Collagen XXIII, novel ligand for integrin alpha2beta1 in the epidermis

Cellular receptors for collagens belong to the family of β(1) integrins. In the epidermis, integrin α(2)β(1) is the only collagen-binding integrin present. Its expression is restricted to basal keratinocytes with uniform distribution on the cell surface of those cells. Although α(2)β(1) receptors localized at the basal surface interact with basement membrane proteins collagen IV and laminin 111 and 332, no interaction partners have been reported for these integrin molecules at the lateral and apical membranes of basal keratinocytes. Solid phase binding and surface plasmon resonance spectroscopy demonstrate that collagen XXIII, a member of the transmembrane collagens, directly interacts with integrin α(2)β(1) in an ion- and conformation-dependent manner. The two proteins co-localize on the surface of basal keratinocytes. Furthermore, collagen XXIII is sufficient to induce adhesion and spreading of keratinocytes, a process that is significantly reduced in the absence of functional integrin α(2)β(1).     

MT-LOOP-dependent Localization of Membrane Type I Matrix Metalloproteinase (MT1-MMP) to the Cell Adhesion Complexes Promotes Cancer Cell Invasion

Localization of membrane type I matrix metalloproteinase (MT1-MMP) to the leading edge is thought to be a crucial step during cancer cell invasion. However, its mechanisms and functional impact on cellular invasion have not been clearly defined. In this report, we have identified the MT-LOOP, a loop region in the catalytic domain of MT1-MMP (¹⁶³PYAYIREG¹⁷⁰), as an essential region for MT1-MMP to promote cellular invasion. Deletion of the MT-LOOP effectively inhibited functions of MT1-MMP on the cell surface, including proMMP-2 activation, degradation of gelatin and collagen films, and cellular invasion into a collagen matrix. This is not due to loss of the catalytic function of MT1-MMP but due to inefficient localization of the enzyme to β1-integrin-rich cell adhesion complexes at the plasma membrane. We also found that an antibody that specifically recognizes the MT-LOOP region of MT1-MMP (LOOP_Ab) inhibited MT1-MMP functions, fully mimicking the phenotype of the MT-LOOP deletion mutant. We therefore propose that the MT-LOOP region is an interface for molecular interactions that mediate enzyme localization to cell adhesion complexes and regulate MT1-MMP functions. Our findings have revealed a novel mechanism regulating MT1-MMP during cellular invasion and have identified the MT-LOOP as a potential exosite target region to develop selective MT1-MMP inhibitors.     

Membrane type-1 matrix metalloproteinase (MT1-MMP) processing of pro-alphav integrin regulates cross-talk between alphavbeta3 and alpha2beta1 integrins in breast carcinoma cells

We have recently demonstrated that in breast carcinoma MCF7 cells MT1-MMP processes the alphav, alpha3, and alpha5 integrin precursors generating the respective mature S-S-linked heavy and light alpha-chains. The precursor of alpha2 integrin subunit was found resistant to MT1-MMP proteolysis. The processing of the alphav subunit by MT1-MMP facilitated alphavbeta3-dependent adhesion, activation of FAK signaling pathway, and migration of MCF7 cells on vitronectin. To elucidate further the effects of MT1-MMP on cellular integrins, we examined the functional activity of alpha5beta1 and alpha2beta1 integrins in MCF7 cells expressing MT1-MMP. Either expression of MT1-MMP alone or its coexpression with alphavbeta3 failed to affect the functionality of alpha5beta1 integrin, and adhesion of cells to fibronectin. MT1-MMP, however, profoundly affected the cross-talk involving alphavbeta3 and alpha2beta1 integrins. In MT1-MMP-deficient cells, integrin alphavbeta3 suppressed the functional activity of the collagen-binding alpha2beta1 integrin receptor and diminished cell adhesion to type I collagen. Coexpression of MT1-MMP with integrin alphavbeta3 restored the functionality of alpha2beta1 integrin and, consequently, the ability of MCF7 cells to adhere efficiently to collagen. We conclude that the MT1-MMP-controlled cross-talk between alphavbeta3 and alpha2beta1 integrins supports binding of aggressive, MT1-MMP-, and alphavbeta3 integrin-expressing malignant cells on type I collagen, the most common substratum of the extracellular matrix.     

Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation,although Furin Cleavage Alone Generates Inactive Proteinase

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD↓L⁵⁰ site initiates the MT1-MMP activation, whereas the ¹⁰⁸RRKR¹¹¹↓Y¹¹² cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.     

Opticin Exerts Its Anti-angiogenic Activity by Regulating Extracellular Matrix Adhesiveness

Opticin is an extracellular matrix glycoprotein that we identified associated with the collagen network of the vitreous humor of the eye. Recently, we discovered that opticin possesses anti-angiogenic activity using a murine oxygen-induced retinopathy model: here, we investigate the underlying mechanism. Using an ex vivo chick chorioallantoic membrane assay, we show that opticin inhibits angiogenesis when stimulated by a range of growth factors. We show that it suppresses capillary morphogenesis, inhibits endothelial invasion, and promotes capillary network regression in three-dimensional matrices of collagen and Matrigel(TM). We then show that opticin binds to collagen and thereby competitively inhibits endothelial cell interactions with collagen via α(1)β(1) and α(2)β(1) integrins, thereby preventing the strong adhesion that is required for proangiogenic signaling via these integrins.     

Mutations in the Catalytic Domain of Human Matrix Metalloproteinase-1 (MMP-1) That Allow for Regulated Activity through the Use of Ca2+

Conditionally active proteins regulated by a physiological parameter represent a potential new class of protein therapeutics. By systematically creating point mutations in the catalytic and linker domains of human MMP-1, we generated a protein library amenable to physiological parameter-based screening. Mutants screened for temperature-sensitive activity had mutations clustered at or near amino acids critical for metal binding. One mutant, GVSK (Gly¹⁵⁹ to Val, Ser²⁰⁸ to Lys), contains mutations in regions of the catalytic domain involved in calcium and zinc binding. The in vitro activity of GVSK at 37 °C in high Ca²⁺ (10 mm) was comparable with MMP-1 (wild type), but in low Ca²⁺ (1 mm), there was an over 10-fold loss in activity despite having similar kinetic parameters. Activity decreased over 50% within 15 min and correlated with the degradation of the activated protein, suggesting that GVSK was unstable in low Ca²⁺. Varying the concentration of Zn²⁺ had no effect on GVSK activity in vitro. As compared with MMP-1, GVSK degraded soluble collagen I at the high but not the low Ca²⁺ concentration. In vivo, MMP-1 and GVSK degraded collagen I when perfused in Zucker rat ventral skin and formed higher molecular weight complexes with α2-macroglobulin, an inhibitor of MMPs. In vitro and in vivo complex formation and subsequent enzyme inactivation occurred faster with GVSK, especially at the low Ca²⁺ concentration. These data suggest that the activity of the human MMP-1 mutant GVSK can be regulated by Ca²⁺ both in vitro and in vivo and may represent a novel approach to engineering matrix-remodeling enzymes for therapeutic applications.     

Cholesterol Sulfate Alters Substrate Preference of Matrix Metalloproteinase-7 and Promotes Degradations of Pericellular Laminin-332 and Fibronectin

Localization of secreted matrix metalloproteinases (MMPs) on the cell surface is required not only for processing of cell surface proteins, but also for controlled degradation of the extracellular matrix (ECM). Our previous study demonstrated that binding of MMP-7 (matrilysin) to cell surface cholesterol sulfate (CS) is essential for the cell membrane-associated proteolytic action of this MMP. In this study, we investigated the role of CS in the MMP-7-catalyzed degradation of protein components of ECM. We found that the degradation of laminin-332 (laminin-5) catalyzed by MMP-7 was accelerated dramatically in the presence of CS, whereas the sulfated lipid inhibited the degradation of casein catalyzed by the protease. The MMP-7-catalyzed degradation of fibronectin was partially inhibited in the presence of low concentrations of CS, whereas it was accelerated significantly at high concentrations of the lipid. Therefore, it is likely that CS alters the substrate preference of MMP-7. We also found that the proteins of which MMP-7-catalyzed degradation were accelerated by CS also had affinities for CS, suggesting that CS facilitates the proteolyses by cross-linking MMP-7 to its substrates. Moreover, MMP-7 tethered to cancer cell surface via CS degraded fibronectin and laminin-332 coated on a culture plate. The degradations of the adhesive proteins led to significant detachment of the cells from the plate. Taken together, our findings provide a novel mechanism in which cell surface CS promotes the proteolytic activities of MMP-7 toward selective substrates in the pericellular ECM, thereby contributing to cancer cell migration and metastasis.     

Noncanonical Activation of Notch1 Protein by Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) Controls Melanoma Cell Proliferation

Notch1 is an evolutionarily conserved signaling molecule required for stem cell maintenance that is inappropriately reactivated in several cancers. We have previously shown that melanomas reactivate Notch1 and require its function for growth and survival. However, no Notch1-activating mutations have been observed in melanoma, suggesting the involvement of other activating mechanisms. Notch1 activation requires two cleavage steps: first by a protease and then by γ-secretase, which releases the active intracellular domain (Notch1^NIC). Interestingly, although ADAM10 and -17 are generally accepted as the proteases responsible of Notch1 cleavage, here we show that MT1-MMP, a membrane-tethered matrix metalloproteinase involved in the pathogenesis of a number of tumors, is a novel protease required for the cleavage of Notch1 in melanoma cells. We find that active Notch1 and MT1-MMP expression correlate significantly in over 70% of melanoma tumors and 80% of melanoma cell lines, whereas such correlation does not exist between Notch1^NIC and ADAM10 or -17. Modulation of MT1-MMP expression in melanoma cells affects Notch1 cleavage, whereas MT1-MMP expression in ADAM10/17 double knock-out fibroblasts restores the processing of Notch1, indicating that MT1-MMP is sufficient to promote Notch1 activation independently of the canonical proteases. Importantly, we find that MT1-MMP interacts with Notch1 at the cell membrane, supporting a potential direct cleavage mechanism of MT1-MMP on Notch1, and that MT1-MMP-dependent activation of Notch1 sustains melanoma cell growth. Together, the data highlight a novel mechanism of activation of Notch1 in melanoma cells and identify Notch1 as a new MT1-MMP substrate that plays important biological roles in melanoma.     

Fibulin-3, -4, and -5 Are Highly Susceptible to Proteolysis,Interact with Cells and Heparin,and Form Multimers

Extracellular short fibulins, fibulin-3, -4, and -5, are components of the elastic fiber/microfibril system and are implicated in the formation and homeostasis of elastic tissues. In this study, we report new structural and functional properties of the short fibulins. Full-length human short fibulins were recombinantly expressed in human embryonic kidney cells and purified by immobilized metal ion affinity chromatography. All three fibulins showed various levels of degradation after the purification procedure. N-terminal sequencing revealed that all three fibulins are highly susceptible to proteolysis within the N-terminal linker region of the first calcium-binding epidermal growth factor domain. Proteolytic susceptibility of the linker correlated with its length. Exposure of these fibulins to matrix metalloproteinase (MMP)-1, -2, -3, -7, -9, and -12 resulted in similar proteolytic fragments with MMP-7 and -12 being the most potent proteases. Fibulin-3 proteolysis was almost completely inhibited in cell culture by the addition of 25 μm doxycycline (a broad spectrum MMP inhibitor). Reducible fibulin-4 dimerization and multimerization were consistently observed by SDS-PAGE, Western blotting, and mass spectrometry. Atomic force microscopy identified monomers, dimers, and multimers in purified fibulin-4 preparations with sizes of ∼10–15, ∼20–25, and ∼30–50 nm, respectively. All short fibulins strongly adhered to human fibroblasts and smooth muscle cells. Although only fibulin-5 has an RGD integrin binding site, all short fibulins adhere at a similar level to the respective cells. Solid phase binding assays detected strong calcium-dependent binding of the short fibulins to immobilized heparin, suggesting that these fibulins may bind cell surface-located heparan sulfate.     

Phospholipid Scramblase 1 Is Secreted by a Lipid Raft-dependent Pathway and Interacts with the Extracellular Matrix Protein 1 in the Dermal Epidermal Junction Zone of Human Skin

We examined the interaction of ECM1 (extracellular matrix protein 1) using yeast two-hybrid screening and identified the type II transmembrane protein, PLSCR1 (phospholipid scramblase 1), as a binding partner. This interaction was then confirmed by in vitro and in vivo co-immunoprecipitation experiments, and additional pull-down experiments with GST-tagged ECM1a fragments localized this interaction to occur within the tandem repeat region of ECM1a. Furthermore, immunohistochemical staining revealed a partial overlap of ECM1 and PLSCR1 in human skin at the basal epidermal cell layer. Moreover, in human skin equivalents, both proteins are expressed at the basal membrane in a dermal fibroblast-dependent manner. Next, immunogold electron microscopy of ultrathin human skin sections showed that ECM1 and PLSCR1 co-localize in the extracellular matrix, and using antibodies against ECM1 or PLSCR1 cross-linked to magnetic immunobeads, we were able to demonstrate PLSCR1-ECM1 interaction in human skin extracts. Furthermore, whereas ECM1 is secreted by the endoplasmic/Golgi-dependent pathway, PLSCR1 release from HaCaT keratinocytes occurs via a lipid raft-dependent mechanism, and is deposited in the extracellular matrix. In summary, we here demonstrate that PLSCR1 interacts with the tandem repeat region of ECM1a in the dermal epidermal junction zone of human skin and provide for the first time experimental evidence that PLSCR1 is secreted by an unconventional secretion pathway. These data suggest that PLSCR1 is a multifunctional protein that can function both inside and outside of the cell and together with ECM1 may play a regulatory role in human skin.     

The Role of Collagen Charge Clusters in the Modulation of Matrix Metalloproteinase Activity

Members of the matrix metalloproteinase (MMP) family selectively cleave collagens in vivo. Several substrate structural features that direct MMP collagenolysis have been identified. The present study evaluated the role of charged residue clusters in the regulation of MMP collagenolysis. A series of 10 triple-helical peptide (THP) substrates were constructed in which either Lys-Gly-Asp or Gly-Asp-Lys motifs replaced Gly-Pro-Hyp (where Hyp is 4-hydroxy-l-proline) repeats. The stabilities of THPs containing the two different motifs were analyzed, and kinetic parameters for substrate hydrolysis by six MMPs were determined. A general trend for virtually all enzymes was that, as Gly-Asp-Lys motifs were moved from the extreme N and C termini to the interior next to the cleavage site sequence, k_cat/K_m values increased. Additionally, all Gly-Asp-Lys THPs were as good or better substrates than the parent THP in which Gly-Asp-Lys was not present. In turn, the Lys-Gly-Asp THPs were also always better substrates than the parent THP, but the magnitude of the difference was considerably less compared with the Gly-Asp-Lys series. Of the MMPs tested, MMP-2 and MMP-9 most greatly favored the presence of charged residues with preference for the Gly-Asp-Lys series. Lys-Gly-(Asp/Glu) motifs are more commonly found near potential MMP cleavage sites than Gly-(Asp/Glu)-Lys motifs. As Lys-Gly-Asp is not as favored by MMPs as Gly-Asp-Lys, the Lys-Gly-Asp motif appears advantageous over the Gly-Asp-Lys motif by preventing unwanted MMP hydrolysis. More specifically, the lack of Gly-Asp-Lys clusters may diminish potential MMP-2 and MMP-9 collagenolytic activity. The present study indicates that MMPs have interactions spanning the P₂₃–P₂₃′ subsites of collagenous substrates.     

Noncanonical Matrix Metalloprotease-1-Protease-activated Receptor-1 Signaling Triggers Vascular Smooth Muscle Cell Dedifferentiation and Arterial Stenosis

Vascular injury that results in proliferation and dedifferentiation of vascular smooth muscle cells (SMCs) is an important contributor to restenosis following percutaneous coronary interventions or plaque rupture. Protease-activated receptor-1 (PAR1) has been shown to play a role in vascular repair processes; however, little is known regarding its function or the relative roles of the upstream proteases thrombin and matrix metalloprotease-1 (MMP-1) in triggering PAR1-mediated arterial restenosis. The goal of this study was to determine whether noncanonical MMP-1 signaling through PAR1 would contribute to aberrant vascular repair processes in models of arterial injury. A mouse carotid arterial wire injury model was used for studies of neointima hyperplasia and arterial stenosis. The mice were treated post-injury for 21 days with a small molecule inhibitor of MMP-1 or a direct thrombin inhibitor and compared with vehicle control. Intimal and medial hyperplasia was significantly inhibited by 2.8-fold after daily treatment with the small molecule MMP-1 inhibitor, an effect that was lost in PAR1-deficient mice. Conversely, chronic inhibition of thrombin showed no benefit in suppressing the development of arterial stenosis. Thrombin-PAR1 signaling resulted in a supercontractile, differentiated phenotype in SMCs. Noncanonical MMP-1-PAR1 signaling resulted in the opposite effect and led to a dedifferentiated phenotype via a different G protein pathway. MMP-1-PAR1 significantly stimulated hyperplasia and migration of SMCs, and resulted in down-regulation of SMC contractile genes. These studies provide a new mechanism for the development of vascular intimal hyperplasia and suggest a novel therapeutic strategy to suppress restenosis by targeting noncanonical MMP-1-PAR1 signaling in vascular SMCs.     

Type III Collagen,a Fibril Network Modifier in Articular Cartilage

The collagen framework of hyaline cartilages, including articular cartilage, consists largely of type II collagen that matures from a cross-linked heteropolymeric fibril template of types II, IX, and XI collagens. In the articular cartilages of adult joints, type III collagen makes an appearance in varying amounts superimposed on the original collagen fibril network. In a study to understand better the structural role of type III collagen in cartilage, we find that type III collagen molecules with unprocessed N-propeptides are present in the extracellular matrix of adult human and bovine articular cartilages as covalently cross-linked polymers extensively cross-linked to type II collagen. Cross-link analyses revealed that telopeptides from both N and C termini of type III collagen were linked in the tissue to helical cross-linking sites in type II collagen. Reciprocally, telopeptides from type II collagen were recovered cross-linked to helical sites in type III collagen. Cross-linked peptides were also identified in which a trifunctional pyridinoline linked both an α1(II) and an α1(III) telopeptide to the α1(III) helix. This can only have arisen from a cross-link between three different collagen molecules, types II and III in register staggered by 4D from another type III molecule. Type III collagen is known to be prominent at sites of healing and repair in skin and other tissues. The present findings emphasize the role of type III collagen, which is synthesized in mature articular cartilage, as a covalent modifier that may add cohesion to a weakened, existing collagen type II fibril network as part of a chondrocyte healing response to matrix damage.     

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.     

Rac1 Recruits the Adapter Protein CMS/CD2AP to Cell-Cell Contacts

Rac1 is a member of the Rho family of small GTPases, which regulate cell adhesion and migration through their control of the actin cytoskeleton. Rho-GTPases are structurally very similar, with the exception of a hypervariable domain in the C terminus. Using peptide-based pulldown assays in combination with mass spectrometry, we previously showed that the hypervariable domain in Rac1 mediates specific protein-protein interactions. Most recently, we found that the Rac1 C terminus associates to the ubiquitously expressed adapter protein CMS/CD2AP. CD2AP is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. We found that CD2AP specifically interacts with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts in epithelial cells, where we found CD2AP to participate in the control of the epithelial barrier function. These data identify CD2AP as a novel Rac1-associated adapter protein that participates in the regulation of epithelial cell-cell contact.     

Angiopoietin-like 4 Interacts with Matrix Proteins to Modulate Wound Healing

A dynamic cell-matrix interaction is crucial for a rapid cellular response to changes in the environment. Appropriate cell behavior in response to the changing wound environment is required for efficient wound closure. However, the way in which wound keratinocytes modify the wound environment to coordinate with such cellular responses remains less studied. We demonstrated that angiopoietin-like 4 (ANGPTL4) produced by wound keratinocytes coordinates cell-matrix communication. ANGPTL4 interacts with vitronectin and fibronectin in the wound bed, delaying their proteolytic degradation by metalloproteinases. This interaction does not interfere with integrin-matrix protein recognition and directly affects cell-matrix communication by altering the availability of intact matrix proteins. These interactions stimulate integrin- focal adhesion kinase, 14-3-3, and PKC-mediated signaling pathways essential for effective wound healing. The deficiency of ANGPTL4 in mice delays wound re-epithelialization. Further analysis revealed that cell migration was impaired in the ANGPTL4-deficient keratinocytes. Altogether, the findings provide molecular insight into a novel control of wound healing via ANGPTL4-dependent regulation of cell-matrix communication. Given the known role of ANGPTL4 in glucose and lipid homeostasis, it is a prime therapeutic candidate for the treatment of diabetic wounds. It also underscores the importance of cell-matrix communication during angiogenesis and cancer metastasis.     

Crystal Structure of the cis-Dimer of Nectin-1: implications for the architecture of cell-cell junctions

In multicellular organisms, cells are interconnected by cell adhesion molecules. Nectins are immunoglobulin (Ig)-like cell adhesion molecules that mediate homotypic and heterotypic cell-cell adhesion, playing key roles in tissue organization. To mediate cell-cell adhesion, nectin molecules dimerize in cis on the surface of the same cell, followed by trans-dimerization of the cis-dimers between the neighboring cells. Previous cell biological studies deduced that the first Ig-like domain of nectin and the second Ig-like domain are involved in trans-dimerization and cis-dimerization, respectively. However, to understand better the steps involved in nectin adhesion, the structural basis for the dimerization of nectin must be determined. In this study, we determined the first crystal structure of the entire extracellular region of nectin-1. In the crystal, nectin-1 formed a V-shaped homophilic dimer through the first Ig-like domain. Structure-based site-directed mutagenesis of the first Ig-like domain identified four essential residues that are involved in the homophilic dimerization. Upon mutating the four residues, nectin-1 significantly decreased cis-dimerization on the surface of cultured cells and abolished the homophilic and heterophilic adhesion activities. These results indicate that, in contrast with the previous notion, our structure represents a cis-dimer. Thus, our findings clearly reveal the structural basis for the cis-dimerization of nectins through the first Ig-like domains.