Murine Vascular Cell Adhesion Molecule-1 (VCAM-1) Proteins Encoded by Alternatively Spliced rnRNAs Are Differentially Targeted in Polarized Cells

VCAM-1 is an immunoglobulin (Ig) superfamily member expressed in endothelial cells that mediates adhesion to a variety of leukocytes in a VLA-4 dependent manner. In the mouse, two distinct forms of VCAM are produced. One form, VCAM(tm), contains seven Ig domains followed by a single transmembrane region and a short cytoplasmic domain. A second form, VCAM^GPI, which is preferentially induced by cytokines and LPS, contains only the first three Ig domains and is attached to the cell surface via a glycosylphosphatidylinositol (GPI) anchor. Both vascular and nonvascular expression of VCAM have been reported in a variety of normal and pathological settings. One possible role for the two VCAM isoforms is to allow for the targeted localization of VCAM to specific cell surface domains of polarized cells. This may be particularly relevant since VCAM is known to be expressed by two different polarized cell types, namely endothelial cells and kidney epithelial cells. In this study, MDCK cells permanently expressing either VCAM(tm) or VCAM^GPI were established and used to examine the targeting of VCAM proteins to different polarized surface domains. VCAM(tm) was primarily located on the basolateral surface while VCAM^GPI was located on the apical surface of polarized MDCK cells. Data is also presented that demonstrates that polarized expression is reversed in endothelial cells where VCAM(tm) was observed primarily on the apical surface. The differential localization of VCAM isoforms on the cell surface has direct implications for the ability of VCAM to mediate cell adhesion and transmigration.     

A dileucine motif targets MCAM-l cell adhesion molecule to the basolateral membrane in MDCK cells

Melanoma cell adhesion molecule (MCAM), an adhesion molecule belonging to the Ig superfamily, is an endothelial marker and is expressed in different epithelia. MCAM is expressed as two isoforms differing by their cytoplasmic domain: MCAM-l and MCAM-s (long and short). In order to identify the respective role of each MCAM isoform, we analyzed MCAM isoform targeting in polarized epithelial Madin-Darby canine kidney (MDCK) cells using MCAM-GFP chimeras. Confocal microscopy revealed that MCAM-s and MCAM-l were addressed to the apical and basolateral membranes, respectively. Transfection of MCAM-l mutants established that a single dileucine motif (41-42) of the cytoplasmic domain was required for MCAM-l basolateral targeting in MDCK cells. Although double labelling experiments showed that MCAM-l is not a component of adherens junctions and focal adhesions, its expression on basolateral membranes suggests that MCAM-l is involved in epithelium insuring.     

Cloning of an alternate form of vascular cell adhesion molecule-1 (VCAM1).

Vascular cell adhesion molecule-1 (VCAM1) of the Ig superfamily is induced by the inflammatory cytokines interleukin-1 and tumor necrosis factor on human umbilical vein endothelial cells (HUVECs). It binds to mononuclear leukocytes via the integrin VLA-4. We have cloned and expressed a cDNA encoding a new form of human VCAM1 containing an additional Ig homologous domain inserted between the third and fourth domains of the original six-domain protein. Characterization of mRNA from HUVECs from three individuals at various time points after induction by tumor necrosis factor indicates that both the long and short VCAM1 mRNAs are made by all three individuals, with the long form predominating quantitatively. Immunoprecipitation of VCAM1 protein from cos7 cells transfected with each cDNA and from cultured endothelial cells followed by deglycosylation suggests that the long form is the major form found on endothelium. The two forms may result from alternate splicing of a precursor mRNA. Both forms support adhesion of VLA-4-expressing cell lines.     

Differential trafficking of transforming growth factor-beta receptors and ligand in polarized epithelial cells

Epithelial cells in vivo form tight cell-cell associations that spatially separate distinct apical and basolateral domains. These domains provide discrete cellular processes essential for proper tissue and organ development. Using confocal imaging and selective plasma membrane domain activation, the type I and type II transforming growth factor-beta (TGFbeta) receptors were found to be localized specifically at the basolateral surfaces of polarized Madin-Darby canine kidney (MDCK) cells. Receptors concentrated predominantly at the lateral sites of cell-cell contact, adjacent to the gap junctional complex. Cytoplasmic domain truncations for each receptor resulted in the loss of specific lateral domain targeting and dispersion to both the apical and basal domains. Whereas receptors concentrate basolaterally in regions of direct cell-cell contact in nonpolarized MDCK cell monolayers, receptor staining was absent from areas of noncell contact. In contrast to the defined basolateral polarity observed for the TGFbeta receptor complex, TGFbeta ligand secretion was found to be from the apical surfaces. Confocal imaging of MDCK cells with an antibody to TGFbeta1 confirmed a predominant apical localization, with a stark absence at the basal membrane. These findings indicate that cell adhesion regulates the localization of TGFbeta receptors in polarized epithelial cultures and that the response to TGFbeta is dependent upon the spatial distribution and secretion of TGFbeta receptors and ligand, respectively.     

Differential modulation of cadherin-mediated cell-cell adhesion by platelet endothelial cell adhesion molecule-1 isoforms through activation of extracellular regulated kinases

The role of platelet endothelial cell adhesion molecule-1 (PECAM-1) in endothelial cell-cell interactions and its contribution to cadherin-mediated cell adhesion are poorly understood. Such studies have been difficult because all known endothelial cells express PECAM-1. We have used Madin-Darby canine kidney (MDCK) cells as a model system in which to evaluate the role of PECAM-1 isoforms that differ in their cytoplasmic domains in cell-cell interactions. MDCK cells lack endogenous PECAM-1 but form cell-cell junctions similar to those of endothelial cells, in which PECAM-1 is concentrated. MDCK cells were transfected with two isoforms of murine PECAM-1, Delta15 and Delta14&15, the predominant isoforms expressed in vivo. Expression of the Delta15 isoform resulted in apparent dedifferentiation of MDCK cells concomitant with the loss of adherens junctions, down-regulation of E-cadherin, alpha- and beta-catenin expression, and sustained activation of extracellular regulated kinases. The Delta15 isoform was not concentrated at cell-cell contacts. In contrast, the Delta14&15 isoform localized to sites of cell-cell contact and had no effect on MDCK cell morphology, cadherin/catenin expression, or extracellular regulated kinase activity. Thus, the presence of exon 14 in the cytoplasmic domain of PECAM-1 has dramatic effects on the ability of cells to maintain adherens junctions and an epithelial phenotype. Therefore, changes in the expression of exon 14 containing PECAM-1 isoforms, which we have observed during development, may have profound functional consequences.     

Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells. The rates of internalization of HA-Y543 and HA-Y543,R546 from the basolateral surface of polarized MDCK cells resembled those in nonpolarized cells, whereas their rates of internalization from the apical cell surface were fivefold slower. However, HA-Y543,R546 was internalized approximately threefold faster than HA-Y543 at both membrane domains, indicating that apical endocytic pits in polarized MDCK cells retained the ability to discriminate between different internalization signals. Slower internalization from the apical surface could not be explained by a limiting number of coated pits: apical membrane contained 0.7 as many coated pits per cell cross-section as did basolateral membranes. 10-14% of HA-Y543 at the apical surface of polarized MDCK cells was found in coated pits, a percentage not significantly different from that observed in apical coated pits of nonpolarized MDCK cells, where internalization was fivefold faster. Thus, there was no lack of binding sites for HA-Y543 in apical coated pits of polarized cells. However, at the apical surface many more shallow pits, and fewer deep, mature pits, were observed than were seen at the basolateral. These results suggest that the slower internalization at the apical surface is due to slower maturation of coated pits, and not to a difference in recognition of internalization signals.     

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.     

Cloning of murine and rat vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen 4 (VLA4). We have cloned the cDNAs for both murine and rat VCAM1 from endotoxin-treated lung libraries. Both sequences encode proteins with seven extracellular Ig-like domains, which show 75.9% and 76.9% identity, respectively, with human VCAM1. Both murine and human cell lines show VLA4-dependent binding to COS cells transiently expressing murine and rat VCAM1. Two mAbs, M-K/1 and M-K/2, which recognize an antigen on murine bone marrow stromal cell lines, bind to murine VCAM1 expressed in COS cells and block VCAM1-dependent adhesion, confirming that these mAbs recognize murine VCAM1.     

The tumor suppressor protein TSLC1 is involved in cell-cell adhesion

TSLC1 is a tumor suppressor gene encoding a member of the immunoglobulin (Ig) superfamily. The significant homology of its extracellular domain with those of other Ig superfamily cell adhesion molecules (IgCAMs) has raised the possibility that TSLC1 participates in cell-cell interactions. In this study, the physiological properties of TSLC1 were investigated in Madin-Darby canine kidney (MDCK) cells expressing TSLC1 tagged with green fluorescent protein (GFP) as well as in the cells that express endogenous TSLC1. Biochemical analysis has revealed that TSLC1 is an N-linked glycoprotein with a molecular mass of 75 kDa and that it forms homodimers through cis interaction within the plane of the cell membranes. Confocal laser scanning microcopy of the cells expressing TSLC1 showed the localization patterns characteristic to adhesion molecules. At the beginning of cell attachment, TSLC1 accumulated in interdigitated structures at cell-cell boundaries, but, when cells reached a confluence, TSLC1 was distributed all along the cell membranes. In polarized cells, TSLC1 was recruited to the lateral membrane, implying trans interaction of TSLC1 between neighboring cells. In support of this notion, MDCK cells expressing TSLC1-GFP showed a significant level of cell aggregation in the absence or presence of Ca(2+) and Mg(2+). Taken together, these results indicate that TSLC1 mediates intracellular adhesion through homophilic interactions in a Ca(2+)/Mg(2+)-independent manner.     

Apical Polarity of N-CAM and EMMPRIN in Retinal Pigment Epithelium Resulting from Suppression of Basolateral Signal Recognition

Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM''s basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.     

Vectorial TGFβ signaling in polarized intestinal epithelial cells

Polarized gastrointestinal epithelial cells form tight junctions that spatially separate apical and basolateral cell membrane domains. These domains harbor functionally distinct proteins that contribute to cellular homeostasis and morphogenesis. Transforming growth factor β (TGFβ) is a critical regulator of gastrointestinal epithelial cell growth and differentiation. Functional assays of vectorial TGFβ signaling and immunofluorescence techniques were used to determine the localization of TGFβ receptors and ligand secretion in polarizing Caco‐2 cells, a colon cancer cell line. Results were compared to the nontransformed MDCK cell line. In both Caco‐2 and MDCK cells, addition of TGFβ1 to the basolateral medium resulted in phosphorylation of Smad2. No phosphorylation was observed when TGFβ1 was added to the apical chamber, indicating that receptor signaling is localized at the basolateral membrane. In support of this, immunofluorescence and biotinylation assays show receptor localization along the basolateral membrane. Secretion of TGFβ1 from MDCK and Caco‐2 cells into the apical or basolateral medium was measured by ELISA. Interestingly, secretion was exclusively apical in the nontransformed MDCK line and basolateral in transformed Caco‐2 cells. Collectively, these results show basolateral domain specificity in localization of the TGFβ receptor signaling apparatus. These observations have important implications for understanding the biology of TGFβ in polarized epithelia, including elements of communication between epithelial and mesenchymal layers, and will prove useful in the design of therapeutics that target TGFβ function. J. Cell. Physiol. 224: 398–404, 2010. © 2010 Wiley‐Liss, Inc.     

Structure/function studies on vascular cell adhesion molecule-1.

Vascular cell adhesion molecule-1 (VCAM1) is a member of the immunoglobulin (Ig) superfamily which interacts with the integrin very late antigen-4 (VLA4). The VCAM1/VLA4 interaction mediates both adhesion and signal transduction and is thought to play an important role in inflammatory and immune responses in vivo. The major form of human VCAM1 contains seven extracellular Ig-like domains, with domain 1 designated as the most N-terminal. We have examined the relationship between human VCAM1 structure and function using a combination of domain truncation mutants and proteolytic fragmentation of recombinant soluble VCAM1. We have characterized two regions of VCAM1, localized to domains 4 and 5, which are highly sensitive to proteolytic cleavage, localized the epitope of the blocking monoclonal antibody 4B9 to domain 1, and found that domains 1-3 are sufficient for both its adhesive function and its ability to initiate T cell activation.     

Apical localization of PMCA2w/b is enhanced in terminally polarized MDCK cells

The “w” splice forms of PMCA2 localize to distinct membrane compartments such as the apical membrane of the lactating mammary epithelium, the stereocilia of inner ear hair cells or the post-synaptic density of hippocampal neurons. Previous studies indicated that PMCA2w/b was not fully targeted to the apical domain of MDCK cells but distributed more evenly to the lateral and apical membrane compartments. Overexpression of the apical scaffold protein NHERF2, however, greatly increased the amount of the pump in the apical membrane of these epithelial cells. We generated a stable MDCK cell line expressing non-tagged, full-length PMCA2w/b to further study the localization and function of this protein. Here we demonstrate that PMCA2w/b is highly active and shows enhanced apical localization in terminally polarized MDCK cells grown on semi-permeable filters. Reversible surface biotinylation combined with confocal microscopy of fully polarized cells show that the pump is stabilized in the apical membrane via the apical membrane cytoskeleton with the help of endogenous NHERF2 and ezrin. Disruption of the actin cytoskeleton removed the pump from the apical actin patches without provoking its internalization. Our data suggest that full polarization is a prerequisite for proper positioning of the PMCA2w variants in the apical membrane domain of polarized cells.     

Surface expression of viral glycoproteins is polarized in epithelial cells infected with recombinant vaccinia viral vectors.

In polarized epithelial cells, maturation sites of enveloped viruses that form by budding at cell surfaces are restricted to particular membrane domains. Recombinant vaccinia viruses were used to investigate the sites of surface expression in the Madin-Darby canine kidney (MDCK) cell line of the hemagglutinin (HA) of influenza virus, the G glycoprotein of vesicular stomatitis virus (VSV), and gp70/p15E of Friend murine leukemia virus (MuLV). These glycoproteins could be demonstrated by immunofluorescence on the surfaces of MDCK cells as early as 4 h post-infection. In intact MDCK monolayers, vaccinia recombinants expressing HA produced a pattern of surface fluorescence typical of an apically expressed glycoprotein. In contrast, cells infected with vaccinia recombinants expressing VSV-G or MuLV gp70/p15E exhibited surface fluorescence only when monolayers were treated with EGTA to disrupt tight junctions, as expected of glycoproteins expressed on basolateral surfaces. Immunoferritin labeling in conjunction with electron microscopy confirmed that MDCK cells infected with the HA recombinant exhibited specific labeling of the apical surfaces whereas the VSV-G and MuLV recombinants exhibited the respective antigens predominantly on the basolateral membranes. Quantitation of surface expression by [125I]protein A binding assays on intact and EGTA-treated monolayers confirmed the apical localization of the vaccinia-expressed HA and demonstrated that 95% of the VSV-G and 97% of the MuLV gp70/p15E glycoproteins were localized on the basolateral surfaces. These results demonstrate that glycoproteins of viruses that normally mature at basolateral surfaces of polarized epithelial cells contain all of the structural information required for their directional transport to basolateral plasma membranes.     

Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells

In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an apical plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells was pulse-labeled, chased, and detected on the plasma membrane with a sensitive trypsin assay. Under all conditions tested, newly made hemagglutinin appeared simultaneously on both domains, with the bulk found in the apical membrane. When trypsin was continuously present on the basolateral surface during the chase, little hemagglutinin was cleaved relative to the amount transported apically. In addition, specific antibodies against the hemagglutinin placed basolaterally had no effect on transport to the apical domain. These observations suggested that most newly synthesized hemagglutinin does not transiently appear on the basolateral surface but rather is delivered directly to the apical surface in amounts that account for its final polarized distribution.     

Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells

Glycosylphosphatidylinositol (GPI) acts as an apical targeting signal in MDCK cells and other kidney and intestinal cell lines. In striking contrast with these model polarized cell lines, we show here that Fischer rat thyroid (FRT) epithelial cells do not display a preferential apical distribution of GPI-anchored proteins. Six out of nine detectable endogenous GPI-anchored proteins were localized on the basolateral surface, whereas two others were apical and one was not polarized. Transfection of several model GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected results. While the ectodomain of decay accelerating factor (DAF) was apically secreted, 50% of the native, GPI-anchored form, of this protein was basolateral. Addition of a GPI anchor to the ectodomain of Herpes simplex gD-1, secreted without polarity, led to basolateral localization of the fusion protein, gD1-DAF. Targeting experiments demonstrated that gD1-DAF was delivered vectorially from the Golgi apparatus to the basolateral surface. These results indicate that FRT cells have fundamental differences with MDCK cells with regard to the mechanisms for sorting GPI-anchored proteins: GPI is not an apical signal but, rather, it behaves as a basolateral signal. The "mutant" behavior of FRT cells may provide clues to the nature of the mechanisms that sort GPI-anchored proteins in epithelial cells.     

SNARE protein trafficking in polarized MDCK cells

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains. This polarity is generated and maintained by the continuous sorting of apical and basolateral components in the secretory and endocytic pathways. Soluble N-ethyl maleimide-sensitive factor attachment protein receptors (SNARE) proteins of vesicle-associated membrane protein (VAMP) and syntaxin families have been suggested to play a role in the biosynthetic transport to the apical and basolateral plasma membranes of polarized cells, where they likely mediate membrane fusion. To investigate the involvement of SNARE proteins in membrane trafficking to the apical and basolateral plasma membrane in the endocytic pathway we have monitored the recycling of various VAMP and syntaxin molecules between intracellular compartments and the two plasma membrane domains in Madin–Darby canine kidney (MDCK) cells. Here we show that VAMP8/endobrevin cycles through the apical but not through the basolateral plasma membrane. Furthermore, we found that VAMP8 localizes to apical endosomal membranes in nephric tubule epithelium and in MDCK cells. This asymmetry in localization and cycling behavior suggests that VAMP8/endobrevin may play a role in apical endosomal trafficking in polarized epithelium cells.     

Polarized apical sorting of guanylyl cyclase C is specified by a cytosolic signal

Receptor guanylyl cyclases respond to ligand stimulation by increasing intracellular cGMP, thereby initiating a variety of cell-signaling pathways. Furthermore, these proteins are differentially localized at the apical and basolateral membranes of epithelial cells. We have identified a region of 11 amino acids in the cytosolic COOH terminus of guanylyl cyclase C (GCC) required for normal apical localization in Madin-Darby canine kidney (MDCK) cells. These amino acids share no significant sequence homology with previously identified cytosolic apical sorting determinants. However, these amino acids are highly conserved and are sufficient to confer apical polarity to the interleukin-2 receptor alpha-chain (Tac). Additionally, we find two molecular weight species of GCC in lysates prepared from MDCK cells over-expressing GCC but observe only the fully mature species on the cell surface. Using pulse-chase analysis in polarized MDCK cells, we followed the generation of this mature species over time finding it to be detectable only at the apical cell surface. These data support the hypothesis that selective apical sorting can be determined using short, cytosolic amino acid motifs and argue for the existence of apical sorting machinery comparable with the machinery identified for basolateral protein traffic.     

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.     

DICAM, a novel dual immunoglobulin domain containing cell adhesion molecule interacts with alphavbeta3 integrin

Immunoglobulin (Ig) superfamily members are abundant with diverse functions including cell adhesion in various tissues. Here, we identified and characterized a novel adhesion molecule that belongs to the CTX protein family and named as DICAM (Dual Ig domain containing cell adhesion molecule). DICAM is a type I transmembrane protein with two V-type Ig domains in the extracellular region and a short cytoplasmic tail of 442 amino acids. DICAM is found to be expressed ubiquitously in various organs and cell lines. Subcellular localization of DICAM was observed in the cell-cell contact region and nucleus of cultured epithelial cells. Cell-cell contact region was colocalized with tight junction protein, ZO-1. The DICAM increased MDCK cell adhesion to 60% levels of fibronectin. DICAM mediated cell adhesion was specific for the alphavbeta3 integrin; other integrins, alpha2, alpha5, beta1, alpha2beta1, alpha5beta1, were not involved in cell adhesion. In identifying the interacting domain of DICAM with alphavbeta3, the Ig domain 2 showed higher cell adhesion activity than that of Ig domain 1. Although RGD motif in Ig domain 2 was engaged in cell adhesion, it was not participated in DICAM-alphavbeta3 mediated cell adhesion. Furthermore, differentially expressing DICAM stable cells showed well correlated cell to cell adhesion capability with integrin beta3-overexpressing cells. Collectively, these results indicate that DICAM, a novel dual Ig domain containing adhesion molecule, mediates cell adhesion via alphavbeta3 integrin.