Hypersensitivity to diphtheria toxin by mouse cells expressing both ...

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Proc. Natl. Acad. Sci. USA Vol. 90, pp. 8184-8188, September 1993 Medical Sciences

Hypersensitivity to diphtheria toxin by mouse cells expressing both diphtheria toxin receptor and CD9 antigen (heparin-binding epidermal growth factor precursor/Vero cells/replica plate assay)

JACQUELINE G. BROWN, BRIAN D. ALMOND, JOSEPH G. NAGLICH*, AND LEON EIDELSt Department of Microbiology, The University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235

Communicated by R. John Collier, June 10, 1993

growth factor (HB-EGF) (6, 7). In addition, we isolated a stable DT-sensitive (DTs) mouse cell line (DTs-II) by transfection of L-M(TK-) cells with the cloned receptor cDNA (4). The DTs-II cells (i) are as highly toxin-sensitive [concentration of DT that results in 50%o inhibition of protein synthesis (ICso) w5 ng/ml] as Vero cells, one of the most toxin-sensitive cell lines (8), and (ii) display =z3-fold more specific toxin receptors (=50,000 vs. %20,000) than Vero cells [which have the largest receptor density of any known cultured cells (9)] but with "40-fold lower affinity than Vero cell receptors. Mekada's laboratory isolated a monoclonal antibody that protected Vero cells from DT-mediated cytotoxicity (10). More recently they cloned a gene whose expressed cellsurface product (Mr ==27,000) is recognized by this monoclonal antibody (11). The deduced amino acid sequence of this protein revealed it to be the monkey homologue of the human CD9 antigen. Interestingly, transfection of mouse L-M(TK-) cells with the cDNA encoding CD9 did not result in DTs mouse cells, suggesting that CD9 is not a DT receptor per se. However, CD9 transfection of moderately toxin-sensitive human-mouse hybrid cells possessing human chromosome 5 [believed to encode the human DT receptor (12)] resulted in increased toxin sensitivity. The number of toxin-binding sites per cell increased from 500 (in the human-mouse hybrid cells) up to 2600 (in the most toxin-sensitive CD9 transfectant), a level of receptor density significantly below that displayed on the surface of Vero cells (4, 9, 11). Furthermore, there appeared to be a correlation between quantity of CD9 expressed on different cell lines and toxin sensitivity (10, 11). It was therefore of interest to investigate whether transfection of the already highly toxin-sensitive DTs-II mouse cells with the gene encoding the monkey CD9 homologue would result in (i) a further increase in toxin sensitivity, (ii) a further increase in the number of DT receptors, and/or (iii) the expression of a class of receptors having an increased toxin affinity comparable to that of Vero cells. We report here that transfection of the highly toxin-sensitive DTs-II cells with the cDNA encoding the monkey CD9 antigen, indeed, results in DT-hypersensitive cells that possess a very large number of specific toxin receptors (p106 per cell) having an unaltered affimity for DT. MATERIALS AND METHODS Materials. All chemicals utilized were of the highest purity and obtained from previously reported sources (4, 5, 13), except for fetal bovine serum and hygromycin B, which were purchased from Hazelton Biologicals, Inc. (Lenexa, KS) and

DTs-ll is a highly diphtheria toxin (DT)ABSTRACT sensitive cell line previously isolated by transfection of wildtype DT-resistant mouse L-M(TK-) cells with the cDNA encoding a monkey Vero cell DT receptor. DTs-II cells are as toxin-sensitive as Vero cells, have =z3-fold more receptors than Vero cells, and have =10-fold lower affinity for DT than Vero cells. We now cotransfected DTs-ll cells with a plasmid contining the Vero cell cDNA coding for CD9 antigen (pCD9) and with a plasmid containing the gene for hygromycin resistance (pHyg). The stably transfected hygromycin-resistant colonies were screened for DT hypersensitivity employing a replica plate system. A DT-hypersensltive colony was isolated and purified. The purified DT-hypersensitive cells, DTs-M, (s) are 10-fold more toxin-sensitive than DTs-ll and Vero cells and (ii) bear -106 DT receptors per cell (i.e., -20-fold and =60-fold more receptors than DTs-ll and Vero cells, respectively), but their receptor affinity is still =10-fold lower than that of Vero cells. Cross-linking experiments employing 12SI-labeled DT demonstrated that DTs-fl and DTs-HI cells have essentially the same profile of DT-binding cell-surface protein(s), suggesting that CD9 antigen, although expressed on the cell surface of DTs-Ill cells, may not be in close proximity to the DT-binding domain of the receptor. CD9 may affect DT receptor expression by increasing receptor density at the cell surface. By employing DTs-II cells it should be possible to puriy and characterize the DT cell-surface receptor protein(s).

Diphtheria toxin (DT) is a M, 58,342 protein, secreted by Corynebacterium diphtheriae, that inhibits protein synthesis in toxin-sensitive eukaryotic cells. The toxin can undergo limited proteolysis to yield a disulfide-linked polypeptide composed of two fragments: the enzymatic A fragment (Mr 21,167) and the receptor-binding and translocation-mediating B fragment (Mr 37,195). The cytotoxic action of DT occurs by the following steps: (i) binding to specific cell-surface receptors, (ii) internalization of the (toxin-receptor) complexes into vesicles, and (iii) translocation of the A fragment from acidified vesicles into the cytosol, where it inhibits protein synthesis by ADP-ribosylation of elongation factor 2 (1-3). Our laboratory has recently used expression cloning to identify a receptor for DT (4). The gene encoding the receptor was cloned by transfecting wild-type toxin-resistant mouse L-M(TK-) cells with a cDNA library prepared from highly toxin-sensitive monkey Vero cells; the transfectants were screened for DT sensitivity employing a replica plate system that allowed for the detection of those mouse cells whose protein synthesis is inhibited upon exposure to DT and that, at the same time, preserved a "replica" of those cells (4, 5). From the deduced amino acid sequence of the cloned DT receptor protein (Mr 20,652), we found a 97% identity with the precursor ofthe human heparin-binding epidermal growth factor-like

Abbreviations: DT, diphtheria toxin; HB-EGF, heparin-binding epidermal growth factor-like growth factor; DTs, DT-sensitive; HygR, hygromycin-resistant; ICso, concentration of DT that results in 50%6 inhibition of protein synthesis. *Present address: Oncology Drug Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb Co., P.O. Box 4000, Princeton, NJ 08543-4000. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Medical Sciences: Brown et al. from Calbiochem, respectively. pcDNA1 vector and Escherichia coli MC1061/P3 were purchased from Invitrogen Corp. pHyg vector was a gift from David Russell (The University of Texas Southwestern Medical Center). Sonicated salmon sperm DNA and horseradish peroxidase-conjugated goat antimouse IgG were purchased from Sigma. DNA restriction enzymes were purchased from Boehringer Mannheim. Dideoxy sequencing was performed by using the Sequenase Version 2.0 sequencing kit (United States Biochemical). Taq I polymerase was from Perkin-Elmer/Cetus. Na'25I (IMS 30, 13-17 ,Ci/pg, 1 Ci = 37 GBq), dATP[a-35S] (>1000 Ci/ mmol), L-[35S]methionine (>800 Ci/mmol), and L-[4,5,3H]leucine (60 Ci/mmol) were from Amersham. Ultrapure agarose was obtained from Bio-Rad. Polyester PeCap HD7-17 membranes (referred to as PeCap membranes in the text) were from Tetko (Elmsford, NY). DT was from Connaught Laboratories, purified and radioiodinated as described (14). Monoclonal anti-human CD9 IgG (BU16, 0.2 mg/ml) was purchased from The Binding Site (Birmingham, U.K.). Disuccinimidyl suberate and 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril (lodo-Gen) were obtained from Pierce. CD9 Cloning and Sequencing. Purified plasmid DNA containing a monkey Vero cell cDNA library (5) was employed for PCR cloning of Vero CD9. Two PCR oligonucleotides (synthesized at the Department of Molecular Cardiology, The University of Texas Southwestern Medical Center) were designed to correspond to the 5' and 3' flanking regions of CD9 based on the human CD9 sequence reported by Lanza et al. (15). The 5' (5'-GGCTGCAGAACAGGCTAAGTTAGCCCTCACC-3') and the 3' (5'-GGGGATCCGGCCTGCTCAGGGATGTAAGCTG-3') oligonucleotides were engineered to include unique Pst I and BamHI restriction sites, respectively. PCR was performed with Taq I polymerase in a Perkin-Elmer DNA thermal cycler 480 utilizing the following reaction conditions: 94°C for 1 min for strand separation, 50°C for 1 min for annealing, and 72°C for 1 min for primer extension (a total of 30 cycles). Following PCR amplification and digestion with Pst I and BamHI restriction enzymes, the fragment was isolated from an agarose gel and subcloned into the pcDNAl vector (pCD9). pCD9 was then transformed into E. coli strain MC1061/P3. To sequence the cloned CD9, pCD9 was digested with Pst I and BamHI restriction enzymes and subcloned into M13mpl8 and M13mpl9, the CD9 antisense and sense strands, respectively. The universal primer and two additional primers that anneal to internal regions of CD9 were required to complete the dideoxy single-stranded DNA sequencing of Vero CD9. Within the 684-nucleotide CD9 open reading frame sequenced, 15 nucleotide differences were identified between Vero and human CD9 (p24/CD9) (15). Most of the differences, however, did not change the deduced amino acid sequence, with the exception of 3 nucleotides, which resulted in two amino acid changes: Vero CD9 contains Asp at amino acid 151, corresponding to an Asn in human CD9, and Vero CD9 contains Ile at amino acid 178, corresponding to Val in human CD9. These two amino acid differences were also found by Mitamura et al. (11). CD9/Hygromycin Cotransfection of L-M(TK-) and DTs-II Cells. Wild-type DT-resistant mouse fibroblast cells [L-M(TK-) (CCL 1.3)] and DTs-II cells (4) were employed for the cotransfection experiment. Cotransfection of pCD9 and pHyg DNA was performed as described by Graham et al. (16). Supercoiled DNA of pCD9 and pHyg was used at 2 ,ug/ml and 0.1 ug/ml, respectively; sonicated salmon sperm DNA (1 pg/ml) was used as nonspecific carrier DNA. Selection for hygromycin-resistant (HygR) transfectants was accomplished in culture medium (Dulbecco's modified Eagle medium containing 10lo fetal bovine serum, 50 units of penicillin per ml, 50 pg of streptomycin per ml, 2.5 .g of amphotericin B per ml, and 2 mM L-glutamine) supplemented

Proc. Natl. Acad. Sci. USA 90 (1993)

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with 1 mg of hygromycin B per ml (selection medium). L-M(TK-)/CD9/HygR cells were isolated and purified directly from the transfection plates, whereas the DTs-II/CD9/ HygR cells were purified employing a replica plate system (as described below). Isolation of L-M(TK-)/CD9 cells. HygR cells were removed from the L-M(TK-) transfection plates by overlaying colonies with BBL blank paper discs saturated with trypsin. The discs were then transferred to six-well plates containing selection medium. The cells were initially screened for CD9 expression by ELISA, employing anti-CD9 IgG (as described below). CD9-positive cells were further purified by the isolation and propagation ofcells from single colonies. Each subsequent cell population was screened employing the ELISA until no further enrichment of CD9 expression was obtained. A purified stable cell line, L-M(TK-)/CD9, obtained in this fashion, was employed in additional ELISA experiments to quantify the concentration of CD9 on the cell surface. Isolation of DTs-H/CD9 Transfectants. Isolation of DThypersensitive DTS-II/CD9 cells proceeded through the use of the replica plate system previously described (4, 5, 13), with slight modifications. Briefly, HygR colonies were overlaid with PeCap membranes and incubated at 37°C in 5% CO2 for 7 days. This allowed the cells to grow into the PeCap membranes, producing a replica of the colonies present on the transfection plate. One membrane was utilized to screen for the presence of colonies hypersensitive to DT by assaying for inhibition of [35S]methionine incorporation into protein. The assay was modified in the following fashion: rather than incubating the PeCap membranes with DT at 2 jg/ml for 18 hr, they were incubated with 0.01 ,g/ml for 1.5 hr, conditions under which DTs-II cells appear to be resistant to DT. Cells from a DT-hypersensitive colony were subsequently subcultured and purified twice with the modified replica plate system. A purified DTs-II/CD9 stable cell line (DTs-III) was employed for all subsequent experiments. Except for the fact that DTs-III cells grow slightly slower than DTs-II cells, they do not appear different than DTs-II cells by light microscopy. Quantification of CD9 by ELISA. To screen for CD9, a whole cell ELISA was developed. Briefly, 96-well plates were seeded at 4 x 104 cells per well. After overnight incubation at 37°C in 5% C02, the medium was removed and the cells were fixed in a 10% formalin solution for 45 min at 4°C. The monolayers were washed three times with phosphate-buffered saline containing Tween 20 (PBST; 8.8 mM Na2HPO4/1.2 mM KH2PO4/140 mM NaCl/10 mM KCI/ 0.05% Tween 20, pH 7.4). To prevent nonspecific adsorption, the monolayers were incubated with blocking solution (PBST containing 0.2% bovine serum albumin) for 1 hr at 4°C. The monolayers were washed once in PBST and then incubated for 1 hr at 4°C with a 1:1000 dilution of anti-CD9 IgG in blocking solution. Following three additional washes in PBST, the monolayers were incubated for 1 hr at 4°C with a 1:1000 dilution (in blocking solution) of horseradish peroxidase-conjugated goat anti-mouse IgG. The monolayers were washed three times with PBST and incubated with 50 pl of substrate solution per well (3 mM o-phenylenediamine dihydrochloride in 0.66 M Na2HPO4/0.347 M citric acid/0.01% H202, pH 5.0). The colorimetric reaction was followed by measuring the change in absorbance at 490 nm. To confirm the cell densities, three control wells per plate were trypsinized and the number of cells per well was determined. The change in absorbance per cell was calculated and plotted vs. time (min); the change in absorbance per cell per min was calculated from the linear portion of the graph. Cytotoxicity Assay. The ability of DT to inhibit protein synthesis was determined by assaying for the incorporation of [3H]leucine into trichloroacetic acid-precipitable material as described (17, 18).

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strating that anti-CD9 reactivity is not acquired upon transfection with pHyg alone. Increased DT Sensitivity Due to CD9 Expression. To determine the extent to which CD9 expression affects DT sensitivity, an in vitro cytotoxicity assay was employed (17, 18). The results obtained (Table 1) demonstrate that Vero and DTs-II cells possess IC50 values of 4.0 and 4.2 ng/ml, respectively, values similar to our previously published results (4). In contrast, DTs-III cells display a 10-fold increase in DT sensitivity. Importantly, the IC50 value of DTs-II/hyg cells does not differ from that of DTs-II cells (Table 1), indicating that hygromycin resistance itself has no effect on DT sensitivity. Furthermore, L-M(TK-)/CD9 cells were not sensitive to DT, confirming the report by Mitamura et al. (11) RESULTS AND DISCUSSION that the expression of CD9 alone, in the absence of a Isolation of DT-Hypersensitive Mouse Cells. In the present DT-specific receptor, does not result in DT sensitivity. From studies, we wanted to determine whether expressing the Vero these results it is clear that coexpression of the DT receptor cell CD9 antigen in highly toxin-sensitive DTs-II cells would and CD9 antigen increases DT sensitivity of DTs-III cells by increase the sensitivity of these cells to DT. A modification about 10-fold as compared to the already highly toxinof the replica plate system previously described (4, 5, 13) was sensitive Vero and DTs-II cells. employed to isolate a hypersensitive cell line. We first Binding of Radiolabeled DT to Cell-Surface Receptors of determined the conditions under which DTs-II cells would DTs-M Cells. In the present studies DTs-III cells were found appear resistant to DT. We found that DTs-II cells appeared to be hypersensitive to DT. To determine whether the resistant when exposed to 0.01 ug of DT per ml for only 1.5 increased sensitivity is due to an increase in the number of hr. DTs-II cells were then cotransfected with pCD9 and receptors and/or to the expression of a class of receptors with pHyg, and HygR cells were screened for DT hypersensitivity higher affinity for DT, the cells were assayed for their ability under the just described conditions. In this fashion, a stable to bind 125I-labeled DT, and the data were subjected to HygR DT-hypersensitive cell line was established. This cell Scatchard analyses. DTs-III cells were found to bind 125I. line, referred to as DTs-III, was used for subsequent experlabeled DT in a highly specific and saturable manner (Fig. iments to determine the level of CD9 expression, the degree 1C). Typically, the level of nonspecific binding in these cells of DT sensitivity, and the number and nature of toxin was 10,000 4.2 ± 1.8 0.49 ± 0.03 >10,000 4.1 ± 1.7

binding sites per cellt 18,000 0§ 48,000 1,040,000 0§ 50,400

Kd, M 1.2 x 10-9 1.5 x 10-8 1.8 x 10-8

ND ND, not determined. *Calculated from the ELISA by utilization of anti-CD9 monoclonal antibody, as described in Materials and Methods; values are the average of seven independent experiments. NR, no anti-human CD9 antibody reactivity was found. tValues are the average of five independent experiments. *The number of 125I-labeled DT-binding sites per cell was determined from the Scatchard plots shown in Fig. 1. §No specific binding could be determined. ¶Stable HygR control cell line established from DTs-II cells transfected with pHyg only.

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FIG. 1. Specific binding of radiolabeled DT to Vero (A), DTs-II (B), and DTs-III (C) cells. Cells were incubated at 4°C with 125I-labeled DT in the absence and in the presence of a 100-fold excess of unlabeled DT in binding medium. After 4 hr, the cells were washed with ice-cold PBS/Ca/Mg, and the cell-associated radioactivity was assayed. The binding data were normalized per 5 x 104 cells. Specific binding (triangles) was determined by calculating the difference between the total binding with 125I-labeled DT (circles) and the nonspecific binding obtained with 125I-labeled DT in the presence of excess unlabeled DT (squares). (Insets) Scatchard analysis of the data presented in each panel. The concentrations of specifically bound 1251-labeled DT (B) are plotted on the abscissa and bound/free toxin (B/F) is on the ordinate. The data were fitted by regression analysis. The experiments shown were performed in triplicate and on the same day. The specific activity of the 1251-labeled DT used was 1.2 x 107 cpm/pg. Note that the ordinate in C differs from that in A and B.

binding sites, we wanted to determine whether CD9 antigen is in close physical association with DT-binding proteins. To study the spatial arrangement of CD9 and 125I-labeled DTbinding surface proteins, we employed a homobifunctional, noncleavable cross-linking reagent (disuccinimidyl suberate). Upon covalent cross-linking of 125I-labeled DT to DTs-II and DTs-III cells and analysis by SDS/PAGE, a number of high molecular weight bands in addition to the major DT band (M, 60,000)

were

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(Fig. 2A). When

DTs-II and DTs-III

cells were analyzed under nonreducing conditions, nearly identical cross-linking patterns were observed. The apparent M,s of the cross-linked, high molecular weight bands were -86,000 (I), -82,000 (II), and -70,000 (III) (Fig. 2A, lanes 1 and 3). These proteins correspond (by difference) to three toxin-binding proteins with Mrs of =26,000 (I), =22,000 (II), and =10,000 (III). Importantly, the addition of a 100-fold excess unlabeled DT during the binding of 125I-labeled DT completely inhibited the appearance of these bands (Fig. 2A, lanes 2 and 4), demonstrating the specificity of binding of 17-5I-labeled DT to DT-binding proteins. When the same crosslinked extracts were analyzed under reducing conditions, the major DT band had a M, of -58,000. The high molecular weight, cross-linked proteins had Mrs of -86,000 (I), %78,000 (II), and "'70,000 (III) (Fig. 2A, lanes 5 and 7). These results are consistent with DT-binding proteins having Mrs of --28,000, -20,000, and =z12,000, respectively. Similar analyses were performed with Vero and DTs-III cell extracts (Fig. 2B). Under nonreducing conditions, the major DT band had a M, of =58,000, and similar cross-linked protein profiles were observed between Vero and DTs-III cells. The appearance of the cross-linked proteins could be inhibited in the presence of a 100-fold excess unlabeled DT. The high molecular weight bands observed in extracts of DTs-III and Vero cells included proteins having Mrs of ==80,000 (I), %72,000 (II), and =66,000 (III) (Fig. 2B, lanes 1 and 3). These correspond to DT-binding proteins having Mrs of --22,000, =14,000, and -"8000, respectively. Under reducing conditions, the major DT band had a Mr of -58,000 and the high molecular weight proteins in DTs-III and Vero cell extracts had Mrs of "'82,000 (I), ''74,000 (II), and =66,000 (III) (Fig. 2B, lanes 5 and 7). These correspond to DT-binding proteins having Mrs of -24,000, =16,000, and =8000, respectively. Taken together, these results demonstrate that minor differences exist in the 125I-labeled DT cross-linked

surface proteins in Vero, DTs-II, and DTs-III cell extracts. However, since the cross-linked patterns from DTS-II and DTs-III cells were essentially identical (in Fig. 2A and in three other similar experiments), it seems unlikely that any of the cross-linked bands constitutes a complex of 125I-labeled DT with the receptor and CD9 or of 125I-labeled DT with CD9. Utilizing the conditions in this study, it seems that CD9 is not in close proximity to the DT-binding site of the receptor. Our original hypothesis was that transfection of DTs-II cells with CD9 would result in increased sensitivity. Indeed, our selection strategy was based on this hypothesis. We have demonstrated that transfection of DTs-II cells with Vero CD9 results in DT-hypersensitive cells, DTs-III, displaying only =2-fold more CD9 antigen than Vero cells and bearing 106 receptors per cell. DTs-III cells have =60-fold more toxinbinding sites than Vero cells but are only '40-fold more sensitive to DT (Table 1). This apparent discrepancy may be due to a 10-fold lower-affinity receptor on DTs-III cells or to the existence of another step in the cytotoxicity process that determines the degree of sensitivity in cells expressing a large excess of toxin-binding sites. Mitamura et al. (11) had previously reported that transfection of human-mouse hybrid cells, possessing human chromosomes 5 and 22, with Vero cell CD9 resulted in cells with affinity similar to that of Vero cells. Based on their results, we expected that expression of Vero cell CD9 in mouse DTs-II cells would result in receptors of increased affinity; however, DTs-III cells displayed receptors having the same affinity as DTs-II cells. The difference between our affinity results and those ofMitamura et al. could be due to the presence of additional factor(s) in Vero cells and human-mouse hybrid cells (possibly encoded by human chromosome 5 or 22) but absent in DTs-III cells. We obtained cross-linking of DT to cell-surface proteins ranging in Mr from 8000 to 28,000 (Fig. 2). The DT-binding proteins may represent differentially modified forms of the DT receptor or subunits of a receptor complex. If these proteins represent subunits of a receptor complex, they do not appear to be covalently linked by disulfide bonds since analyses of the cross-linked samples in the absence and in the presence of reducing agent were essentially the same (Fig. 2). If there are subunits in the receptor complex, our results do not provide direct evidence that CD9 is one of the subunits since analysis of the cross-linked proteins derived from DTs-II and DTs-III cells appears essentially identical (Fig. 2A). Since

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FIG. 2. Cross-linking of 125I-labeled DT to surface proteins of Vero, DTs-II, and DTs-III cells. Cells were incubated at 4°C for 4 hr with 125I-labeled DT (250 ng/ml) alone (-) or in the presence of a 100-fold excess of unlabeled DT (+) in binding medium. The cells were chemically cross-linked using disuccinimidyl suberate and the cross-linked proteins were separated by SDS/PAGE (8%) and analyzed by autoradiography. A (DTs-II and DTs-III cells) and B (Vero and DTs-III cells) are representative but independent experiments. The samples were analyzed without prior reduction (lanes 1-4) and following reduction with 5% 2-mercaptoethanol prior to electrophoresis (lanes 5-8). The lanes represent lysates containing an equivalent amount of radioactivity (cpm). The molecular weight markers are given on the left margin of each panel (Mr X 10-3): myosin (200), ,-galactosidase (116), phosphorylase b (97), bovine serum albumin (66), and ovalbumin (43). Positions of the cross-linked DT-binding proteins are denoted by I, II, and III.

anti-CD9 antibodies have been shown to protect cells against DT (10, 11), we propose that this protection could be the result of steric hindrance due to binding of the antibodies to cell-surface CD9 epitopes in the vicinity of the DT receptor. Except for the fact that CD9 is involved in platelet activation (19), possibly due to its association with small GTPbinding proteins (20), the role of CD9 is poorly understood.

The role of CD9 in DT receptor expression, in particular, is also not clear. Possible roles can be envisioned: (i) CD9 may allow for the increased translocation of DT receptors to the cell surface or (ii) CD9 may extend the half-life of DT receptors at the cell surface. The latter could be accomplished either by inhibiting the normal proteolytic processing that the DT receptor undergoes as a precursor of HB-EGF or by affecting the rate of DT receptor/HB-EGF precursor recycling. Regardless, DTs-III cells-the most DT-sensitive cells hitherto described-will be valuable in further studies of toxin-receptor interactions due to their very low nonspecific binding. Furthermore, the fact that these cells bear =w106 receptors per cell will facilitate the purification and further characterization of the cell-surface protein(s) that DT appropriates in order to gain access into eukaryotic cells. Note Added in Proof. Preliminary experiments indicate that the levels of DT receptor mRNA in DTs-II and DTs-III cells are essentially the same, suggesting that CD9 does not affect transcription of DT receptor mRNA. We thank Robert S. Munford and Kyle P. Hooper for critical review of the manuscript. The editorial assistance of Eleanor R. Eidels is greatly appreciated. This research was supported by U.S. Public Health Service Grant AI-16805. 1. Collier, R. J. (1975) Bacteriol. Rev. 39, 54-85. 2. Eidels, L., Proia, R. L. & Hart; D. A. (1983) Microbiol. Rev. 47, 5%-620. 3. Middlebrook, J. L. & Dorland, R. B. (1984) Microbiol. Rev. 48, 199-221. 4. Naglich, J. G., Metherall, J. E., Russell, D. W. & Eidels, L. (1992) Cell 69, 1051-1061. 5. Naglich, J. G., Rolf, J. M. & Eidels, L. (1992) Proc. Natl. Acad. Sci. USA 89, 2170-2174. 6. Higashiyama, S., Abraham, J. A., Miller, J., Fiddes, J. C. & Klagsbrun, M. (1991) Science 251, 936-939. 7. Higashiyama, S., Lau, K., Besner, G. E., Abraham, J. A. & Klagsbrun, M. (1992) J. Biol. Chem. 267, 6205-6212. 8. Middlebrook, J. L. & Dorland, R. B. (1977) Can. J. Microbiol. 23, 183-189.9. Middlebrook, J. L., Dorland, R. B. & Leppla, S. H. (1978) J. Biol. Chem. 253, 7325-7330. 10. Iwamoto, R., Senoh, H., Okada, Y., Uchida, T. & Mekada, E. (1991) J. Biol. Chem. 266, 20463-20469. 11. Mitamura, T., Iwamoto, R., Umata, T., Yomo, T., Urabe, I., Tsuneoka, M. & Mekada, E. (1992) J. Cell Biol. 118, 1389-1399. 12. Creagan, R. P., Chen, S. & Ruddle, F. H. (1975) Proc. Natl. Acad. Sci. USA 72, 2237-2241. 13. Naglich, J. G. & Eidels, L. (1990) Proc. Natl. Acad. Sci. USA 87, 7250-7254. 14. Cieplak, W., Gaudin, H. M. & Eidels, L. (1987) J. Biol. Chem. 262, 13246-13253. 15. Lanza, F., Wolf, D., Fox, C. F., Kieffer, N., Seyer, J. M., Fried, V. A., Coughlin, S. R., Phillips, D. R. & Jennings, L. K. (1991) J.

Biol. Chem. 266, 10638-10645. 16. Graham, F. L., Bacchetti, S., McKinnon, R., Stanners, C., Cordell, B. & Goodman, H. M. (1980) in Introduction of Macromolecules into Viable Mammalian Cells, eds. Baserga, R., Croce, C. & Rovera, G. (Liss, New York), Vol. 1, pp. 3-25. 17. Proia, R. L., Eidels, L. & Hart, D. A. (1981) J. Biol. Chem. 256, 4991-4997. 18. Eidels, L. & Hart, D. A. (1982) Infect. Immun. 37, 1054-1058. 19. Slupsky, J. R., Seehafer, J. G., Tang, S.-C., Masellis-Smith, A. & Shaw, A. R. E. (1989) J. Biol. Chem. 264, 12289-12293. 20. Seehafer, J. G. & Shaw, A. R. E. (1991) Biochem. Biophys. Res. Commun. 179, 401-406.