The αE(CD103)ß7 integrin interacts with oral and skin keratinocytes in an E-cadherin-independent manner*.

The integrin αE(CD103)ß7 (αEß7) is expressed by intraepithelial lymphocytes, dendritic cells and regulatory T cells. It plays an important role in the mucosal immune system by retaining lymphocytes within the epithelium and is involved in graft rejection, immunity against tumours and the generation of gut-homing effector cells. In gut and breast, the ligand for αEß7 is E-cadherin but in human oral mucosa and skin, there is evidence that lymphocytes use an alternative, unknown, ligand. In the present study, the I domain of the human αE subunit, which contains the E-cadherin-binding site, was locked in a highly active, 'open' and an inactive, 'closed' conformation by the introduction of disulphide bonds and these domains were expressed as IgG Fc fusion proteins. αE fusion proteins recognize E-cadherin, the only known ligand for αEß7. This interaction was inhibited by an antibody that blocks the αE-binding site on E-cadherin and by the omission of Mn(2+) , which is essential for integrin function in vitro. The locked 'open' conformation of αE adhered to human oral and skin keratinocytes, including the E-cadherin-negative H376 cell line, and this was not inhibited by blocking antibody against the αEß7-binding site on E-cadherin, providing further evidence for the existence of an alternative ligand for αEß7 in skin and oral mucosa. The interaction with E-cadherin and the alternative ligand was Mn(2+) dependent and mediated by the metal ion-dependent coordination site (MIDAS) of the locked 'open'αE I domain, independently of the ß7 subunit.

Endothelin-1-stimulated InsP3-induced Ca2+ release is a nexus for hypertrophic signaling in cardiac myocytes.

Ca(2+) elevations are fundamental to cardiac physiology-stimulating contraction and regulating the gene transcription that underlies hypertrophy. How Ca(2+) specifically controls gene transcription on the background of the rhythmic Ca(2+) increases required for contraction is not fully understood. Here we identify a hypertrophy-signaling module in cardiac myocytes that explains how Ca(2+) discretely regulates myocyte hypertrophy and contraction. We show that endothelin-1 (ET-1) stimulates InsP(3)-induced Ca(2+) release (IICR) from perinuclear InsP(3)Rs, causing an elevation in nuclear Ca(2+). Significantly, we show that IICR, but not global Ca(2+) elevations associated with myocyte contraction, couple to the calcineurin (CnA)/NFAT pathway to induce hypertrophy. Moreover, we found that activation of the CnA/NFAT pathway and hypertrophy by isoproterenol and BayK8644, which enhance global Ca(2+) fluxes, was also dependent on IICR and nuclear Ca(2+) elevations. The activation of IICR by these activity-enhancing mediators was explained by their ability to stimulate secretion of autocrine/paracrine ET-1.

Cadherin adhesion depends on a salt bridge at the N-terminus.

There is now considerable evidence that cell adhesion by cadherins requires a strand exchange process in which the second amino acid at the N-terminus of the cadherin molecule, Trp2, docks into a hydrophobic pocket in the domain fold of the opposing cadherin. Here we show that strand exchange depends on a salt bridge formed between the N-terminal amino group of one cadherin molecule and the acidic side chain of Glu89 of the other. Prevention of this bond in N-cadherin by introducing the mutation Glu89Ala or by extending the N-terminus with additional amino acids strongly inhibited strand exchange. But when the two modifications were present in opposing cadherin molecules respectively, they acted in a complementary manner, lowering activation energy for strand exchange and greatly increasing the strength of the adhesive interaction. N-cadherin that retained an uncleaved prodomain or lacked Trp2 adhered strongly to the Glu89Ala mutant but not to wild-type molecules. Similarly, N-cadherin in which the hydrophobic acceptor pocket was blocked by an isoleucine side chain adhered to a partner that had an extended N-terminus. We explain these results in terms of the free energy changes that accompany strand exchange. Our findings provide new insight into the mechanism of adhesion and demonstrate the feasibility of greatly increasing cadherin affinity.

The mechanism of cell adhesion by classical cadherins: the role of domain 1.

The mechanism by which classical cadherins mediate cell adhesion and, in particular, the roles played by calcium and Trp2, the second amino acid in the N-terminal domain, have long been controversial. We have used antibodies to investigate the respective contributions of Trp2 and calcium to the stability of the N-terminal domain of N-cadherin. Using a peptide antibody to the betaB strand in domain 1, which detects a disordered structure, we show that both Trp2 and calcium play crucial parts in regulating stability of the domain. The epitope for another antibody, mAb GC4, has been mapped to the base of domain 1. Binding of GC4 to this epitope was shown to depend on intramolecular 'docking' of Trp2 into the domain 1 structure. Using this property, we provide evidence that calcium regulates a dynamic equilibrium between docked and undocked Trp2. Finally, a novel technique has been developed to test whether Trp2 cross-intercalation between cadherin molecules from adjacent cells (strand exchange) is central to cadherin-mediated cell adhesion. Guided by crystal structures showing strand exchange, we have introduced single cysteine point mutations into N-cadherin domain 1 in such a way that a disulphide bond will form between opposing N-cadherin molecules during cell adhesion if strand exchange occurs. The bond requires complementary cysteines to be precisely juxtaposed according to the strand exchange model. Our results demonstrate that the disulphide bond forms as predicted. This provides compelling evidence that strand exchange is indeed a primary event in cell adhesion by classical cadherins.

Role of the alphaI domain in ligand binding by integrin alphaEbeta7.

The I domain of integrin alphaE was modeled on the crystal structure of that in CD11b and mutated to produce an open (high affinity) or closed (low affinity) conformation. K562 transfectants expressing mutant alphaE and wild-type beta7 were tested for adhesion to E-cadherin-Fc. Downward displacement of the C terminus of the alphaI domain with a disulfide bridge enhanced adhesion and Mn(2+) dependency. Adhesion greatly exceeded that observed using wild type integrin under similar conditions. The closed integrin gave poor adhesion which was greatly improved by PMA-induced clustering. Blocking beta7 function with a betaI domain-specific antibody inhibited the wild-type but not the locked open integrin. Isolated open alphaI domain expressed on K562 cells showed strong Mn(2+)-dependent adhesion to E-cadherin, whereas the wild-type version was ineffective. alphaEbeta7 was shown to bind to monomeric E-cadherin but to only one component of dimeric E-cadherin. Finally, we report that M290, a function-blocking antibody, bound to a conformation-sensitive epitope near the rim of the alphaI domain MIDAS and recognized wild-type and closed alphaI domain but not the open conformation. The results broadly support the paradigm for affinity regulation by conformational change that has been established for beta2 integrins. Nevertheless, for alphaE, the fully open conformation may represent an extreme situation that does not occur physiologically.