medline] [Order article via Infotrieve]
  34. Currier, S. J., Kane, S. E., Willingham, M. C., Cardarelli, C. O., Pastan, I., and Gottesman, M. M. (1992) J. Biol. Chem. 267, 25153-25159 [Abstract/Free Full Text]
  35. Bushman, E., and Gros, P. (1991) Mol. Cell. Biol. 11, 595-603 [Medline] [Order article via Infotrieve]
  36. Carey, T. E., Toshitada, T., Resnick, L. A., Oettgen, H. F., and Old, L. J. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3278-3282 [Abstract]
  37. Zhang, X. P., Ritke, M. K., Yalowich, J. C., Slovak, M. L., Ho, J. P., Collins, K. I., Annable, T., Arceci, R. J., and Greenberger, L. M. (1994) Oncol. Res. 6, 291-301 [Medline] [Order article via Infotrieve]
  38. Shen, D., Cardarelli, C., Hwang, J., Cornwell, M., Richert, N., Ishii, S., Pastan, I., and Gottesman, M. M. (1986) J. Biol. Chem. 261, 7762-7770 [Abstract/Free Full Text]
  39. Akrigg, A., Wilkinson, G. W., and Oram, J. D. (1985) Virus Res. 2, 107-121 [CrossRef][Medline] [Order article via Infotrieve]
  40. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 488-492 [Abstract]
  41. Pastan, I., Gottesman, M. M., Udea, K., Lovelace, E., Rutherford, A. V., and Willingham, M. C. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 4486-4490 [Abstract]
  42. Choi, K., Chen, C., Kriegler, M., and Roninson, I. B. (1988) Cell 53, 519-529 [Medline] [Order article via Infotrieve]
  43. Van der Bliek, A. M., Baas, F., Houte de Lange, T. T., Kooiman, P. M., Van der Velde-Koerts, I., and Borst, P. (1987) EMBO J. 6, 3325-3331 [Abstract]
  44. Chen, C., and Okayama, H. (1987) Mol. Cell. Biol. 7, 2745-2752 [Medline] [Order article via Infotrieve]
  45. Arceci, R. J., Stieglitz, K., Bras, J., Schinkel, S., Baas, F., and Croop, J. M. (1993) Cancer Res. 53, 310-317 [Abstract]
  46. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol Chem. 193, 265-275 [Free Full Text]
  47. Moore, R. E., Shepherd, J. W., and Hoskins, J. (1990) Nucleic Acids Res. 18, 1921 [Medline] [Order article via Infotrieve]
  48. Loo, T. W., and Clarke, D. M. (1993) J. Biol. Chem. 268, 19965-19972 [Abstract/Free Full Text]
  49. Glossmann, H., Ferry, D. R., Striessnig, J., Goll, A., and Moosburger, K. (1987) Trends Pharmacol. Sci. 8, 95-100 [CrossRef]
  50. Loo, T. W., and Clarke, D. M. (1993) J. Biol. Chem. 268, 3143-3149 [Abstract/Free Full Text]
  51. Gros, P., Dhir, R., and Talbot, F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7289-7293 [Abstract]
  52. Devine, S. E., Ling, V., and Melera, P. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 4564-4568 [Abstract]
  53. Anderson, M., P., Gregory, R., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991) Science 253, 202-205 [Medline] [Order article via Infotrieve]
  54. Foote, S. J., Kyle, D. E., Martin, R. K., Oduola, A. M. J., Forsyth, K., Kemp, D. J., and Cowman, A. F. (1990) Nature 345, 255-258 [CrossRef][Medline] [Order article via Infotrieve]
  55. Ewart, G. D., Cannell, D., Cox, G., and Howells, A. J. (1994) J. Biol. Chem. 269, 10370-10377 [Abstract/Free Full Text]
  56. Lothstein, L., Hsu, S. I.-H., Horwitz, S. B., and Greenberger, L. M. (1989) J. Biol. Chem 264, 16054-16058 [Abstract/Free Full Text]
  57. Wells, J. A. (1990) Biochemistry 29, 8509-8517 [Medline] [Order article via Infotrieve]
  58. Kajiji, S., Talbot, F., Grizzuti, K., Van Dyke-Phillips, V., Agresti, M., Safa, A. R., and Gros, P. (1993) Biochemistry 32, 4185-4194 [Medline] [Order article via Infotrieve]
  59. Safa, A. R., Stern, R. K., Choi, K., Agresti, M., Tamai, I., Mehta, N. D., and Roninson, I. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7225-7229 [Abstract]
  60. Welsh, M. J., and Smith, A. E. (1993) Cell 73, 1251-1254 [Medline] [Order article via Infotrieve]
  61. Zhang, J.-T., and Ling, V. (1993) Biochim. Biophys. Acta 1153, 191-202 [Medline] [Order article via Infotrieve]
  62. Altenberg, G. A., Vanoye, C. G., Han, E. S., Deitmer, J. W., and Reuss, L. (1994) J. Biol. Chem. 269, 7145-7149 [Abstract/Free Full Text]
  63. Zhang, J.-T., Duthie, M., and Ling, V. (1993) J. Biol. Chem. 268, 15101-15110 [Abstract/Free Full Text]
  64. Skach, W. R., Calayag, M. C., and Lingappa, V. R. (1993) J. Biol. Chem. 268, 6903-6908 [Abstract/Free Full Text]

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