RGD cadherins and α2β1 integrin in cancer metastasis: A dangerous liaison

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2β1 integrin, these cadherins promote integrin β1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2β1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2β1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2β1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment.     

Direct and Regulated Interaction of Integrin αEβ7 with E-Cadherin

The cadherins are a family of homophilic adhesion molecules that play a vital role in the formation of cellular junctions and in tissue morphogenesis. Members of the integrin family are also involved in cell to cell adhesion, but bind heterophilically to immunoglobulin superfamily molecules such as intracellular adhesion molecule (ICAM)–1, vascular cell adhesion molecule (VCAM)–1, or mucosal addressin cell adhesion molecule (MadCAM)–1. Recently, an interaction between epithelial (E-) cadherin and the mucosal lymphocyte integrin, α_Eβ₇, has been proposed. Here, we demonstrate that a human E-cadherin–Fc fusion protein binds directly to soluble recombinant α_Eβ₇, and to α_Eβ₇ solubilized from intraepithelial T lymphocytes. Furthermore, intraepithelial lymphocytes or transfected JY′ cells expressing the α_Eβ₇ integrin adhere strongly to purified E-cadherin–Fc coated on plastic, and the adhesion can be inhibited by antibodies to α_Eβ₇ or E-cadherin.

The binding of α_Eβ₇ integrin to cadherins is selective since cell adhesion to P-cadherin–Fc through α_Eβ₇ requires >100-fold more fusion protein than to E-cadherin–Fc. Although the structure of the α_E-chain is unique among integrins, the avidity of α_Eβ₇ for E-cadherin can be regulated by divalent cations or phorbol myristate acetate. Cross-linking of the T cell receptor complex on intraepithelial lymphocytes increases the avidity of α_Eβ₇ for E-cadherin, and may provide a mechanism for the adherence and activation of lymphocytes within the epithelium in the presence of specific foreign antigen. Thus, despite its dissimilarity to known integrin ligands, the specific molecular interaction demonstrated here indicates that E-cadherin is a direct counter receptor for the α_Eβ₇ integrin.

     

Integrin α3β1 Binding to Fibronectin Is Dependent on the Ninth Type III Repeat

Fibronectin (Fn) is a promiscuous ligand for numerous cell adhesion receptors or integrins. The vast majority of Fn-integrin interactions are mediated through the Fn Arg-Gly-Asp (RGD) motif located within the tenth type III repeat. In the case of integrins αIIbβ3 and α5β1, the integrin binds RGD and the synergy site (PHSRN) located within the adjacent ninth type III repeat. Prior work has shown that these synergy-dependent integrins are exquisitely sensitive to perturbations in the Fn integrin binding domain conformation. Our own prior studies of epithelial cell responses to recombinant fragments of the Fn integrin binding domain led us to hypothesize that integrin α3β1 binding may also be modulated by the synergy site. To explore this hypothesis, we created a variety of recombinant variants of the Fn integrin binding domain: (i) a previously reported (Leu → Pro) stabilizing mutant (FnIII9′10), (ii) an Arg to Ala synergy site mutation (FnIII9^R→^A10), (iii) a two-Gly (FnIII9^2G10) insertion, and (iv) a four-Gly (FNIII9^4G10) insertion in the interdomain linker region and used surface plasmon resonance to determine binding kinetics of integrin α3β1 to the Fn fragments. Integrin α3β1 had the highest affinity for FnIII9′10 and FnIII9^2G10. Mutation within the synergy site decreased integrin α3β1 binding 17-fold, and the four-Gly insertion decreased binding 39-fold compared with FnIII9′10. Cell attachment studies demonstrate that α3β1-mediated epithelial cell binding is greater on FnIII9′10 compared with the other fragments. These studies suggest that the presence and spacing of the RGD and synergy sites modulate integrin α3β1 binding to Fn.     

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB signaling

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5β1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1β, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5β1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1β, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5β1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5β1 as a promising target for treating vascular inflammation in COVID-19.     

Entamoeba histolytica Cysteine Proteinase 5 Binds Integrin on Colonic Cells and Stimulates NFκB-mediated Pro-inflammatory Responses

Integrins are important mammalian receptors involved in normal cellular functions and the pathogenesis of inflammation and disease. Entamoeba histolytica is a protozoan parasite that colonizes the gut, and in 10% of infected individuals, causes amebic colitis and liver abscess resulting in 10⁵ deaths/year. E. histolytica-induced host inflammatory responses are critical in the pathogenesis of the disease, yet the host and parasite factors involved in disease are poorly defined. Here we show that pro-mature cysteine proteinase 5 (PCP5), a major virulent factor that is abundantly secreted and/or present on the surface of ameba, binds via its RGD motif to α_Vβ₃ integrin on Caco-2 colonic cells and stimulates NFκB-mediated pro-inflammatory responses. PCP5 RGD binding to α_Vβ₃ integrin triggered integrin-linked kinase(ILK)-mediated phosphorylation of Akt-473 that bound and induced the ubiquitination of NF-κB essential modulator (NEMO). As NEMO is required for activation of the IKKα-IKKβ complex and NFκB signaling, these events markedly up-regulated pro-inflammatory mediator expressions in vitro in Caco-2 cells and in vivo in colonic loop studies in wild-type and Muc2^−/− mice lacking an intact protective mucus barrier. These results have revealed that EhPCP5 RGD motif is a ligand for α_Vβ₃ integrin-mediated adhesion on colonic cells and represents a novel mechanism that E. histolytica trophozoites use to trigger an inflammatory response in the pathogenesis of intestinal amebiasis.     

Foot-and-mouth disease virus forms a highly stable, EDTA-resistant complex with its principal receptor, integrin alphavbeta6: implications for infectiousness

The initial stage of foot-and-mouth disease virus (FMDV) infection is virus binding to cell surface integrins via the RGD motif in the GH loop of the VP1 capsid protein. As for all ligand/integrin interactions, the initial contact between FMDV and its integrin receptors is cation dependent and hence inhibited by EDTA. We have investigated this binding process with RGD-containing peptides derived from the VP1 capsid protein of FMDV and discovered that, upon binding, some of these peptides form highly stable, EDTA-resistant associations with integrin αvβ6. Peptides containing specific substitutions show that this stable binding is dependent on a helical structure immediately C terminal to the RGD and, specifically, two leucine residues at positions RGD +1 and RGD +4. These observations have a biological consequence, as we show further that stable, EDTA-resistant binding to αvβ6 is a property also exhibited by FMDV particles. Thus, the integrin-binding loop of FMDV appears to have evolved to form very stable complexes with the principal receptor of FMDV, integrin αvβ6. An ability to induce such stable complexes with its cellular receptor is likely to contribute significantly to the high infectiousness of FMDV.     

Discoidin Domain Receptors Promote α1β1- and α2β1-Integrin Mediated Cell Adhesion to Collagen by Enhancing Integrin Activation

The discoidin domain receptors, DDR1 and DDR2, are receptor tyrosine kinases that bind to and are activated by collagens. Similar to collagen-binding β1 integrins, the DDRs bind to specific motifs within the collagen triple helix. However, these two types of collagen receptors recognize distinct collagen sequences. While GVMGFO (O is hydroxyproline) functions as a major DDR binding motif in fibrillar collagens, integrins bind to sequences containing Gxx’GEx”. The DDRs are thought to regulate cell adhesion, but their roles have hitherto only been studied indirectly. In this study we used synthetic triple-helical collagen-derived peptides that incorporate either the DDR-selective GVMGFO motif or integrin-selective motifs, such as GxOGER and GLOGEN, in order to selectively target either type of receptor and resolve their contributions to cell adhesion. Our data using HEK293 cells show that while cell adhesion to collagen I was completely inhibited by anti-integrin blocking antibodies, the DDRs could mediate cell attachment to the GVMGFO motif in an integrin-independent manner. Cell binding to GVMGFO was independent of DDR receptor signalling and occurred with limited cell spreading, indicating that the DDRs do not mediate firm adhesion. However, blocking the interaction of DDR-expressing cells with collagen I via the GVMGFO site diminished cell adhesion, suggesting that the DDRs positively modulate integrin-mediated cell adhesion. Indeed, overexpression of the DDRs or activation of the DDRs by the GVMGFO ligand promoted α1β1 and α2β1 integrin-mediated cell adhesion to medium- and low-affinity integrin ligands without regulating the cell surface expression levels of α1β1 or α2β1. Our data thus demonstrate an adhesion-promoting role of the DDRs, whereby overexpression and/or activation of the DDRs leads to enhanced integrin-mediated cell adhesion as a result of higher integrin activation state.     

The Arg-Gly-Asp Motif in the Cell Adhesion Molecule L1 Promotes Neurite Outgrowth via Interaction with the αvβ3 Integrin

The cell adhesion molecule L1 is a potent inducer of neurite outgrowth and it has been implicated in X-linked hydrocephalus and related neurological disorders. To investigate the mechanisms of neurite outgrowth stimulated by L1, attempts were made to identify the neuritogenic sites in L1. Fusion proteins containing different segments of the extracellular region of L1 were prepared and different neuronal cells were assayed on substrate-coated fusion proteins. Interestingly, both immunoglobulin (Ig)-like domains 2 and 6 (Ig2, Ig6) promoted neurite outgrowth from dorsal root ganglion cells, whereas neural retinal cells responded only to Ig2. L1 Ig2 contains a previously identified homophilic binding site, whereas L1 Ig6 contains an Arg-Gly-Asp (RGD) sequence. The neuritogenic activity of Ig6 was abrogated by mutations in the RGD site. The addition of RGD-containing peptides also inhibited the promotion of neurite outgrowth from dorsal root ganglion cells by glutathione S-transferase-Ig6, implicating the involvement of an integrin. The monoclonal antibody LM609 against α_vβ₃ integrin, but not an anti-β₁ antibody, inhibited the neuritogenic effects of Ig6. These data thus provide the first evidence that the RGD motif in L1 Ig6 is capable of promoting neurite outgrowth via interaction with the α_vβ₃ integrin on neuronal cells.     

Analysis of the α4β1 Integrin–Osteopontin Interaction

The integrin α4β1 is involved in mediating exfiltration of leukocytes from the vasculature. It interacts with a number of proteins up-regulated during the inflammatory response including VCAM-1 and the CS-1 alternatively spliced region of fibronectin. In addition it binds the multifunctional protein osteopontin (OPN), which can act as both a cytokine and an extracellular matrix molecule. Here we map the region of human OPN that supports cell adhesion via α4β1 using GST fusion proteins. We show that α4β1 expressed in J6 cells interacts with intact OPN when the integrin is in a high activation state, and by deletion mapping that the α4β1 binding region in OPN lies between amino acid residues 125 and 168 (aa125–168). This region contains the central RGD motif of OPN, which also interacts with integrins αvβ3, αvβ5, αvβ1, α8β1, and α5β1. Mutating the RGD motif to RAD had no effect on the interaction with α4β1. To define the binding site the region incorporating aa125–168 was divided into 5 overlapping peptides expressed as GST fusion proteins. Two peptides supported adhesion via α4β1, aa132–146, and aa153–168; of these only a synthetic peptide, SVVYGLR (aa162–168), derived from aa153–168 was able to inhibit α4β1 binding to CS-1. These data identify the motif SVVYGLR as a novel peptide inhibitor of α4β1, and the primary α4β1 binding site within OPN.     

Use of a β1 Integrin-deficient Human T Cell to Identify β1 Integrin Cytoplasmic Domain Sequences Critical for Integrin Function

T cell activation rapidly and transiently regulates the functional activity of integrin receptors. Stimulation of CD3/T cell receptor, CD2 or CD28, as well as activation with phorbol esters, can induce within minutes an increase in β1 integrin-mediated adhesion of T cells to fibronectin. In this study, we have produced and utilized a mutant of the Jurkat T cell line, designated A1, that lacks protein and mRNA expression of the β1 integrin subunit but retains normal levels of CD2, CD3, and CD28 on the cell surface. Activation-dependent adhesion of A1 cells to fibronectin could be restored upon transfection of a wild-type human β1 integrin cDNA. Adhesion induced by phorbol 12-myristate 13-acetate-, CD3-, CD2-, and CD28 stimulation did not occur if the carboxy-terminal five amino acids of the β1 tail were truncated or if either of two well-conserved NPXY motifs were deleted. Scanning alanine substitutions of the carboxy-terminal five amino acids demonstrated a critical role for the tyrosine residue at position 795. The carboxy-terminal truncation and the NPXY deletions also reduced adhesion induced by direct stimulation of the β1 integrin with the activating β1 integrin-specific mAb TS2/16, although the effects were not as dramatic as observed with the other integrin-activating signals. These results demonstrate a vital role for the amino-terminal NPXY motif and the carboxy-terminal end of the β1 integrin cytoplasmic domain in activation-dependent regulation of integrin-mediated adhesion in T cells. Furthermore, the A1 cell line represents a valuable new cellular reagent for the analysis of β1 integrin structure and function in human T cells.     

Calcium and Calmodulin-dependent Serine/Threonine Protein Kinase Type II (CaMKII)-mediated Intramolecular Opening of Integrin Cytoplasmic Domain-associated Protein-1 (ICAP-1α) Negatively Regulates β1 Integrins

Focal adhesion turnover during cell migration is an integrated cyclic process requiring tight regulation of integrin function. Interaction of integrin with its ligand depends on its activation state, which is regulated by the direct recruitment of proteins onto the β integrin chain cytoplasmic domain. We previously reported that ICAP-1α, a specific cytoplasmic partner of β1A integrins, limits both talin and kindlin interaction with β1 integrin, thereby restraining focal adhesion assembly. Here we provide evidence that the calcium and calmodulin-dependent serine/threonine protein kinase type II (CaMKII) is an important regulator of ICAP-1α for controlling focal adhesion dynamics. CaMKII directly phosphorylates ICAP-1α and disrupts an intramolecular interaction between the N- and the C-terminal domains of ICAP-1α, unmasking the PTB domain, thereby permitting ICAP-1α binding onto the β1 integrin tail. ICAP-1α direct interaction with the β1 integrin tail and the modulation of β1 integrin affinity state are required for down-regulating focal adhesion assembly. Our results point to a molecular mechanism for the phosphorylation-dependent control of ICAP-1α function by CaMKII, allowing the dynamic control of β1 integrin activation and cell adhesion.     

Activation of Distinct α5β1-mediated Signaling Pathways by Fibronectin's Cell Adhesion and Matrix Assembly Domains

The interaction of cells with fibronectin generates a series of complex signaling events that serve to regulate several aspects of cell behavior, including growth, differentiation, adhesion, and motility. The formation of a fibronectin matrix is a dynamic, cell-mediated process that involves both ligation of the α₅β₁ integrin with the Arg-Gly-Asp (RGD) sequence in fibronectin and binding of the amino terminus of fibronectin to cell surface receptors, termed “matrix assembly sites,” which mediate the assembly of soluble fibronectin into insoluble fibrils. Our data demonstrate that the amino-terminal type I repeats of fibronectin bind to the α₅β₁ integrin and support cell adhesion. Furthermore, the amino terminus of fibronectin modulates actin assembly, focal contact formation, tyrosine kinase activity, and cell migration. Amino-terminal fibronectin fragments and RGD peptides were able to cross-compete for binding to the α₅β₁ integrin, suggesting that these two domains of fibronectin cannot bind to the α₅β₁ integrin simultaneously. Cell adhesion to the amino-terminal domain of fibronectin was enhanced by cytochalasin D, suggesting that the ligand specificity of the α₅β₁ integrin is regulated by the cytoskeleton. These data suggest a new paradigm for integrin-mediated signaling, where distinct regions within one ligand can modulate outside-in signaling through the same integrin.     

Variation in One Residue Associated with the Metal Ion-Dependent Adhesion Site Regulates αIIbβ3 Integrin Ligand Binding Affinity

The Asp of the RGD motif of the ligand coordinates with the β I domain metal ion dependent adhesion site (MIDAS) divalent cation, emphasizing the importance of the MIDAS in ligand binding. There appears to be two distinct groups of integrins that differ in their ligand binding affinity and adhesion ability. These differences may be due to a specific residue associated with the MIDAS, particularly the β3 residue Ala²⁵² and corresponding Ala in the β1 integrin compared to the analogous Asp residue in the β2 and β7 integrins. Interestingly, mutations in the adjacent to MIDAS (ADMIDAS) of integrins α4β7 and αLβ2 increased the binding and adhesion abilities compared to the wild-type, while the same mutations in the α2β1, α5β1, αVβ3, and αIIbβ3 integrins demonstrated decreased ligand binding and adhesion. We introduced a mutation in the αIIbβ3 to convert this MIDAS associated Ala²⁵² to Asp. By combination of this mutant with mutations of one or two ADMIDAS residues, we studied the effects of this residue on ligand binding and adhesion. Then, we performed molecular dynamics simulations on the wild-type and mutant αIIbβ3 integrin β I domains, and investigated the dynamics of metal ion binding sites in different integrin-RGD complexes. We found that the tendency of calculated binding free energies was in excellent agreement with the experimental results, suggesting that the variation in this MIDAS associated residue accounts for the differences in ligand binding and adhesion among different integrins, and it accounts for the conflicting results of ADMIDAS mutations within different integrins. This study sheds more light on the role of the MIDAS associated residue pertaining to ligand binding and adhesion and suggests that this residue may play a pivotal role in integrin-mediated cell rolling and firm adhesion.     

Lipid Raft Is Required for PSGL-1 Ligation Induced HL-60 Cell Adhesion on ICAM-1

P-selectin glycoprotein ligand-1 (PSGL-1) and integrins are adhesion molecules that play critical roles in host defense and innate immunity. PSGL-1 mediates leukocyte rolling and primes leukocytes for integrin-mediated adhesion. However, the mechanism that PSGL-1 as a rolling receptor in regulating integrin activation has not been well characterized. Here, we investigate the function of lipid raft in regulating PSGL-1 induced β2 integrin-mediated HL-60 cells adhesion. PSGL-1 ligation with antibody enhances the β2 integrin activation and β2 integrin-dependent adhesion to ICAM-1. Importantly, with the treatment of methyl-β-cyclodextrin (MβCD), we confirm the role of lipid raft in regulating the activation of β2 integrin. Furthermore, we find that the protein level of PSGL-1 decreased in raft fractions in MβCD treated cells. PSGL-1 ligation induces the recruitment of spleen tyrosine kinase (Syk), a tyrosine kinase and Vav1 (the pivotal downstream effector of Syk signaling pathway involved in cytoskeleton regulation) to lipid raft. Inhibition of Syk activity with pharmacologic inhibitor strongly reduces HL-60 cells adhesion, implicating Syk is crucial for PSGL-1 mediated β2 integrin activation. Taken together, we report that ligation of PSGL-1 on HL-60 cells activates β2 integrin, for which lipid raft integrity and Syk activation are responsible. These findings have shed new light on the mechanisms that connect leukocyte initial rolling with subsequent adhesion.     

α-Catenin Localization and Sarcomere Self-Organization on N-Cadherin Adhesive Patterns Are Myocyte Contractility Driven

The N-cadherin (N-cad) complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. α-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that α-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that α-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.     

The regulation of integrin function by divalent cations

Integrins are a family of α/β heterodimeric adhesion metalloprotein receptors and their functions are highly dependent on and regulated by different divalent cations. Recently advanced studies have revolutionized our perception of integrin metal ion-binding sites and their specific functions. Ligand binding to integrins is bridged by a divalent cation bound at the MIDAS motif on top of either α I domain in I domain-containing integrins or β I domain in α I domain-less integrins. The MIDAS motif in β I domain is flanked by ADMIDAS and SyMBS, the other two crucial metal ion binding sites playing pivotal roles in the regulation of integrin affinity and bidirectional signaling across the plasma membrane. The β-propeller domain of α subunit contains three or four β-hairpin loop-like Ca²⁺-binding motifs that have essential roles in integrin biogenesis. The function of another Ca²⁺-binding motif located at the genu of α subunit remains elusive. Here, we provide an overview of the integrin metal ion-binding sites and discuss their roles in the regulation of integrin functions.     

Protocadherins branch out: Multiple roles in dendrite development

The proper formation of dendritic arbors is a critical step in neural circuit formation, and as such defects in arborization are associated with a variety of neurodevelopmental disorders. Among the best gene candidates are those encoding cell adhesion molecules, including members of the diverse cadherin superfamily characterized by distinctive, repeated adhesive domains in their extracellular regions. Protocadherins (Pcdhs) make up the largest group within this superfamily, encompassing over 80 genes, including the ∼60 genes of the α-, β-, and γ-Pcdh gene clusters and the non-clustered δ-Pcdh genes. An additional group includes the atypical cadherin genes encoding the giant Fat and Dachsous proteins and the 7-transmembrane cadherins. In this review we highlight the many roles that Pcdhs and atypical cadherins have been demonstrated to play in dendritogenesis, dendrite arborization, and dendritic spine regulation. Together, the published studies we discuss implicate these members of the cadherin superfamily as key regulators of dendrite development and function, and as potential therapeutic targets for future interventions in neurodevelopmental disorders.     

Structure of the cadherin-related neuronal receptor/protocadherin-alpha first extracellular cadherin domain reveals diversity across cadherin families

The recent explosion in genome sequencing has revealed the great diversity of the cadherin superfamily. Within the superfamily, protocadherins, which are expressed mainly in the nervous system, constitute the largest subgroup. Nevertheless, the structures of only the classical cadherins are known. Thus, to broaden our understanding of the adhesion repertoire of the cadherin superfamily, we determined the structure of the N-terminal first extracellular cadherin domain of the cadherin-related neuronal receptor/protocadherin-alpha4. The hydrophobic pocket essential for homophilic adhesiveness in the classical cadherins was not found, and the functional significance of this structural domain was supported by exchanging the first extracellular cadherin domains of protocadherin and classical cadherin. Moreover, potentially crucial variations were observed mainly in the loop regions. These included the protocadherin-specific disulfide-bonded Cys-X(5)-Cys motif, which showed Ca(2+)-induced chemical shifts, and the RGD motif, which has been suggested to be involved in heterophilic cell adhesion via the active form of beta1 integrin. Our findings reveal that the adhesion repertoire of the cadherin superfamily is far more divergent than would be predicted by studying the classical cadherins alone.     

Force Measurements of the α5β1 Integrin–Fibronectin Interaction

The interaction of the α₅β₁ integrin and its ligand, fibronectin (FN), plays a crucial role in the adhesion of cells to the extracellular matrix. An important intrinsic property of the α₅β₁/FN interaction is the dynamic response of the complex to a pulling force. We have carried out atomic force microscopy measurements of the interaction between α₅β₁ and a fibronectin fragment derived from the seventh through tenth type III repeats of FN (i.e., FN7-10) containing both the arg-gly-asp (RGD) sequence and the synergy site. Direct force measurements obtained from an experimental system consisting of an α₅β₁ expressing K562 cell attached to the atomic force microscopy cantilever and FN7-10 adsorbed on a substrate were used to determine the dynamic response of the α₅β₁/FN7-10 complex to a pulling force. The experiments were carried out over a three-orders-of-magnitude change in loading rate and under conditions that allowed for detection of individual α₅β₁/FN7-10 interactions. The dynamic rupture force of the α₅β₁/FN7-10 complex revealed two regimes of loading: a fast loading regime (>10,000 pN/s) and a slow loading regime (<10,000 pN/s) that characterize the inner and outer activation barriers of the complex, respectively. Activation by TS2/16 antibody increased both the frequency of adhesion and elevated the rupture force of the α₅β₁/wild type FN7-10 complex to higher values in the slow loading regime. In experiments carried out with a FN7-10 RGD deleted mutant, the force measurements revealed that both inner and outer activation barriers were suppressed by the mutation. Mutations to the synergy site of FN, however, suppressed only the outer barrier activation of the complex. For both the RGD and synergy deletions, the frequency of adhesion was less than that of the wild type FN7-10, but was increased by integrin activation. The rupture force of these mutants was only slightly less than that of the wild type, and was not increased by activation. These results suggest that integrin activation involved a cooperative interaction with both the RGD and synergy sites.     

Novel α2β1 Integrin Inhibitors Reveal That Integrin Binding to Collagen under Shear Stress Conditions Does Not Require Receptor Preactivation

The interaction between α2β1 integrin (GPIa/IIa, VLA-2) and vascular collagen is one of the initiating events in thrombus formation. Here, we describe two structurally similar sulfonamide derivatives, BTT-3033 and BTT-3034, and show that, under static conditions, they have an almost identical effect on α2-expressing CHO cell adhesion to collagen I, but only BTT-3033 blocks platelet attachment under flow (90 dynes/cm²). Differential scanning fluorimetry showed that both molecules bind to the α2I domain of the recombinant α2 subunit. To further study integrin binding mechanism(s) of the two sulfonamides, we created an α2 Y285F mutant containing a substitution near the metal ion-dependent adhesion site motif in the α2I domain. The action of BTT-3033, unlike that of BTT-3034, was dependent on Tyr-285. In static conditions BTT-3034, but not BTT-3033, inhibited collagen binding by an α2 variant carrying a conformationally activating E318W mutation. Conversely, in under flow conditions (90 dynes/cm²) BTT-3033, but not BTT-3034, inhibited collagen binding by an α2 variant expressing E336A loss-of-function mutation. Thus, the binding sites for BTT-3033 and BTT-3034 are differentially available in distinct integrin conformations. Therefore, these sulfonamides can be used to study the biological role of different functional stages of α2β1. Furthermore, only the inhibitor that recognized the non-activated conformation of α2β1 integrin under shear stress conditions effectively blocked platelet adhesion, suggesting that the initial interaction between integrin and collagen takes place prior to receptor activation.