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3ß1 integrin–CD151, a component of the cadherin–catenin complex, regulates PTPµ expression and cell–cell adhesion

¹ Department of Medicine, Children's Hospital
² Department of Pediatrics, Harvard Medical School, Boston, MA 02115
³ School of Biomedical Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
⁴ Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, OH 44106

Address correspondence to Jordan Kreidberg, Division of Nephrology, Hunnewell 3, Children's Hospital, 300 Longwood Ave., Boston, MA 02115. Tel.: (617) 247-5194. Fax: (617) 232-4315. email: Jordan.Kreidberg{at}tch.harvard.edu

	Abstract

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The ß1 family of integrins has been primarily studied as a set of receptors for the extracellular matrix. In this paper, we define a novel role for 3ß1 integrin in association with the tetraspanin CD151 as a component of a cell–cell adhesion complex in epithelial cells that directly stimulates cadherin-mediated adhesion. The integrin–tetraspanin complex affects epithelial cell–cell adhesion at the level of gene expression both by regulating expression of PTPµ and by organizing a multimolecular complex containing PKCßII, RACK1, PTPµ, ß-catenin, and E-cadherin. These findings demonstrate how integrin-based signaling can regulate complex biological responses at multiple levels to determine cell morphology and behavior.

Key Words: cell contact; tetraspanin; phosphatase; tyrosine phosphorylation; PKCßII

Abbreviation used in this paper: shRNA, small hairpin RNA.

	Introduction

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Most, if not all, cellular morphogenetic events result from modulation in cell–cell or cell–substratum adhesion. Traditionally, integrins are thought to mediate cell–matrix adhesion and cadherins cell–cell adhesion. There are well-known exceptions to this rule, such as the leukocyte–endothelium cell–cell interactions mediated by the ß2 family of integrins (Carter et al., 1990; Larjava et al., 1990). Nevertheless, integrins of the ß1 family have primarily been studied as receptors for the ECM. However, certain members of the ß1 family of integrins, when complexed with another family of transmembrane proteins, known as tetraspanins, have been hypothesized to be involved in cell–cell adhesion (Fitter et al., 1999).

Tetraspanins are a group of cell surface molecules that have four transmembrane domains. Tetraspanins are thought to play an important role in a variety of normal and pathological processes, such as cell differentiation, cell motility, egg–sperm fusion, and tumor cell metastasis (Berditchevski, 2001; Boucheix and Rubinstein, 2001). In vivo, 3ß1 integrin and CD151 are coexpressed in a variety of epithelial cells, including basal keratinocytes of the skin and glomerular epithelial cells of the kidney (Sterk et al., 2000, 2002). It has been found that the 3ß1–CD151 complex is very stable and can withstand conditions that disrupt all other integrin–tetraspanin and tetraspanin–tetraspanin interactions (Yauch et al., 1998; Serru et al., 1999). Notably, the interaction of CD151 and 3ß1 integrin has been found to affect cell motility and signaling (Zhang et al., 2001; Yang et al., 2002). Yauch et al. (1998)( 2000) reported that the stalk region of the 3 extracellular domain (between 570 and 705 aa) and a region of large extracellular domain of CD151 (between 186 and 217 aa) is important for stable association of these two molecules (Berditchevski et al., 2001). Tetraspanins are not found in focal contacts, nor do they appear to have any effect on ECM adhesion mediated by integrins (Berditchevski et al., 1996; Berditchevski and Odintsova, 1999). However, numerous reports have described the localization of tetraspanins at sites of cell–cell contact, suggesting a possible role in cell–cell adhesion tetraspanins in promoting cell–cell interactions (Fitter et al., 1999; Yanez-Mo et al., 2001).

Several studies have demonstrated that disruption of integrin function in epithelial cells leads to a loss of the cortical cytoskeleton and the assembly of actin stress fibers (Carter et al., 1990; Hodivala-Dilke et al., 1998; Wang et al., 1999). In Wang et al. (1999), it was shown that 3ß1 integrin–deficient collecting duct epithelial cells assembled actin stress fibers instead of a subcortical cytoskeleton, and that there was reduced association of the cadherin–catenin complex with -actinin in 3ß1-deficient cells. Here, we show that association of 3ß1 integrin with CD151 does indeed promote association of the cadherin–catenin complex with the actin cytoskeleton and cadherin-mediated cell–cell adhesion. Two levels of regulation were evident. First, the integrin–tetraspanin complex regulates the gene expression of PTPµ, a transmembrane protein tyrosine phosphatase previously shown to be involved in cadherin-mediated adhesion (Brady-Kalnay et al., 1995, 1998; Hellberg et al., 2002). Second, we observed a unique complex involving PKCßII, RACK1, and PTPµ used by integrins to regulate cadherin-mediated cell–cell adhesion, possibly by modulating tyrosine phosphorylation of ß-catenin. It was possible to identify a large multimolecular complex containing 3ß1 integrin–CD151–PKCßII–RACK1–PTPµ–E-cadherin–ß-catenin, whose presence in epithelial cells was dependent on the integrin–tetraspanin interaction. The 3ß1 integrin receptors associated with the cadherin–catenin complex could be distinguished from those involved in binding laminin. Therefore, two distinct populations of integrins are present in epithelial cells, one involved in cell–matrix adhesion and another involved in cell–cell adhesion.

	Results

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Expression of chimeric integrin receptors on the surface of 3ß1-deficient cells and their association with CD151
Yauch et al. (2000) previously characterized a set of chimeric integrin subunits, used here, containing the 6 extracellular domain and the 3 cytoplasmic domain (Fig. 1 A). Under their conditions, and confirmed in our studies (Fig. 1 B), CD151 was only able to interact with subunits containing the 3-stalk region. 3ß1-Deficient cells were stably transfected with 6/3 chimeric integrins, and heterodimeric expression of chimeric subunits with ß1 integrin was confirmed (Fig. 1 B); henceforth, transfected cell lines are designated as 3-stalk and 6-stalk. As in previously published studies (Yauch et al., 2000), only wild-type 3 or 3-stalk subunits interacted with CD151 (Fig. 1 C), the amount of CD151 being equal in all cell lines (Fig. 1 C).

View larger version (81K): [in this window] [in a new window]   Figure 1. Association of integrins and CD151. In all panels, the cell lines are designated below the panel showing the first blot; when shown, reblots are below the cell line designations. In all figures: WT, wild-type cells; KO, 3ß1 integrin-deficient cells; 3-stalk, cells expressing 6/3 chimeric integrin that has 3-stalk region; 6-stalk, cells expressing 6/3 chimeric integrin that has 6-stalk region. (A) A schematic representation of the chimeric integrin subunits, showing the inclusion of either the 3- or 6-stalk region. (B) Immunoprecipitation of surface-labeled cells using a polyclonal antibody to the 3 integrin cytoplasmic domain or an 6 mAb. Cells were labeled with biotin, lysed using 1% Brij 96 buffer, and lysates were subjected to immunoprecipitation either with the 3 cytoplasmic domain antibody or with the 6 mAb. (C) Coimmunoprecipitation of integrins and CD151. Cell lines were extracted in 1% Triton X-100 buffer and immunoprecipitated with either the 3 or 6 antibody or with the CD151 antibody and immunoblotted with CD151 antibody. The immunoprecipitation and immunoblot antibodies are designated above or below the panels. (D) CD151 shRNA. Wild-type cells were infected with lentivirus containing CD151 shRNA. Cells were extracted in 1% Triton X-100 buffer and tested for CD151 expression and for their association with 3ß1 integrin. One of the CD151 shRNAs (CD151 shRNA) blocked the expression and the other one did not (control shRNA) and was used in subsequent experiments as a negative control. (E) Adhesion assay. Plates were coated with laminin-1 or human placental laminin (contains mainly laminin-10 and -11), and adhesion of the cells was analyzed by an MTT assay. Each bar represents the mean of five wells, and SEM bars are shown. 100% adhesion is defined as the adhesion of 6-stalk cells for laminin-1 and wild-type cells for laminin-10 and -11.

Multiple tetraspanins interact with 3ß1 integrin. Lentiviral-based small hairpin RNA (shRNA; Rubinson et al., 2003) vectors were constructed to allow specific inhibition of CD151 expression (Fig. 1 D). Two of three constructs sufficiently inhibited CD151 expression (Fig. 1 D), no CD151 was found in association with 3ß1 integrin. The shRNA vector that did not inhibit CD151 was used in further experiments as a negative control.

6/3 chimeric integrins mediate adhesion to laminin-1, -10, and -11
Although 3ß1 and 6ß1 are both receptors for laminin, 3ß1 preferentially binds laminins 5, 10, and 11, whereas 6ß1 has been shown to have a less restricted laminin-binding repertoire (Delwel et al., 1994; Kikkawa et al., 1998; Yauch et al., 2000). Cells expressing the 6 extracellular domain (3-stalk and 6-stalk) showed increased adhesion to laminin-1 in comparison with wild-type and mutant cells (Fig. 1 E). Consistent with the ability of both 3ß1 and 6ß1 to bind laminin-10 and -11, wild-type and chimeric integrin-expressing cell lines showed increased adhesion to laminin-10 and -11 in comparison with 3ß1 integrin–deficient cells. Importantly, the heterodimer of ß1 and the 6-stalk–containing subunit mediated adhesion to laminin-10 and -11 despite its inability to interact with CD151. This finding is consistent with observations that integrin–tetraspanin interactions are not involved in modulating the cell–ECM adhesive functions.

3ß1–CD151 complex stimulates E-cadherin–mediated cell–cell adhesion
Cells were trypsinized in the presence of calcium to preserve cadherins (Takeichi, 1977) and kept in suspension, such that the ability of cells to aggregate could be studied independently of cell migration. 50% of the input wild-type cells and 3-stalk–expressing cells became aggregated within 1 h in suspension, and after 3 h, nearly 80% of the cells were found in clusters. In contrast, only 20% of mutant and 6-stalk–expressing cells were found in clusters at 1 h, and only 30% after 3 h in suspension (Fig. 2 A). Similarly, expression of CD151 shRNA decreased the aggregation of wild-type cells down to levels observed for mutant or 6-stalk–expressing cells (Fig. 2 A). Thus, cell aggregation appears to be stimulated by the integrin–tetraspanin interaction.

View larger version (59K): [in this window] [in a new window]   Figure 2. Cadherin-mediated cell–cell adhesion. (A) Cell aggregation assay. Cells were kept in suspension for the designated time periods in complete medium, in medium with the addition of the HAV hexapeptide, or in the presence of E-cadherin–blocking antibody. The number of single cells and the number of cells in clusters were counted. The standard curve was used to determine the optimum concentration of HAV hexapeptide. (B) Binding assay. 96-well plates were coated with E-cadherin/Fc recombinant protein and binding of control, E-cadherin–blocking antibody–treated and HAV peptide–treated cells were measured by the MTT assay. Binding of wild-type cells to E-cadherin was considered as 100%, representing binding of over 95% of the input cells. Each bar is the mean of five wells and SEMs are shown.

The integrin–tetraspanin interaction is required for the interaction of the cadherin–catenin complex with the subcortical cytoskeleton
Cadherin-mediated cell–cell interactions progress from weak to strong interactions through the assembly of cadherin–catenin complexes and the association of these complexes with the subcortical actin cytoskeleton (Braga, 2000). E-cadherin and ß-catenin were present at cell–cell contacts in all cell lines, although cell–cell contacts appeared more diffuse in mutant and 6-stalk cells (Fig. 3 A). -Actinin is an actin-binding protein shown to be involved in associating the cadherin–catenin complex with the cytoskeleton (Knudsen et al., 1995; Nieset et al., 1997). -Actinin was present at the cell–cell junction of wild-type and 3-stalk–containing cells but stained diffusely in cells where integrin–tetraspanin interaction was absent, suggesting that the cadherin–catenin complex does not colocalize with -actinin in these cells. Moreover, a subcortical actin cytoskeleton was present in both wild-type and 3-stalk cells, whereas actin stress fibers were more prominent in mutant and 6-stalk cells (Fig. 3 A) or in cells expressing shRNA for CD151 (Fig. 3 A).

View larger version (175K): [in this window] [in a new window]   Figure 3. Cadherin–catenin complexes. (A) Immunocytochemistry. Cells were stained with different antibodies indicated at the top of each panel and cell lines are designated at the left side of each panel. Phalloidin-Texas red was used to stain for actin. (B) E-cadherin immunoprecipitation. Cells were extracted in 1% Triton X-100 buffer and equal amounts of cell extract from each cell lines were immunoprecipitated and blotted. Cell lines and antibodies for immunoprecipitation and immunoblot are shown at the bottom and top of each panel, respectively. (C) Inhibition of CD151 expression with shRNA dissociated -actinin from the cadherin–catenin complex. (D) Tyrosine phosphorylation of ß-catenin. Cells were treated with 1 mM sodium orthovanadate before extraction. All buffers used in this experiment were supplemented with 1 mM sodium orthovanadate. The top panel is the phosphotyrosine blot, and the bottom panel is the reblot with the same antibody used for immunoprecipitation. (E) A model summarizing the results of this figure.

ß-Catenin tyrosine phosphorylation is regulated by the integrin–tetraspanin interaction
The association of E-cadherin with ß-catenin can be regulated by the tyrosine phosphorylation of ß-catenin (Muller et al., 1999; Roura et al., 1999), and tyrosine phosphorylation of ß-catenin generally has been correlated with a loss of epithelial morphology (Ozawa and Kemler, 1998; Provost and Rimm, 1999). Consistent with these observations, loss of the 3ß1–CD151 interaction leads to increased tyrosine phosphorylation of ß-catenin (Fig. 3 D). It was also observed that the total amount of ß-catenin present in wild-type and 3-stalk cells is higher than that of mutant and 6-stalk cells (Fig. 3 D), even though the amount of ß-catenin associated with E-cadherin does not differ (Fig. 3 B). However, our work does not support the hypothesis that tyrosine phosphorylation of ß-catenin dissociates it from E-cadherin, at least in the cells under study. This is because when cells are pretreated with orthovanadate to preserve phosphorylation (as in Fig. 3 D but not Fig. 3 B), the reblot with an anti–ß-catenin antibody shows all the ß-catenin in 3ß1-deficient and 6-stalk chimera–expressing cells as a higher molecular mass band than that observed in wild-type and 3-stalk chimera–expressing cells (Fig. 3 D), presumably reflecting a hyperphosphorylated state. Combined with our additional observation that the E-cadherin–ß-catenin association did not differ depending on the integrin–tetraspanin interaction, our results suggest that tyrosine-phosphorylated ß-catenin remains associated with E-cadherin in these cell lines. The results are summarized in a model (Fig. 3 E).

PTPµ expression is regulated by integrin–tetraspanin association
The increased tyrosine phosphorylation observed in mutant cells led us to examine the possibility that a phosphatase activity was decreased in the absence of the integrin–tetraspanin interaction. Several tyrosine phosphatases have been shown to be associated with E-cadherin (Lilien et al., 2002). PTPµ is a transmembrane protein tyrosine phosphatase that can interact with several classical cadherins, including E-cadherin, and can regulate E-cadherin–mediated cell–cell adhesion (Brady-Kalnay et al., 1995, 1998; Hellberg et al., 2002). The proteolytic form of PTPµ (detected at 100 kD) was found to be associated with cadherin–catenin complex only in wild-type and 3-stalk–expressing cells, suggesting a potential role for PTPµ in regulating the tyrosine phosphorylation of the cadherin–catenin complex. Indeed, upon further analysis, it was determined that the full-length (200 kD) and proteolytic (100 kD) forms of PTPµ are expressed only in the cells where the integrin–tetraspanin interaction is present, neither form of PTPµ or PTPµ RNA was found in mutant or 6-stalk–expressing cells (Fig. 4, A and B). This finding indicates a role for the 3ß1–CD151 complex in modulating PTPµ gene expression, an area of future studies.

View larger version (33K): [in this window] [in a new window]   Figure 4. Association of the cadherin–catenin complex with PTPµ. Antibodies used for immunoprecipitation and immunoblotting are described at the top of each panel and cell lines are designated at the bottom. (A) PTPµ coimmunoprecipitation. PTPµ is expressed only in cells where 3ß1–CD151 complex was present and coimmunoprecipitated with E-cadherin and ß-catenin. (B) RT-PCR. Total RNA was extracted from the cells and RT-PCR using primers for PTPµ. No-RT, negative control using wild-type RNA but no reverse transcriptase. ß-Actin RT-PCR was used as a control. (C) A model summarizing the results of this figure.

View larger version (184K): [in this window] [in a new window]   Figure 5. PTPµ baculovirus infection. (A) Cell aggregation assays. Cells were treated with either 1 mM phosphatase inhibitor (bpV [Phen]) or infected with baculovirus expressing either wild type or the C-S mutant of PTPµ, each as a GFP fusion protein. (B) E-cadherin/Fc adhesion assay after baculovirus infection. Each bar is the mean of five wells and SEMs are shown. (C) FACS® analysis after baculovirus expression. The shaded curve shows uninfected cells, and the unshaded curve shows infected cells. (D) Immunoprecipitation. Cells were extracted in 1% Triton X-100 buffer after baculovirus infection. Cadherin–catenin immunoprecipitations and the ß-catenin tyrosine phosphorylation assay were performed as described in Materials and methods. (E) Phalloidin staining of baculovirus-infected cells. (F) A model summarizing the results of this figure. When wild-type PTPµ is present in 6-stalk cells, it is not associated with the cadherin–catenin complex (middle model); hence, cadherin-mediated cell–cell adhesion is weak. When the C-S form of PTPµ is overexpressed in wild-type cells (right model), it displaces wild-type PTPµ from its association with the cadherin–catenin complex; ß-catenin becomes phosphorylated; and there is weak cadherin-mediated adhesion.

The 3ß1–tetraspanin complex stabilizes the interaction of PTPµ with the cadherin–catenin complex
The failure of wild-type PTPµ to rescue mutant or 6-stalk cells suggested that the presence of the integrin–tetraspanin complex on the cell membrane could also affect the association of PTPµ with the cadherin–catenin complex. Previous studies demonstrated an interaction of the 3ß1–CD151 complex with PKCßII (Zhang et al., 2001) and an interaction between PTPµ and the adaptor protein RACK1 (Mourton et al., 2001). Because activated PKCßII binds RACK1 (Ron et al., 1994; Mochly-Rosen, 1995; Csukai et al., 1997), these results led us to investigate the possibility that the integrin–tetraspanin complex could stabilize the interaction of PTPµ with the cadherin–catenin complex through the establishment of a complex that contains PKCßII, RACK1, and PTPµ. After cross-linking, PKCßII, RACK1, and PTPµ could be coimmunoprecipitated with 3ß1 or the 3-stalk–containing integrin heterodimer, but they were not present in immunoprecipitates from the 6-stalk–expressing cells (Fig. 6 A). Expression of CD151 shRNA also abrogated the association of RACK1 with the integrin–tetraspanin complex (Fig. 6 A). Sequential immunoprecipitations of cross-linked cells were used to demonstrate that the same pool of PTPµ associated with 3ß1–CD151 and with the cadherin–catenin complex (Fig. 6 B). As negative controls, none of these components could be coimmunoprecipitated from wild-type cells with 6ß1 integrin. As additional negative controls, neither CD44, an abundant membrane protein, nor Ezrin, an abundant peripheral membrane protein associated with the cytoskeleton, could be coimmunoprecipitated with 3ß1 integrin, PTPµ, E-cadherin (Fig. 6 A), or RACK1 and PKCßII (not depicted). Additionally, the epidermal growth factor receptor, although present in all cells, was not coimmunoprecipitated with 3ß1–CD151 (unpublished data).

View larger version (162K): [in this window] [in a new window]   Figure 6. Interactions of the integrin–tetraspanin complex. (A) Coimmunoprecipitations of the integrin–tetraspanin complex. Cell lines and antibodies for immunoprecipitation and immunoblot are shown at the top and bottom of each panel, and cell lines are designated as in Fig. 1. In A–C, cells were cross-linked with 1 mM DSP at 4°C for 1 h before extraction. Cells were extracted in 1% Triton X-100 buffer and subjected to immunoprecipitation either with 3 or 6 antibody. The control 3 reblot of the 3 immunoprecipitation is shown in Fig. 1 C, as this required nonreducing conditions and was run in a separate gel. An 6 immunoprecipitation was used as a negative control. CD151 RNAi experiment. CD151shRNA or control shRNA was expressed in wild-type cells before immunoprecipitation of 3ß1 integrin and immunoblotting for RACK1. (B) Sequential immunoprecipitations showing the same pool of PTPµ is associated with 3ß1–CD151 complex and the cadherin–catenin complex. Cells were cross-linked before extraction. Proteins were extracted from 3 immunoprecipitates with 1% Triton X-100 buffer containing 0.5% SDS. The second immunoprecipitation was done with PTPµ antibody and the immunoblot was developed either with E-cadherin or ß-catenin antibodies. The negative control in the rightmost lane of each panel is a wild-type extract in which the primary antibody is omitted from the second immunoprecipitation. The reblots with PTPµ serve as positive controls for the immunoprecipitations. After obtaining the first blots, the membrane was stripped in buffer containing 62.5 mM Tris-HCl, pH 6.7, 0.7% ß-mercaptoethanol, and 2% SDS for 30 min at 50°C and reprobed with a PTPµ antibody. (C and D) Inhibition of PKC–RACK1 interaction. Cells were treated with 1 µM of carrier or PKCßII translocation inhibitor peptide, as designated above the panels, 1 h before beginning the aggregation assay or before extraction. (C) Translocation inhibitor effects on the 3ß1 integrin–E-cadherin–PTPµ association. (D) Effect of translocation inhibitor on cell aggregation and cadherin complex association. The inhibitor decreased aggregation of wild-type and 3-stalk cells. Each bar of the aggregation assay histogram is the mean of five wells, and SEMs are shown. Association with -actinin is lost, but the E-cadherin–ß-catenin association is not affected.

3ß1 integrins are expressed on the lateral surface of the epithelial cells and can act as members of the cell–cell adhesion complex
Some integrins, including 3ß1 integrin, are often found in a basolateral distribution, particularly in developing epithelia. Confocal microscopy demonstrated the lateral localization of 3ß1 in 3-stalk and 6-stalk–expressing cells (Fig. 7 A). Together with the preceding data, this suggests a model in which 3ß1 in basal membranes mediates cell–matrix adhesion, whereas 3ß1–CD151 in lateral membranes modulates cell–cell adhesion. To further examine the hypothesis that 3ß1 molecules associated with cadherin–catenin complexes were separate from those binding laminin, and the converse situation, sequential immunoprecipitations were performed. DiPersio et al. (1995) had shown that it is possible to cross-link 3ß1 integrin to the underlying ECM before immunofluorescent staining. Using a similar approach, cells plated on a laminin-5–rich matrix were cross-linked and extracted, and the immunoprecipitate obtained with an anti–3 integrin antibody was reimmunoprecipitated with anti–laminin-5, and then blotted for E-cadherin, which was not detected (Fig. 7 B). Laminin-5 could be detected after reblot, serving as a positive control for the immunoprecipitations. In the converse experiment, cells were sequentially immunoprecipitated with anti–3 integrin and anti–E-cadherin, and then blotted for laminin-5, which was not detected, with the reblotting for E-cadherin serving as a positive control (Fig. 7 B). Both experiments showed that distinct pools of 3ß1 integrin receptors were either binding laminin-5 or associated with E-cadherin, but not both. To further determine whether the ECM ligand-binding properties of 3ß1 integrin affected its association with E-cadherin and PTPµ, cells were plated on fibronectin instead of laminin-5 or -10 and -11. Plating on fibronectin had no effect on the association of 3ß1 with CD151, PKCßII, RACK1, or PTPµ, nor did it affect the expression or activity of total PTPµ or that associated with 3ß1 integrin (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200306067/DC1), supporting the conclusion that cell–cell interaction is regulated by 3ß1 situated at the cell–cell junction and not dependent on interaction of 3ß1 with components of the ECM.

View larger version (33K): [in this window] [in a new window]   Figure 7. Expression of 3ß1 integrin on the lateral surface of epithelial cells. (A) Immunofluorescence staining of 3ß1. The xz axis is shown. Staining for 3ß1 integrin showed lateral expression along the entire z-axis. Cell lines are shown above each panel. (B) Sequential immunoprecipitations. Cells were treated with 1 mM DSP before extraction. Cell lines and antibodies for immunoprecipitations and immunoblots are shown above each panel. Lane C is the negative control in which the primary antibody is omitted from the second immunoprecipitation.

	Discussion

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View larger version (25K): [in this window] [in a new window]   Figure 8. A model for 3ß1 as a component of the cell–cell adhesion complex. The integrin–tetraspanin complex present on the lateral surface of the cells induced expression of PTPµ and can organize a multimolecular complex containing 3ß1–CD151–PKCßII–RACK1–PTPµ–ß-catenin–E-cadherin.

There has been relatively little study of how expression of specific integrin repertoires may generate specific patterns of gene expression. Previously, the expression of MMP9 was shown to be activated in immortalized keratinocytes in the absence of 3ß1 integrin (DiPersio et al., 2000), providing at least one example of a gene expression difference related to 3ß1 expression. The dependence of PTPµ expression on the 3ß1–tetraspanin interaction demonstrates how epithelial morphology and adhesive behavior can be dramatically affected by differences in gene expression. Activation of phosphatase and kinase expression based on interactions of integrins with ECM ligands or other cell surface molecules, as shown here, provides an indication of how the integrin repertoire may affect cell migration or cell–cell interaction during development or tumorigenesis.

Regulation of cadherin–catenin association
Cadherin-mediated adhesion is regulated through the assembly of cadherin–catenin complexes at the cadherin cytoplasmic domain. The assembly of these complexes is essential for the transition from weak to strong cell–cell contacts. There are different observations with regard to the specific molecular interactions that are affected by signaling events regulating cell–cell interaction and cell morphology. For example, several studies that either increased kinase activity through stimulation with EGF or decreased phosphatase activity using pervanadate or phosphatase mutants demonstrated decreased interaction between a cadherin–catenin complex and the cytoskeleton (Balsamo et al., 1998; Hazan and Norton, 1998; Ozawa and Kemler, 1998). This decreased interaction was correlated with increased tyrosine phosphorylation of ß-catenin. Other studies have shown that tyrosine phosphorylation of ß-catenin results in decreased interaction of ß-catenin with E-cadherin (Muller et al., 1999; Roura et al., 1999). In Wang et al. (1999), we observed yet another level of regulation between the cadherin–catenin complex and -actinin shown here to be dependent on the integrin–tetraspanin interaction. Wang et al. also detected no integrin-dependent difference in the -catenin–ß-catenin association, confirmed in this work. It is possible that these different observations reflect the distinct cell types used in the respective studies and the different kinase and phosphatase activities present therein. As discussed previously, our paper does not support the hypothesis that tyrosine phosphorylation of ß-catenin dissociates it from E-cadherin. However, it is important to note that there are several tyrosine residues in ß-catenin, and it is not known if the tyrosine residues phosphorylated in our cell lines are the same identified in a previous paper (Roura et al., 1999).

The HAV sequence is conserved among several members of the cadherin family, and HAV-containing peptides have historically been used to block homophilic interaction between cadherin molecules. Renaud-Young and Gallin recently published a paper in which mutation of the HAV sequence did not affect homophilic adhesion, leading them to suggest that the HAV sequence may not be involved in the initial cadherin homophilic interaction (Renaud-Young and Gallin, 2002). This possibility is consistent with our results because the HAV peptide and the phosphatase inhibitor blocked adhesion of wild-type cells to E-cadherin/Fc down to levels observed for knockout cells, whereas the E-cadherin–blocking antibody entirely blocked adhesion to E-cadherin/Fc.

Integrins, tetraspanins, and cell transformation
Our results lead us to hypothesize that neoplastic transformation of a cell is due to both the activation of specific oncogenes and the loss of signaling molecules from integrin–tetraspanin complexes. In a normal epithelial cell, integrin–tetraspanin complexes direct expression of PTPµ, which binds RACK1 and establishes an integrin–tetraspanin-dependent link to the cadherin–catenin complex, thereby stimulating cadherin-mediated cell–cell adhesion. The RACK1 scaffolding protein binds to the Src tyrosine kinase, and binding of RACK1 to PTPµ or Src is mutually exclusive (Mourton et al., 2001). In a neoplastic cell, increased levels of activated Src may displace PTPµ from RACK1 and suppress cadherin-mediate adhesion, or loss of 3ß1 integrin expression may result in a complete loss of PTPµ expression, exacerbating the affect of activated oncogenes.

Hellberg et al. (2002) recently studied the role of PTPµ in conferring cadherin-dependent cell–cell adhesion in prostate carcinoma cells. Both Hellberg et al. (2002) and the present work demonstrate the importance of PTPµ in regulating E-cadherin–mediated adhesion. In contrast to the observations of Hellberg et al., we demonstrated that the phosphatase activity of PTPµ is required to increase E-cadherin–dependent adhesion. Because that work used prostate carcinoma cells, this difference may reflect different levels of activated tyrosine kinases or different integrin–tetraspanin complexes within the respective cell types that rendered the cells more or less sensitive to the phosphatase activity of PTPµ. In support of this hypothesis, N-cadherin–dependent neurite outgrowth does require PTPµ tyrosine phosphatase activity (Burden-Gulley and Brady-Kalnay, 1999). As demonstrated in Hellberg et al., PTPµ expression is variable among carcinoma cells lines. It will be of interest to examine various transformed cell lines and determine if expression of PTPµ correlates with the presence of integrin–tetraspanin complexes. Not all epithelial cells in mammals express 3ß1 integrin, and it is likely that other closely related integrins that are also known to associate with tetraspanins may function similarly in other cell types.

In summary, this paper identifies a new role for 3ß1 and perhaps other integrins as components of cell–cell adhesion complexes. Association with tetraspanins appears essential for this function, and integrin–tetraspanin complexes may direct specific patterns of gene expression in addition to directing protein–protein interactions at the membrane. In the future, consideration of the role of integrins in disease processes that involved changes in cell morphology, such as epithelial to mesenchymal transitions in fibrosis, or tumor progression, will need to consider this new role for integrins.

	Materials and methods

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Antibodies, peptides, and other materials
Rabbit polyclonal anti-3ß1 antibody was obtained from R. Hynes (Massachusetts Institute of Technology, Cambridge, MA; DiPersio et al., 1995); rabbit polyclonal anti-GFP was obtained from P. Silver (Dana-Farber Cancer Center, Boston, MA); monoclonal anti-CD151 11b1-G4 (Sincock et al., 1997). mAbs against intracellular domain of PTPµ (SK15 and SK18; Brady-Kalnay et al., 1993). Anti–integrin 6 A6ELE was obtained from M. Hemler (Dana-Farber Cancer Center; Lee et al., 1995). E-cadherin antibody ECCD-2 for immunofluorescence was purchased from Zymed Laboratories; E-cadherin–blocking antibody (monoclonal antiuvomorulin, clone DECMA-1) and anti–-actinin clone BM-75.2 antibody was purchased from Sigma-Aldrich; E-cadherin (clone 36), ß-catenin (clone 14), -catenin (clone 5), and RACK1 (clone 20) antibodies were purchased from BD Biosciences; antiphosphotyrosine antibody 4G10 was purchased from Upstate Biotechnology; and anti-PTPµ (C-20), anti-6 (N-19), anti-PKCßII (C-18), and anti-3ß1 (Ralph 3.2) were purchased from Santa Cruz Biotechnology, Inc. Anti-6 (MA6) for immunoprecipitation and monoclonal anti–laminin-5 (epiligrin, clone P3H9–2) were obtained from Chemicon International. All secondary antibodies were obtained from Jackson ImmunoResearch Laboratories.

E-cadherin–blocking peptide Ac-SHAVAS-NH₂ and control peptide (Ac-SVAHAS-NH₂) were obtained from New England Peptide, Inc. PKC regulator peptides pp94 and pp96 were obtained from the D. Mochly-Rosen (Stanford University, Stanford, CA).

NHS biotinylation reagent and DSP were obtained from Pierce Chemical Co.; recombinant mouse E-cadherin/Fc chimeric protein, Trichostatin A, and MTT were obtained from Sigma-Aldrich; matrigel was obtained from BD Biosciences; tyrosine phosphatase inhibitor bpV(phen) was obtained from Calbiochem; human placental laminin (contains mainly laminin-10 and -11) was obtained from Chemicon International; and laminin-1 was obtained from BD Biosciences. N-Cadherin/Fc recombinant protein plasmid was obtained from P. Doherty (Kings College, London, UK; Utton et al., 2001).

Oligonucleotides for PTPµ RT-PCR were obtained from Invitrogen. CD151 RNAi oligonucleotides were obtained from IDT. All other common chemicals were obtained from Sigma-Aldrich and Bio-Rad Laboratories.

cDNA constructs
Construction of 6/3 chimeric integrins was performed as described in Yauch et al. (2000). 6/3 chimeric integrins were subcloned in pcDNA3.1 hygro (-) vector.

Cell lines
Generation of wild-type and knockout immortalized cell lines from wild-type and 3 mutant mouse kidney collecting ducts was performed as described previously (B7 and B12 cells in Wang et al., 1999). To obtain the 3- and 6-stalk cells, knockout cells were transfected with 6/3 chimeric integrins in pCDNA3.1 hygro using calcium phosphate transfections and selected for hygromycin resistance. Pools of transfected cells were FACS^® sorted using anti–human 6 (A6-ELE) antibody. To culture cells on laminin-5, SCC25 cells (which produce a laminin-5–rich matrix) were grown to confluence and removed (Xia et al., 1996) before plating cells under study.

Cell lysis and immunoprecipitation
Cells were grown in 10-cm dishes precoated with a laminin-5–rich matrix. For immunoprecipitation and blotting of cadherin–catenin complexes, cells were washed with PBS and lysed in lysis buffer (20 mM Tris, pH 7.6, 1% Triton X-100, 2 mM CaCl₂, 1 mM benzamidine, 0.1 mM ammonium molybdate, 1 mM PMSF, 20 µg/ml aprotinin, and 10 µg/ml leupeptin). For ß-catenin tyrosine phosphorylation assay, cells were pretreated with 1 mM sodium orthovanadate before lysis, and all the buffers for immunoprecipitation and immunoblot were supplemented with 2 mM sodium orthovanadate.

In experiments where the interaction of 3ß1–CD151 complex with PKCßII, RACK1, and PTPµ was studied (Fig. 6) and in sequential immunoprecipitations (Figs. 6 and 7), cells were incubated in 1 mM DSP for 1 h at 4°C (to cross-link the proteins) and treated as described by Berditchevski et al. (1995). Integrin surface labeling and immunoprecipitations were conducted as described previously (Wang et al., 1999).

Laminin adhesion assay
96-well plates were coated with human placental laminin (predominantly laminin-10 and -11) or laminin-1 for 2 h at RT, and blocked with 1% BSA in PBS containing 100 mM Ca²⁺ and 100 mM Mg²⁺ for 1 h. 2.5 x 10⁴ cells prepared by trypsinization in 200 µl of medium containing 1% FCS were added in each well for 1 h at RT, this being the maximal number of cells that can adhere to a coated well. After washing out nonadherent cells, adherent cells were incubated 3 h in medium containing 800 µg/ml MTT solution. The reaction product was measured at 595 nm. Each data point is the mean of five wells, and SEMs are shown at the top of each bar in the figures. The background level of binding, defined as the number of wild-type cells adhering to a BSA-only well, usually 1–2% of the level of wild-type cells binding to placental laminin, was subtracted from all results. In pilot experiments, absorbance at 595 nm was directly proportional to the number of adherent cells.

E-cadherin/Fc adhesion assay
Cells were treated as previously described (Higgins et al., 1998), 96-well plates were coated with 1.5 µg/ml recombinant mouse E-cadherin/Fc chimeric or with N-cadherin/Fc chimeric proteins and blocked with 1% BSA. To test the effect of E-cadherin–blocking antibody or HAV peptide on cell binding, cells were incubated in 5 µg/ml antibody or 400 µM HAV peptide before adding to the wells. Adhesion was measured as described above for the laminin binding assay.

Cell aggregation assay
Cells were trypsinized in the presence of calcium as described in the preceding section. A single cell suspension was obtained and 2.5 x 10⁴ cells were placed in a 0.2-ml tube and incubated on a rotation apparatus for 0, 1, or 3 h at RT. At the end of the incubation, cells were diluted into single wells of a 6-well plate to prevent further aggregation. After allowing cells to settle for 10 min at 33°C, the number of single cells and cells in clusters were manually counted, counting 10 low-power fields using an inverted tissue culture microscope. The percentage of cells in clusters was calculated as the number in clusters of five or more cells, divided by the total number of single cells and cells in clusters. To study the effect of phosphatase inhibitor or PKC inhibitor peptides on cell–cell aggregation, cells were treated with 1 mM bpV (phen) or 1 µM PKC-regulating or control peptide before trypsinization. In the case of HAV peptide (or control peptide) or antibody treatment, cells were kept in suspension in the presence of 5 µg/ml anti–E-cadherin–blocking antibody or 400 µM HAV peptide (or control peptide).

Immunofluorescent staining
For immunofluorescent staining, the cells were grown overnight in 8-well glass chamber slides coated with human placental laminin (source of laminin-10 and -11). Cells were washed, fixed in 3% PFA, permeabilized with 5% NP-40, and blocked with 10% sheep serum. After blocking, the cells were incubated with E-cadherin, ß-catenin, or -actinin antibodies, followed by FITC-coupled IgG. For actin staining, cells were reacted with Texas red–coupled phalloidin.

RT-PCR
Total RNA was isolated from cells as described previously (Chomczynski and Sacchi, 1987). 7 µg of total RNA was used for reverse transcription reaction using Prostar first stand RT-PCR kit. First strand cDNA was synthesized as described by the manufacturer (Stratagene). The resulting cDNA was subjected to PCR amplification reaction using primers 5'-ACCTCCTCCAACACATCAC-3' and 5'-TCACGGACACTGTAGAACTC-3' and following protocol supplied by QIAGEN. The PCR product was visualized using ethidium bromide in 1% agarose gel.

Baculovirus production
Using the pBacMam-2 vector obtained from Novagen, the following constructs were generated: wild-type PTPµ and a catalytically inactive (C-S) mutant form of PTPµ. This vector system allows expression of exogenous genes in mammalian cells using recombinant baculoviruses. The wild-type PTPµ tagged with the GFP at the COOH terminus and the catalytically inactive (C-S) mutant PTPµ-GFP have been described previously (Burden-Gulley and Brady-Kalnay, 1999). The pBPSTR1 plasmids were digested with NotI and the PTPµ-GFP–encoding DNA was ligated into the pBacMAM-2 vector (Novagen) that had been digested with NotI. The recombinant baculoviruses were made using the BaculoGold Transfection System (Invitrogen). In brief, recombinant baculoviruses were generated by calcium phosphate-mediated cotransfection of Sf9 cells with plasmid and viral DNA. Four rounds of virus amplification were performed. The virus was harvested from the Sf9 cells 4 d after infection. To infect mammalian cells, 500 µl of viral supernatant was added to a 10-cm dish of cells containing 4 ml of media and incubated at 37°C for 2 h. After incubation all the culture media was removed and fresh media containing a final concentration of 150 nM Trichostatin A (Sigma-Aldrich) was added to the cultures. 16 to 24 h after virus addition, PTPµ expression was analyzed by FACS^®. Exogenous PTPµ expression was also verified by immunoblotting lysates from infected cells with antibodies to PTPµ.

CD151 RNAi
Three sequences were selected from mouse CD151 gene, which were predicted to form CD151 shRNA. The oligos were inserted into pLentilox3.7 vector obtained from L. van Parijs (Massachusetts Institute of Technology; Rubinson et al., 2003) to generate pLL CD151–1, pLL-CD151–2, and pLL-CD151–3, and cotransfected with packaging vectors into 293T cells. This vector expresses shRNA and GFP, each from distinct promoters. The lentivirus in the supernatant was collected after 24 h and used directly to infect the wild-type cell line; GFP expression was maximum after 48 h of infection. Immunoprecipitation, immunofluorescence, and cell–cell aggregation assay were done after 48 h of infection.

Online supplemental material
Fig. S1 shows in vitro phosphatase assays demonstrating the activity of endogenous and baculoviral PTPµ. Fig. S2 demonstrates similar behavior of cells plated on fibronectin or laminin. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200306067/DC1.

	Acknowledgments

 
S.M. Brady-Kalnay acknowledges the technical assistance of Tracy Mourton and Carol Luckey. The authors thank Douglas Cotanche for assistance with confocal microscopy and Mary Taglienti for technical assistance. The confocal microscope facility is supported by the Sarah Fuller Fund. Daria Mochly-Rosen is acknowledged for the PKC inhibitor peptides, Martin Hemler for chimeric integrin constructs, and Luk van Parijs for the Lentiviral shRNA vectors. The authors have no commercial interest in this work.

This work was supported by a National Institute of Diabetes and Digestive and Kidney Diseases grant DK57604 to J.A. Kreidberg and National Institutes of Health grant EY12251 to S.M. Brady-Kalnay. S.M. Brady-Kalnay acknowledges additional support from a Visual Sciences Research Center Core grant from the National Eye Institute (grant P0-EY11373).

Submitted: 13 June 2003

Accepted: 29 October 2003

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