1 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, Maryland 21201; and 2 Austin Research Institute, Austin and Repeat Medical Center, Heidelberg, Victoria 3084, Australia
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ABSTRACT |
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MUC1 mucin is a transmembrane glycoprotein that is highly expressed in various cancer cell lines and is also present in most of the glandular epithelial cells including the airway. Although the presence of numerous phosphorylation sites in its cytoplasmic domain suggests its potential role as a receptor, the unavailability of a ligand for MUC1 mucin has limited our understanding of its function. In this paper, we tried to circumvent this problem by constructing a chimeric receptor containing the cytoplasmic domain of MUC1 mucin, which can be phosphorylated on activation. To this end, we constructed a chimeric plasmid vector (pCD8/MUC1) by replacing the extracellular and transmembrane domains of human MUC1 mucin with those of human CD8. Transient transfection of the vector into COS-7 cells resulted in expression of the chimeric receptor on the surface of the COS-7 cells as judged by immunologic assays with various antibodies as well as by fluorescence-activated cell-sorting analysis. Treatment of the transfected COS-7 cells with an anti-CD8 antibody resulted in a significant increase in phosphorylation of tyrosine moieties of the chimeric receptor. This chimeric receptor will serve as a powerful tool in elucidating the signaling mechanism as well as the functional role of MUC1 mucin in the airway.
CD8; phosphorylation; signal transduction
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INTRODUCTION |
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MUCUS LINING THE AIRWAY EPITHELIAL SURFACE provides a protective barrier against various pathogens and toxins. The major component of mucus and the primary contributor to its viscoelastic property is a highly glycosylated, large-molecular-mass macromolecule known as mucin. To date, there have been nine mucin genes identified, most of which encode secreted mucins. MUC1, which encodes a membrane-associated mucin (1, 6, 11), was initially detected in various cancer cell lines and later in secretory epithelial cells lining the respiratory, reproductive, and gastrointestinal tracts (2-5). The deduced amino acid sequence of MUC1 mucin reveals four distinct domains: the NH2-terminal domain consisting of a hydrophobic signal sequence, a highly O-glycosylated tandem-repeat domain, a transmembrane (TM) domain, and a cytoplasmic (CT) domain (1). The extracellular (EC) domain of MUC1 mucin consists of a variable number of tandem repeats and is heavily glycosylated with O-linked oligosaccharides (1, 6). A high degree of sialylation of the sugar moieties of the EC domain thus confers an antiadhesion activity between cells as well as between the cell and the matrix (12). The EC domain of MUC1 mucin varies among species as well as among tissues within the same species (2).
On the other hand, the CT domain of MUC1 mucin is highly conserved among species (9, 10). The CT domain of MUC1 mucin contains several tyrosine residues, and the presence of phosphorylation on some of these residues was recently demonstrated by Zrihan-Licht et al. (15). In addition, Pandy et al. (8) reported that phosphorylation of one of the tyrosine residues in the CT domain of MUC1 mucin resulted in binding to Grb2, which, in turn, binds to Sos, a guanine nucleotide exchange factor. Association of Sos with Ras at the plasma membrane is known to initiate a kinase cascade, eventually leading to the activation of various transcription factors (7). Therefore, in light of the structure of the CT domain, it is highly likely that MUC1 mucin is a typical receptor that recognizes an extracellular signal and transmits it into the cell.
The ligand for MUC1 mucin, however, is unknown. The unavailability of the specific ligand has made it extremely difficult to study the molecular mechanism of signal transduction of MUC1 mucin. In addition, the search for a ligand has been hampered by the high degree of heterogeneity of the EC domain, most likely due to its heavy glycosylation. In this paper, we attempted to circumvent such a problem. Recently, Yao et al. (14) demonstrated that a chimeric CD8 molecule, which is also a transmembrane glycoprotein, could be activated by binding to its own antibody by using a cell line. In this paper, we describe construction of a chimeric receptor containing the EC and TM domains of CD8 and the CT domain of MUC1 mucin. Incubation of an anti-CD8 antibody with COS-7 cells expressing this chimera resulted in a significant increase in phosphorylation of the chimera, indicating a means to activate the CT domain of MUC1 mucin without a "natural" ligand. This system is not only simple but also highly reproducible and, therefore, will be extremely useful in studying the signal transduction of MUC1 mucin.
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METHODS |
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Materials. All materials used in this study were purchased from Sigma (St. Louis, MO) unless otherwise stated.
Construction of a chimeric receptor. A 627-bp fragment encoding the EC and TM domains of human CD8 was generated by PCR with oligonucleotides containing either an EcoR I or BamH I restriction site as a primer and human CD8 cDNA as a template. The human CD8 cDNA was kindly provided by Dr. Silvio Gutkind (National Institute of Dental Research, Bethesda, MD). The nucleotide sequences of the two primers are 5'-GGGAATTCCACCATGGCCTTACCAGTGACCGCCTT-3' and 5'-GGGGATCCTTCCTGTGGTTGCAGTAA-3'. The forward primer contains an additional sequence corresponding to the Kozak sequence to optimize the initiation of translation. Likewise, a 207-bp fragment encoding the CT domain of human MUC1 was also amplified by PCR with MUC1 cDNA as a template and oligonucleotides containing either a BamH I or Kpn I restriction site as a primer to allow easy ligation to the vector. In addition, two nucleotides (shown in boldface in the sequence below) were added to the reverse primer to ensure a continuous open reading frame through the myc epitope and histidine tags contained within the vector. The MUC1 cDNA was a generous gift from Dr. Sandra Gendler (Mayo Clinic Scottsdale, Scottsdale, AZ). The nucleotide sequences of the primers are 5'-GGGGATCCCCGCCGAAAGAACTACGG-3' and 5'-CGGGTACCAGCAAGTTGGCAGAAGTGGC-3'.
The following PCR conditions were used: 95°C for 3 min; 30 cycles at 94°C for 1 min, 55°C for 45 s, and 72°C for 1 min; and a final extension at 72°C for 7 min. The amplified PCR products were digested with the appropriate enzymes, checked on a 1% agarose gel, and then eluted and ligated appropriately. The resulting chimera was finally ligated into a pcDNA3.1 myc-His expression vector (Invitrogen, San Diego, CA) such that the COOH-terminal of the chimera is continuous with the nucleotide sequence of myc. This vector contains the nucleotide sequence corresponding to both a myc epitope and a histidine tag and is referred to as pCD8/MUC1 throughout the remaining text. An additional chimeric receptor lacking the myc epitope and histidine tag was constructed by eliminating the additional two nucleotides (AG) in the reverse primer. This generated a peptide sequence downstream of the MUC1 CT domain, which was totally different from the myc epitope. This mutant construct is referred to as the pCD8/MUC1 mutant. The validity of the constructs, both pCD8/MUC1 and pCD8/MUC1 mutant, was confirmed by nucleotide sequence analysis.
Antibodies. The antibodies used in this study were 1) anti-CD8 antibody 1: a rabbit polyclonal antibody raised against the extracellular domain of human CD8 (Santa Cruz Biotechnology, Santa Cruz, CA); 2) anti-CD8 antibody 2: a mouse monoclonal antibody raised against the extracellular domain of human CD8 (Serotec, Raleigh, NC); 3) CT1 antibody: a rabbit polyclonal antibody raised against the cytoplasmic 17-mer COOH-terminal peptide sequence of human MUC1 mucin (a generous gift from Dr. Sandra Gendler) (10); 4) CT91 antibody: a rat monoclonal antibody raised against the same sequence as in the CT1 antibody (13); 5) anti-myc antibody: a mouse monoclonal antibody raised against the myc epitope present in the vector (Invitrogen, San Diego, CA); and 6) anti-phosphotyrosine antibody: a mouse monoclonal antibody (Upstate Biotechnology, Lake Placid, NY). Two different antibodies were used for CD8, mainly for economical reasons: anti-CD8 antibody 1 was used for both immunoprecipitation and immunoblotting, whereas antibody 2 was used for "activation" of the chimera.
Transient transfection and immunoblotting of the chimeric receptor. COS-7 cells (American Type Culture Collection, Manassas, VA) were grown in F-12 medium-DMEM supplemented with 10% fetal bovine serum. After reaching ~70% confluence, the cells were transiently transfected with the above vectors with SuperFect (Qiagen, Santa Clarita, CA) according to the manufacturer's protocol. After 48 h, transiently transfected COS-7 cells were lysed on ice for 20 min in a lysis buffer [1% Nonidet P-40 (NP-40), 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 10 mM benzamidine, 6.25 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, and 200 µM sodium orthovanadate], and the cell lysate was then centrifuged to remove the detergent-insoluble material. An aliquot of the supernatant was incubated at 4°C for 2 h with either the anti-CD8 antibody 2 or the CT1 antibody. The mixture was then incubated again with protein A agarose (Life Technologies, Gaithersburg, MD) for 2 h at 4°C. After several washes with a buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 1% NP-40), the mixture was eluted in SDS sample buffer. The immunoprecipitates were separated on a 10% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membrane. The membrane was blocked for 1 h at room temperature in 10 mM Tris-Cl, pH 7.5, and 150 mM NaCl [Tris-buffered saline (TBS)], and 0.05% (vol/vol) Tween 20 (TBS-T) containing 5% nonfat dry milk and then incubated for 2 h at room temperature with the three different antibodies: anti-CD8 antibody 1, CT1 antibody, and anti-myc antibody. The resulting immunoblot was incubated with the secondary antibody conjugated with horseradish peroxidase and then visualized by enhanced chemiluminescence (Pierce, Rockford, IL).
Fluorescence-activated cell-sorting analysis. COS-7 cells transfected with either vector (control) or pCD8/MUC1 were harvested and incubated with the anti-CD8 antibody followed by FITC-conjugated goat anti-mouse IgG (Sigma). After being washed with PBS, the cells were then fixed in a PBS solution containing 1% paraformaldehyde, and fluorescence was read on a FACScan (Becton Dickinson) at 488 nm.
Phosphorylation of the CT domain. COS-7 cells that had been transiently transfected with either pCD8/MUC1 or its mutant were first treated with a mixture of hydrogen peroxide (2 mM) and sodium orthovanadate (2 mM) for 15 min at 37°C and then incubated with 5 µg of anti-CD8 antibody 2 for 10 min at 37°C. At the end of the treatment, the cells were lysed immediately on ice for 20 min in a lysis buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 10 mM benzamidine, 6.25 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, 200 µM sodium orthovanadate, and 1% NP-40), and the cell lysates were then centrifuged at 12,000 g for 10 min at 4°C to remove the detergent-insoluble material. The lysates were subjected to immunoprecipitation with anti-CD8 antibody 1 or CT1 antibody, and the immunoprecipitated protein was separated on a 10% SDS-polyacrylamide gel and then transferred to polyvinylidene difluoride membrane. The membrane was blocked with the TBS-T containing 5% BSA by incubation for 1 h at room temperature. A 1:20,000 dilution of the anti-phosphotyrosine antibody was prepared in TBS-T containing 1% BSA and then incubated with the membrane for 2 h at room temperature. The resulting immunoblot was incubated with the secondary antibody conjugated with horseradish peroxidase, and then visualization was carried out as described in Transient transfection and immunoblotting of the chimeric receptor.
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RESULTS AND DISCUSSION |
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After constructing the entire chimera (pCD8/MUC1) as described in
METHODS, we confirmed the nucleotide sequence of the final construct. The structure of pCD8/MUC1 as well as of the
nucleotide and amino acid sequences of the entire construct is shown in
Fig. 1.
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We then determined whether the chimeric receptor could be produced
through transient transfection of pCD8/MUC1 into COS-7 cells. COS-7
cells alone express neither CD8 nor MUC1 mucin (Fig. 2). After transient transfection, total
cell extracts were subjected first to immunoprecipitation with anti-CD8
antibody 1 and then to immunoblotting of the immunoprecipitate
with three different antibodies: anti-CD8 antibody 2, CT1
antibody, and anti-myc antibody. Figure 2 reveals the presence
of a common band with a molecular mass of ~34 kDa. This size was
consistent with that deduced from the nucleotide sequence of the
construct, which was calculated to be 33.9 kDa on the basis of a
computer program (www.expasy.ch). Thus the results indicate that COS-7
cells transfected with pCD8/MUC1 produced a chimeric receptor
containing both the EC domain of CD8 and the CT domain of MUC1 mucin.
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In the next experiment, we intended to verify the presence of the EC
domain of CD8 on the surface of the COS-7 cells. Our fluorescence-activated cell-sorting analysis revealed that the COS-7
cells transfected with pCD8/MUC1 were positive in ~17% of the total
population, whereas the COS-7 cells transfected with the control vector
were completely negative (Fig. 3). The result indicates that a significant number of COS-7 cells
transfected with pCD8/MUC1 expressed the EC domain of
CD8 on the surface of the cells.
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Having obtained COS-7 cells that express the chimeric receptor in the
plasma membrane, we then determined whether the chimeric receptor could
be activated by the anti-CD8 antibody. We hypothesized that activation of the chimeric receptor would result in tyrosine phosphorylation of the CT domain. Cell lysates from both
"unactivated" and activated cells were immunoprecipitated with
anti-CD8 antibody 1 and then immunoblotted with the
anti-phosphotyrosine antibody. Two bands appeared in both of the
samples; however, the intensity of the upper band was much greater in
the unactivated sample than in the activated sample (Fig.
4A, lanes 2 and 3,
respectively). Untransfected cells did not show either of these two
bands even under the activated condition (Fig. 4A, lane
1). The identical result was obtained when the CT1 antibody instead
of the anti-CD8 antibody was used for the immunoprecipitation (data not
shown). Thus the results indicate that the anti-CD8 antibody can
activate the CD8/MUC1 chimera, which results in an increase in the
level of tyrosine phosphorylation on the CT domain of the chimera.
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In a parallel study, the same cell lysates were immunoprecipitated with anti-CD8 antibody 1 and then immunoblotted with CT91. As seen in Fig. 4B, an additional band appeared in the activated cells at a location identical to the upper band in Fig. 4A. Therefore, it is most likely that the lower band is a major form of CD8/MUC1 chimera that is "weakly" phosphorylated on a tyrosine residue(s) and that activation of the EC domain of the chimera causes an increase in phosphorylation on a tyrosine residue(s) that results in a shift to the upper band. The presence of the constitutive phosphorylation on tyrosine residues in MUC1 mucins was previously reported by others (15). The mechanism(s) responsible for the increased tyrosine phosphorylation by the CD8 antibody remains to be investigated.
Because the myc epitope does not contain any tyrosine moieties, the increased phosphorylation must be on the tyrosine moieties of the CT domain of MUC1 mucin. However, it might be possible that the presence of the myc epitope in the CT domain of the chimera may have contributed to the increased tyrosine phosphorylation on the CT domain of MUC1 mucin after activation of the chimera. In an attempt to examine such a possibility, we constructed another mutant chimera in which the myc epitope is absent and then repeated the above experiment. As seen in Fig. 4C, activation of the mutant chimera gave exactly the same result as the "intact" chimera, thus ruling out the possible involvement of the myc epitope in the increased tyrosine phosphorylation on the CT domain after the activation of the EC domain of the chimera.
Collectively, the present experiments demonstrate that 1) the CD8/MUC1 protein can be expressed on the surface of COS-7 cells through transient transfection of its chimeric plasmid and 2) the chimeric receptor thus expressed on the cells can be activated by the anti-CD8 antibody, resulting in phosphorylation on the tyrosine moieties of the CT domain of MUC1 mucin. This system will circumvent the need for an extracellular ligand that is still unknown in activating the MUC1 mucins. The availability of the present system will allow us to investigate not only the function of MUC1 mucin in the airway but also the molecular basis of signal transduction after phosphorylation of the cytoplasmic domain of this protein.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Paul Shapiro (Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD) and Dr. Natalie Ahn (Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO) for critical review of this manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-47125 and a Designated Research Incentive Fund from the University of Maryland School of Pharmacy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: K. C. Kim, Dept. of Pharmaceutical Sciences, Univ. of Maryland School of Pharmacy, 20 N. Pine St., Room 446, Baltimore, MD 21201.
Received 9 April 1999; accepted in final form 8 December 1999.
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