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Molecular Biology and Genetics |
Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978 [A. B., M-l. H., M. Y., Y. A., S. G., J. Z., N. I. S., I. K., D. H. W.], and Department of Surgery A, Tel Aviv Medical Center [Y. St., Y. Sk.], Israel
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The best characterized MUC1 gene product, MUC1/REP (variously referred to as episialin, PEM, H23Ag, EMA, CA15–3, MCA, and others), is a polymorphic, type 1 transmembrane mucin-like protein (Fig. 1)
that contains a large extracellular domain, primarily consisting of a 20-amino acid repeat motif as well as a transmembrane domain and a 72-amino acid cytoplasmic tail (10, 11, 12, 13)
. During its biosynthesis, the MUC1/REP protein is modified to a large extent, and a considerable number of O-linked sugar moieties confer mucin-like characteristics on the mature protein (14)
. Soon after translation, the MUC1/REP protein undergoes a proteolytic cleavage event, generating two cleavage products (15)
. These form a tight heterodimer complex that is composed of the large repeat array-containing (+ repeat) extracellular domain, linked by noncovalent, SDS-sensitive bonds to the much smaller protein molecule, which includes the cytoplasmic and transmembrane domains. The MUC1/REP extracellular domain (+ repeat) can be shed from the cell by an as yet obscure mechanism (16)
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. By using mAbs4
that are specific to the MUC1/SEC protein, it was shown that this protein isoform is also present in the serum of breast cancer patients (17)
. Thus, both the MUC1/SEC protein and the MUC1/REP extracellular cleavage product can be secreted from breast cancer cells. The repeat array-containing mucin-like MUC1 proteins play a distinctive role in reducing cell-cell and cell-extracellular matrix interactions, and it has been postulated that they may be involved in the metastatic spread of cancer cells from the initial tumor site (18, 19, 20) . Using knockout mice, it was also demonstrated that MUC1 expression enhances tumor initiation and progression, suggesting that the MUC1 gene products are in some way involved in the oncogenetic process (21) .
In recent years, there is a growing body of evidence that mucin-like proteins not only passively mediate cellular processes but that they also have an active signaling function. It is well documented now that endothelium mucin-like molecules (e.g., CD34, GlyCAM1, PSGL1, and MadCAM1) are actively involved in lymphocyte trafficking and can transfer signals to the cell nucleus on binding to their ligands/counter receptors. These recent findings place mucin proteins, in general, and the mucin-like MUC1 proteins, in particular, in a different light and raise the intriguing possibility that the MUC1 proteins may also participate in receptor/ligand interactions.
We recently identified a supplementary and unique MUC1 isoform, MUC1/Y (22)
, which is generated by differential splicing. Although MUC1/Y is a transmembrane protein that shares identical transmembrane and cytoplasmic domains with the MUC1/REP protein, it contains neither the repeat array domain nor its flanking region and, hence, is devoid of the hallmark mucin-like features (Fig. 1)
. By using antibodies specifically directed against MUC1/Y, it was clearly shown that the MUC1/Y protein is expressed by various human secretory epithelial tumors, but is not detectable in the adjacent normal tissue(22, 23, 24)
. Moreover, the involvement of the MUC1/Y protein in the oncogenetic process was established by demonstrating its potential to enhance tumor initiation and progression in vivo (24)
.
Previously reported results have demonstrated that MUC1/Y undergoes tyrosine and serine phosphorylation and can potentially bind second messenger proteins, such as GRB2, thereby initiating a signaling cascade (25) . The MUC1/Y membrane-located protein, thus, has features reminiscent of receptor molecules.
In the present study, we describe the identification of cognate binding proteins for the MUC1/Y protein. We show that the secreted mucin-like MUC1 protein, namely MUC1/SEC, interacts with the MUC1/Y protein. Coimmunoprecipitation analyses clearly revealed the presence of these complexes in primary BT tissue, thus, underscoring their in vivo significance to breast cancer. Evidence is presented that the interaction of MUC1/Y with MUC1/SEC induces the phosphorylation of the MUC1/Y protein and profoundly affects cell morphology. These findings suggest that the interaction of the two proteins MUC1/SEC and MUC1/Y, both generated from the same MUC1 locus, represents a bona fide receptor/cognate binding protein interaction that can elicit biological functions. Furthermore, using purified MUC1/SEC as a probe, we were able to target MUC1/Y-expressing tumor cells within the intact animal, thereby suggesting the clinical use of this interaction.
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MATERIALS AND METHODS |
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-32P]ATP, 125I conjugated Bolton-Hunter reagent, [32P]Pi, goat antimouse antibody conjugated to horseradish peroxidase, and goat antirabbit antibody conjugated to horseradish peroxidase were purchased from Amersham Corp. Ni+2 Agarose Ni-NTA was purchased from Qiagen, Inc. Imidazole was purchased from Sigma Chemical Co. Affigel-10 was purchased from Bio-Rad.
Cells.
MUC1/Y transfectants were generated by cotransfecting an expression plasmid harboring MUC1/Y cDNA with the G418-resistant plasmid (pSV2 neo) into 3T3 ras-transformed fibroblasts or HBL100 human epithelial breast cells (24)
. MUC1/REP and MUC/SEC stable DA3 transfectants were generated by cotransfecting an expression plasmid harboring MUC1/SEC-(DA3/SEC) or MUC1/REP-(DA3/TM) cDNA, together with pSV2 neo, into the DA3 mouse mammary-transformed epithelial cell line (24)
. MUC1/Y + MUC1/REP and MUC1/Y + MUC1/SEC double transfectants were generated by transfecting 3T3 mouse fibroblasts (or DA3) together with the pSV2 neo plasmid. T47D-Cl11, T47D-Cl10, T47D-Cl8, are cloned cell lines derived from a human mammary adenocarcinoma.
cDNA Constructs.
An expression vector harboring the MUC1/Y extracellular domain cDNA was generated by using the pET16b expression system (Novagene). MUC1/Y extracellular fragment (nucleotides 127–526; numbering as in Ref. 22
) was PCR amplified, using the full-length MUC1/Y cDNA as template. The downstream primer was designed to contain a sequence coding for a PKA phosphorylation site. The 453 bp PCR product was ligated into predigested pET16b vector, using upstream NdeI and downstream BamHI restriction sites. BL21-competent bacteria were transformed with the MUC1/Y extracellular domain expressing plasmid by electroporation. The generation of expression vectors harboring either the full-length transmembrane MUC1/REP, MUC1/SEC, or the novel MUC1/Y cDNA driven by the HMG CoA reductase promoter (expression vector pCL642) has been described (22)
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Antibodies.
Western blot and coimmunoprecipitation analyses were performed with: (a) mAbs (H23 mAb) that recognize an epitope contained within the tandem repeat domain of the MUC1/REP protein (26)
; (b) a polyclonal antibody directed against the MUC1 cytoplasmic domain; (c) an affinity-purified polyclonal antibody (designated anti-MUC1/Yex) prepared by injecting rabbits with the MUC1/Y extracellular domain synthesized as a recombinant protein using the pET expression system according to the protocol described below; and (d) unique mAbs, designated 6E6, which specifically recognize an epitope present within the MUC1/Y extracellular domain and do not interact with any other MUC1 isoform.
Purification and Labeling of the Recombinant MUC1/Y Extracellular Domain.
N-terminal histidine-tagged recombinant MUC1/Yex protein was generated and purified as described (24)
. For 32P labeling and factor Xa digestion of the recombinant MUC1/Y extracellular domain, 5 µg of the purified recombinant protein were incubated with 3 units of PKA, 2 units of factor Xa, and 150 µCi of [-32P]ATP in labeling buffer [20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 2 mM CaCl2, 5 mM MgCl2, and 2 mM DTT] for 5 h at 30°C. The labeling mixture was then loaded onto a 5-ml Sephadex G-50 colum prequilibrated with Buffer E [20 mM Tris-HCl (pH 7.4), 50 mM NaCl, 1% Triton X-100, 5 mM MgCl2, and 50 µg/ml BSA], and fractions were eluted against the same buffer.
Gel Overlay Assay.
The following procedure is a modification of the previously described assay (27)
. The 32P-labeled purified MUC1/Y extracellular domain (described above) was used as a probe for the screening of proteins present in primary human BT tissue lysates, human breast tumor cell line lysates, and human breast tumor-conditioned media. Samples were separated on 6% SDS-PAGE, and the gels were subsequently washed against protein renaturation buffer [50 mM Tris-HCl (pH7.4), 2.5% Triton X-100, and 0.1% CHAPS) at 25°C with agitation for 3 h and with several changes. Gels were incubated with the probe at a final concentration of 106 cpm/ml in probe buffer (50 mM Tris-HCl, 100 mM NaCl, 2.5% Triton X-100, 0.1% CHAPS, and 4 mM MgCl2) for 16 h at 4°C, followed by washing of the gel in probe buffer for 32 h with several changes.
Preparation of Conditioned Media.
DA3 MUC1 transfectants were grown to subconfluency in a 75-cm2 dish (Costar) in DMEM medium containing 10% FCS. Cells were then washed once with serum-free medium and incubated for 3 h in a serum-free medium. The medium was removed and replaced with fresh serum-free medium, and the cells were reincubated against this medium for 72 h. The conditioned medium was cleared from the cells, filtered through a 0.2 micron filter, and used directly or concentrated 100-fold in an ultrafiltration cell through a 3 kDa or 100 kDa cutoff Centriprep membrane (Amicon).
Purification and Labeling of MUC1/SEC.
MUC1/SEC was purified from conditioned medium of DA3/SEC transfectants by affinity chromatography. A 500-ml volume of conditioned medium was passed through a 5-ml H-23 antirepeat mAb affinity column (1 mg of antibodies/ml Affigel-10 beads). The column was washed extensively with PBS containing 0.05% Tween 20, followed by elution with 3.5 M sodium thiocyanate (pH 4.5). Peak fractions containing purified MUC1/SEC (assayed by dot blot) were pooled and immediately transferred through a Sephadex G-50 column pre-equilibrated with 10 mM HEPES and 30 mM NaCl. Purified MUC1/SEC (2 µg) was incubated with 125I conjugated Bolton-Hunter reagent (200 microcurie) for 15 min on ice. The reaction was terminated by the addition of glycine to a final concentration of 0.2 M, and the solution was run on a Sephadex G-50 gel to remove free iodine.
Cell Binding Assay.
Ras-transformed 3T3 cells or their MUC1/Y-expressing transfectants were harvested using 10 mM EDTA, and 106 cells were incubated with 5 nM125I-labeled purified MUC1/SEC (specific activity, 120 Ci/mmol) diluted in serum-free DMEM for 3 h at 4°C with gentle shaking. Unbound protein was removed by spinning cells through a prechilled binding oil column. Bound protein, as well as the unbound fraction, was counted in a gamma counter.
32P Metabolic Labeling of Cells.
Cells were incubated overnight in phosphate-free medium supplemented with 10% FCS that had been dialyzed against saline. The following day, radioactive carrier-free inorganic phosphate was added to a final concentration of 1 mCi/ml, and incubation was continued for another 8 h. Thirty minutes before harvesting, the cells were treated with the tyrosine phosphatase inhibitors sodium orthovanadate and hydrogen peroxide (200 µM; Ref. 28
) or with conditioned medium prepared from DA3 MUC1/SEC transfectants, as described above.
Western Blot Analyses.
Cell lysates were prepared by adding lysis buffer [50 mM NaCl, 20 mM Hepes (pH 7.4), 1 mM EGTA, 1 mM MgCl2, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1% Triton X-100, 1 mM NaVO4, and 100 mM NaF] to cell pellets, followed by vortex mixing and sonication (3 x 10 s bursts, using a Branson sonicator). Cell debris was removed by centrifugation at 12,000 x g for 10 min. All procedures were performed on ice or at 4°C. Protein samples were denatured by boiling in SDS buffer containing mercaptoethanol and analyzed on SDS-PAGE. The gel was electrotransferred for 2 h at 0.5 Amps to nitrocellulose filter papers that were blocked in PBS containing 5% skimmed milk and 0.1% Brij, followed by incubation with primary antibody overnight at 4°C. The filters were washed in PBS containing 0.1% Brij and then incubated with a secondary antimouse antibody or antirabbit antibodies conjugated to horseradish peroxidase, followed by enhanced chemiluminescence detection (Amersham Corp.).
Immunoprecipitations.
Cell lysates prepared as described above were precleared by incubating proteinA-agarose bound to normal serum antibodies for 2 h at 4°C. Supernatants were then added to proteinA-agarose antibody complexes and incubated for 2 h at 4°C. The immunocomplexes were washed four times with cell lysis buffer, and 2 x SDS sample buffer was added.
In Vivo Targeting of MUC1/Y-expressing Cells.
DA3 mouse mammary carcinoma transfectants (105 cells) were injected into the mammary pads of BALB/c mice to produce mammary tumors. Three weeks later, purified 125I-labeled, MUC1/SEC (8 x 105 cpm) was injected into the tail vein of each mouse. The animals were sacrificed 5 days later and were exposed to imager analyses; subsequently, the tumors were excised and counted in a gamma counter.
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RESULTS |
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To identify molecules that can interact with MUC1/Y and may regulate its function, a recombinant histidine-tagged MUC1/Y extracellular domain (Yex) was expressed in bacteria and purified to homogeneity. A protein kinase A (PKA) phosphorylation site was inserted at the COOH terminus of the recombinant protein, thus, enabling efficient labeling using PKA and [- 32P]ATP of the MUC1/Y extracellular domain (Fig. 2A
, Lane a, black arrow). The histidine tail could then be cleaved with factor Xa protease (Fig. 2A
, Lane b, open arrow). Conditioned media from breast tumor cell lines were analyzed using the 32P-labeled extracellular domain (Yex*) of the MUC1/Y protein as a probe in a receptor gel overlay assay. This analysis revealed the presence of high molecular weight (>200 kDa) reacting bands in the conditioned media of the breast tumor cell lines (Fig. 2B, a)
. The histidine-tailed Yex* probe did not react with these bands (Fig. 2B, b)
, probably due to allosteric interference. Competition experiments with a 200-fold excess of unlabeled Yex in relation to the labeled Yex probe abolished binding (Fig. 2B, c)
, thereby confirming specificity.
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. For example, probing with the antirepeat mAbs demonstrated that the Cl11 and Cl8 cell lines (clonal derivatives of the T47D breast cancer cell line) are heterozygous and express two different-sized MUC1/REP alleles (Fig. 2C, a and b
, Lanes 3 and 4, respectively), whereas the T47D/Cl10 cell line abnormally expresses three alleles (Fig. 2C, a and b
, Lane 2). This pattern tallies with the pattern observed in the receptor gel overlay assay. Of note, when the Cl11 breast cancer cell line whole lysate was examined, only the mature MUC1(+ repeat) proteins bound the labeled MUC1/Y probe, whereas the immunoreactive immature MUC1(+ repeat) proteins were completely nonreactive in the receptor gel overlay asssay (Fig. 2C, a and b
, Lane 1). This indicates that posttranslational modifications within the MUC1(+ repeat) isoforms may be required for the interaction with Yex. To exclude the possibility that binding to Yex is a cell line artifact, lysates prepared from primary human tumor tissue were analyzed, using the gel overlay assay. Identical results to those seen with in vitro grown breast cancer cells were obtained (Fig. 2D, a and b)
. Coomassie blue staining of a gel run in parallel demonstrated very low protein concentrations in the high molecular weight binding region. Conversely, many low molecular weight Coomassie-stained bands that did not react with the Yex* probe were visible (data not shown).
The MUC1/SEC Protein Binds to the Extracellular Domain of the MUC1/Y Protein.
MUC1-reacting bands were present not only in the cell lysates (Fig. 2C, a and b Lane 1)
, but also in the conditioned media. This suggests that the MUC1/Y extracellular domain interacts with the extracellular cleavage product of the MUC1/REP proteins and/or the secreted MUC1(+ repeat) isoforms, namely MUC1/SEC (Fig. 1B)
. To investigate these possibilities, stable DA3 mouse mammary carcinoma cell transfectants, expressing the MUC1/REP or MUC1/SEC proteins, were analyzed by Yex gel overlay assay. Both MUC1 isoforms bound the Yex probe (Fig. 3A)
. The Yex binding pattern correlates precisely with the expression patterns of MUC1/SEC and the MUC1/REP extracellular cleavage product both at the protein and at the RNA level (Fig. 3, B and C)
. The binding site for the extracellular domain of MUC1/Y is, thus, common to both MUC1/SEC and the MUC1/REP extracellular cleavage product (Fig. 1B)
. By performing the receptor gel overlay assay with different concentrations of labeled Yex, the kD value of MUC1/Y binding for MUC1/SEC was estimated as 1.2 nM, indicating a high affinity between the interacting proteins.
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To demonstrate direct binding of MUC1/SEC to MUC1/Y-expressing cells, the MUC1/SEC protein, derived from conditioned medium of MUC1/SEC-transfected DA3 cells, was purified to apparent homogeneity, as assayed by silver staining (Fig. 4A)
, radioactively iodinated (Fig. 4B)
, and applied either to stable MUC1/Y-expressing ras-transformed 3T3 transfectants (3T3sub5/Y) or, as a control, to the parental cells that do not express the MUC1/Y protein (3T3sub5). Significant MUC1/SEC binding was observed to the MUC1/Y-expressing cells; only background levels of radiolabeled MUC1/SEC were associated with the control cells (Fig. 4C)
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. Gamma counter readings of dissected tumors confirmed imager analyses (Fig. 5B)
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, Lane 1) and MUC1/SEC (Fig. 6A
, Lane 5). Immunoprecipitation of cells expressing both MUC1/REP and MUC1/Y, using the H23 antirepeat mAb also revealed, as expected, the MUC1/REP cytoplasmic cleavage products (C.P. MUC1/REP). A control immunoprecipitation performed with mAbs (6E6) that specifically recognize the MUC1/Y isoform, and subsequent immunoblotting with anti-MUC1/Yex antibodies demonstrated the presence of the MUC1/Y protein (Fig. 6A
, Lanes 3 and 7). The coimmunoprecipitation analyses unequivocally showed that MUC1/Y can exist within the intact cell as a complex, in which it is bound to the repeat array-containing proteins.
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. This analysis clearly demonstrated that, within the primary BT tissue, MUC1/Y forms a complex with the MUC1(+ repeat) proteins.
MUC1/SEC and MUC1/Y Form a Receptor Complex That Alters Cellular Morphology and Initiates Cell Signaling.
Having demonstrated the specific interaction between MUC1/Y and the MUC1(+ repeat) isoforms, we proceeded to investigate the biological significance of this interaction. HBL100 normal breast epithelial cells transfected with the neomycin-resistant gene alone (NEO cells) or MUC1/Y-transfected HBL100 cells (Y cells) were incubated with MUC1/SEC containing conditioned media prepared from DA3 cell transfectants (Fig. 7)
. These experiments demonstrate that treatment with DA3/SEC-conditioned medium (Y cells + SEC C.M.) elicits a pronounced morphological change in the growth pattern of the HBL/Y cells (Fig. 7B)
. The cells assume an elongated shape, forming "Indian file" rows of cells circumscribing large lumens. In contrast, cells treated with the control medium (Y cells + DMEM) formed large clusters (Fig. 7A)
. The altered morphological pattern of cell growth was not observed when parental (non-MUC1/Y-transfected) neomycin-resistant HBL100 were used as target cells (NEO cells + SEC C.M; Fig. 7C
), nor when the cells were treated with conditioned medium from DA3 cells transfected with the neomycin-resistant gene (data not shown). Significantly, the morphological change observed in DA3/SEC-conditioned medium-treated cells was reversed by the addition of a 100-fold-excess of the recombinant soluble MUC1/Y extracellular domain [Ycells + (SEC C.M + REC. Yex); Fig. 7D
]. This probably saturated the soluble MUC1/SEC in solution, such that it could no longer exert its effect by binding to the MUC1/Y-expressing cells. Furthermore, depletion of the MUC1/SEC protein from the conditioned medium by passage through an antitandem-repeat array mAb affinity columns abrogated the morphological changes observed with the MUC1/SEC containing conditioned medium (Y cells + SEC depleted C.M; Fig. 7E
). Collectively, these results demonstrate that the morphological changes in the growth pattern of the HBL/Y cells are attributable to the formation of the MUC1/Y-MUC1/SEC complex.
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. Immunoprecipitation of MUC1/Y with anticytoplasmic domain antibodies revealed that the addition of MUC1/SEC-containing medium (Fig. 8
, Lane 4), as opposed to the control conditioned medium (Fig. 8
, Lane 3), induced MUC1/Y phosphorylation. Phosphoamino acid analysis demonstrated the presence of phosphorylated MUC1/Y serine residues with lower levels of phosphotyrosine (data not shown).
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Characterization of the MUC1/Y and MUC1/SEC Binding Sites.
Truncations, deletions, and point mutations of the bacterial recombinant MUC1/Y extracellular domain demonstrated that the MUC1/SEC binding site maps to the MUC1/Y 44 amino acids NH2-terminal to the transmembrane domain. Notably, this region shows similarity to the ligand binding sites of cytokine receptors (see Fig. 3
in Ref. 25
). Deletion of both the decapeptide sequence INVHDVETQF and the pentapeptide INVHD (amino acids 112–121 and 112–116, respectively; numbering as in Ref. 22
) abrogated all binding; Fig. 9A
). Within this sequence, aspartic acid D116 is conserved in the mouse MUC1 homologue; in other species it is conservatively changed to glutamic acid (E). Mutation of this aspartic acid residue to alanine drastically reduced binding, whereas the D to E mutation had no effect. Significantly, the COOH-terminal region of the MUC1/Y extracellular domain contains the "signature" motif of serine, hydrophobic and acidic amino acid residues (ISDVS), which is found at an identical location in cytokine receptors (30)
. All COOH-terminal truncations of the MUC1/Y extracellular domain completely abrogated MUC1/SEC binding (Fig. 9A)
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Bacterially generated recombinant MUC1/SEC was devoid of MUC1/Y binding activity (data not shown). This suggests that, in contrast to MUC1/Y, posttranslational modifications occurring on MUC1/SEC are integral components of the interaction. Moreover, synthesis in some cell lines (such as the kidney cell line HK293) led to significant levels of posttranslationally modified MUC1/SEC, which, nonetheless, were devoid of all MUC1/Y binding activity (Fig. 9B)
, indicating that cell-type-specific modifications are required to generate the MUC1/Y binding site within MUC1/SEC.
We do not as yet know whether N-linked or O-linked glycosylations occurring on the MUC1/SEC molecule are required for a productive interaction to proceed between the MUC1/SEC and MUC1/Y proteins; indeed, the possibility exists that a cell-type-specific glycosylation event occurring on MUC1/SEC is an essential requirement. Protease treatment of the MUC1/SEC protein did, however, abolish all binding activity, and the binding site, thus, obviously encompasses a protein component.
The notion that the tandem repeats contained within MUC1/SEC are not responsible for binding was supported by analyzing the effect of COOH-terminal truncations of the protein. Deletion of the COOH-terminal 26 amino acids abrogated all binding (Fig. 9C)
, thereby implicating that this region, when appropriately posttranslationally modified, contributes an essential component for the interaction with MUC1/Y.
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DISCUSSION |
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Previously, we demonstrated that MUC1/Y can be transphosphorylated, within the intact cell, on tyrosine and serine residues within the cytoplasmic domain (25) . This finding, along with recent data demonstrating a specific interaction between the cytoplasmic domain of MUC1 and the adaptor protein GRB2, supports the notion that the MUC1 proteins may be involved in cell signaling (25 , 31) . Moreover, a marked homology between sequences within the extracellular region of MUC1/Y and cytokine receptor sequences involved in ligand binding led to the hypothesis that MUC1/Y functions in a similar manner to cytokine receptors and is regulated by a secreted ligand (25) .
We now demonstrate a specific interaction between the MUC1/Y transmembrane protein and the secreted MUC1(+ repeat) isoforms. Furthermore, we show here that the MUC1/SEC present in the conditioned medium forms an active receptor complex with MUC1/Y, resulting in the phosphorylation of MUC1/Y and a concomitant change in cellular morphology.
The morphological alterations we observed could be attributed to the presentation on the cell surface of the MUC1/SEC isoform via complex formation with the extracellular domain of the membrane-bound MUC1/Y molecule (18) . As MUC1/Y undergoes serine and tyrosine phosphorylation after MUC1/SEC binding, it is possible that the morphological change is driven by an additional mechanism that embraces cell signaling. To phosphorylate its own serine and tyrosine residues, MUC1/Y must recruit (as yet unidentified) transacting tyrosine and/or serine/threonine kinases. The regulatory controls determining the precise MUC1/Y serine/threonine and tyrosine phosphorylation patterns remain obscure and probably depend on the presence of specific transacting kinases present in the cytoplasmic milieu. In accordance with the recent finding that ß-catenin interacts with the cytoplasmic domain of MUC1 (32) , MUC1/Y could alter cellular morphology by serving as a link between the recruited transacting kinases and ß-catenin, thereby regulating catenin function (33 , 34) .
The novelty of our present findings is that the MUC1/SEC and MUC1/Y isoforms, which are both derived from the MUC1 gene, interact with each other, and in so doing generate a receptor complex capable of eliciting cellular responses. Strong evidence for this interaction is provided by published findings and our experimental data: (a) MUC1/SEC and MUC1/Y are generated from two different alternatively spliced mRNA species (10 , 17 , 22 , 29 , 35) ; (b) MUC1/SEC and MUC1/Y localize to different cellular compartments (the former is secreted from the cell, and the latter is membrane bound; Refs. 17 , 22 ); (c) coimmunoprecipitation analyses demonstrate, both in double transfectants and in primary BT tissue, the presence of MUC1/SEC and MUC1/Y complexes; and (d) this interaction elicits biological effects, as reflected by changes in cellular morphology and MUC1/Y phosphorylation status.
We, therefore, postulate that the binding of MUC1/SEC to MUC1/Y represents a bona fide receptor/cognate binding protein interaction, and propose a variation to the receptor-ligand interaction "canon," according to which the interacting membrane-bound receptor and soluble ligand proteins derive from different genes. Precedents for variations on this theme do exist. For example, both the ligand (or counter-receptor) and receptor can be bound to the membrane of different cells (36) or, as in the case of GDNF and Ret, a membrane glycosylphosphatidylinositol-linked cell surface receptor (GDNFR) bound to its cognate binding protein (GDNF-GDNFR) acts as the ligand for a different membrane receptor, Ret (37) . Perhaps most relevant to the findings described here is the unmasking of a ligand by thrombin cleavage of the thrombin receptor (38) . The new NH2-terminal amino acid sequence thereby formed represents a "tethered" ligand that then acts by intramolecular interactions with the thrombin receptor; thus, the receptor and its cognate binding protein/ligand reside in the one and same protein molecule and are derived from the same gene (38) . These examples highlight the difficulty in defining the terms "receptor" and "ligand" and in relating to "receptor-ligand" interactions in a simplistic fashion.
The results presented here suggest a mechanism whereby alternative splicing generates, from the one and same gene, both a membrane-bound receptor and its secreted, soluble cognate binding protein. Our findings, thus, imply that alternative splicing regulates both the relative levels of the cognate binding protein and receptor and the biological effects elicited by the interaction of these two isoforms. Indeed, some cells express the MUC1/Y protein in preference to the tandem repeat array-containing MUC1 isoforms and vice versa (24) . An individual cell may express both isoforms (data not shown), thereby suggesting the involvement of an autocrine mechanism in regulating the biological effects elicited by the interaction of the receptor with its cognate binding proteins. This does not exclude the possibility that paracrine mechanisms also apply.
Several molecules with mucin-like features have been shown recently to act as ligands. For example, P-selectin glycoprotein ligand-1 is a mucin-like glycoprotein that acts as the counter-receptor for P-selectin, but only following appropriate posttranslational modifications (39 , 40) . Similarly, the cognate binding proteins of MUC1/Y are mucin-like proteins that, to support binding, require posttranslational modification in a cell-type-specific manner. Our results suggest that the region COOH-terminal to the repeats, which undergoes extensive posttranslational modification, is a critical component for a productive interaction to proceed. Indeed, deletion of the COOH-terminal 26 amino acids from MUC1/SEC abolished all binding activity. It is noteworthy that the pentapeptide sequence "YYQEL" present in these COOH-terminal 26 amino acids is conserved in the mouse, hamster, guinea pig, rabbit, cow, gibbon, and human MUC1 homologues (41) . Cells transfected with constructs in which the two tyrosine residues were mutated to phenylalanine synthesized MUC1/SEC, which did not bind to MUC1/Y (data not shown). We do not know whether part or all of the MUC1/Y binding site resides in this 26 amino acid sequence. Alternatively, this stretch may be required for the correct targeting of the nascent protein to a specific cellular compartment, thereby permitting an essential and specific posttranslational modification to take place.
Having demonstrated the interaction of MUC1/Y with MUC1/SEC, we proceeded to investigate its possible clinical applications. Using purified labeled MUC1/SEC, we were able to efficiently target MUC1/Y-expressing tumors in mice. For several years, attempts have been made to target the MUC1/REP protein, using mAbs directed against the repeat array (42 , 43) . Two difficulties are inherent in this approach: (a) a lack of specificity due to an unstable pattern of modification within the repeat array region; and (b) the circulation of high levels of the soluble repeat array containing proteins that will sequester the injected antibodies. It is now clear that the use of naturally occurring molecules may be preferable for cell targeting; previous studies applying receptor-ligand interaction for gene delivery have proven successful (44) . MUC1/Y could serve as an ideal target for cancer treatment for the following reasons. First, MUC1/Y is expressed by breast and ovarian carcinoma cells and, in some cases, especially in ovarian carcinoma, is the only MUC1 isoform expressed (22 , 24) . Second, MUC1/Y, in contrast to MUC1/REP, does not undergo proteolytic cleavage and, thus, consists of a continuous extracellular domain that is not shed from the cell surface (22) . Finally, the MUC1/Y-MUC1/SEC interaction is a naturally occurring one, thereby circumventing the inherent difficulties in the use of nonself molecules for targeting. Our results indicate that it is not the repeat array region but the COOH-terminal portion of MUC1/SEC that is involved in MUC1/Y binding. Thus, a small, posttranslationally modified polypeptide sequence derived from the MUC1/SEC protein, which does not include the large repeat array, may lead to improved specificity in targeting MUC1/Y-expressing cancer cells both for diagnostic and therapeutic purposes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Israel Cancer Association, the Israel Cancer Research Fund, the Israel Academy of Sciences, the Israel Ministry of Science and Technology (to D. H. W.), the Simko Chair for Breast Cancer Research, the Federico Fund for Tel Aviv University, and the Barbara Friedman Fund (to I. K.). 
2 These authors contributed equally to these studies.
3 To whom requests for reprints should be addressed, at Department of Cell Research and Immunology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv 69978, Israel. Phone: 972-3-6407425; Fax: 972-3-6422046; E-mail: danielhw{at}post.tau.ac.il
4 The abbreviations used are: mAb, monoclonal antibody; BT, breast tumor; PKA, protein kinase A.
Received 10/19/98. Accepted 2/ 2/98.
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