Compromised E-cadherin adhesion and epithelial barrier function with activation of G protein-coupled receptors is rescued by Y-to-F mutations in β-catenin

Abstract

vation of the type 1 histamine (H1) or the type 2 protease-activated (PAR-2) G protein-coupled receptors interrupts E-cadherin adhesion and decreases the transepithelial resistance (TER) of epithelium. Several reports suggest that cadherin adhesive function depends on the association of cadherin with ␤-catenin and that this association is regulated by phosphorylation of tyrosines in ␤-catenin. We tested the hypothesis that loss of cadherin adhesion and compromise of TER on activation of the H1 or PAR-2 receptor is due to phosphorylation of tyrosines in ␤-catenin. L cells were stably transfected to express E-cadherin (L-E-cad cells) and H1 (L-H1-E-cad cells). L cells and Madin-Darby canine kidney (MDCK) cells constitutively express PAR-2. Stably transfected L-E-cad, L-H1-E-cad, and MDCK cells were also stably transfected with FLAG-tagged wild-type (WT) or mutant ␤-catenin, converting tyrosine 142, 489, or 654 to the nonphosphorylatable mimetic, phenylalanine (WT, Y142F, Y489F, or Y654F). Activation of H1 or PAR-2 interrupted adhesion to an immobilized E-cadherin-Fc fusion protein of L-H1-E-cad, L-E-cad, and MDCK cells expressing WT or Y142F ␤-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant ␤-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F ␤-catenin 40 -45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F ␤-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates ␤-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT ␤-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F ␤-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT ␤-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F ␤-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in ␤-catenin is a necessary step in the process by which G protein-coupled H1 and PAR-2 receptors interrupt Ecadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion.AIRWAY EPITHELIA CREATE an important barrier between the tissues of the lung and the environment. This barrier is dependent on molecules that bind the epithelial cells to each other and to molecules of the tight junction that act as resistors to paracellular molecular traffic. E-cadherin mediates calciumdependent homotypic cell-cell adhesion in epithelia. We (24, 27) previously reported that activation of the type 1 histamine (H1) or the type 2 protease-activated receptor (PAR-2) decreased the transepithelial resistance (TER) of airway epithelium by interrupting E-cadherin adhesion. In these reports, activation of the H1 or PAR-2 receptor interrupted E-cadherindependent adhesion of E-cadherin expressing L cells and of primary airway epithelium to immobilized E-cadherin. Activation of H1 or ...

The Integrin β1 Subunit Regulates Paracellular Permeability of Kidney Proximal Tubule Cells

“…Consistent with our data, overexpression of claudin-7 in porcine PTCs markedly increases TER, decreases paracellular chloride conductance and increases paracellular sodium conductance (26). E-cadherin expression has also been shown to regulate TER in MDCK cells (27,28). These previous studies support our hypothesis that the increase in TER in the ␤1…”

HER2 activation results in β-catenin-dependent changes in pulmonary epithelial permeability

“…Although it is very possible that these two events are independent of each other and that HER2 impacts cell adhesion through a process independent of Ecadherin, a role for ␤-catenin in regulating E-cadherin cell-cell adhesion is supported by existing literature. ␤-Catenin phosphorylation can be associated with a decrease in ␤-catenin-Ecadherin interaction and E-cadherin-mediated cell-cell adhesion (36,47). Indeed Winter et al (47) demonstrated that G protein receptors influence E-cadherin-mediated adhesion though a process dependent on ␤-catenin tyrosine phosphorylation.…”

“…␤-Catenin phosphorylation can be associated with a decrease in ␤-catenin-Ecadherin interaction and E-cadherin-mediated cell-cell adhesion (36,47). Indeed Winter et al (47) demonstrated that G protein receptors influence E-cadherin-mediated adhesion though a process dependent on ␤-catenin tyrosine phosphorylation. Although the exact mechanisms of how loss of ␤-catenin interaction with E-cadherin regulates its adhesive properties is unknown and not a focus of our study, it has been postulated that loss of catenin-cadherin binding leads to a conformational change in E-cadherin that alters the extracellular E-cadherin-E-cadherin bond (19).…”

Epithelium: sticking it out, together

“…Indeed, recent work by Miyahara et al (6) suggest Src kinase inhibition prevents the ventilatorinduced increase in lung vascular permeability. Overall, the insights provided by Winter et al (10) are significant and can help to guide our forthcoming studies to provide ever more direct insight into the functional control of cell communities, as in the case of adhesion strength among lung epithelial cells.…”

“…L cells endogenously express PAR-2, and so this receptor was not transfected. Using this model system, Winter et al (10) demonstrated that H1 receptor ligation and PAR-2 activation (e.g., G protein-coupled receptor activation) decreased E-cadherin-dependent adhesion. In both cases, the response required the phosphorylation of ␤-catenin Y489 and Y654 but not Y142.…”