Compromised E-cadherin adhesion and epithelial barrier function with activation of G protein-coupled receptors is rescued by Y-to-F mutations in beta-catenin.
Activation 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 beta-catenin and that this association is regulated by phosphorylation of tyrosines in beta-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 beta-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 beta-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 beta-catenin but did not interrupt adhesion of L-H1-E-cad, L-E-cad, and MDCK cells expressing the Y489F or Y654F mutant beta-catenins. PAR-2 activation decreased the TER of monolayers of MDCK cells expressing WT or Y142F beta-catenin 40-45%. However, PAR-2 activation did not decrease the TER of monolayers of MDCK cells expressing Y489F or Y654F beta-catenin. The protein tyrosine phosphatase PTP1B binds to the cadherin cytoplasmic domain and dephosphorylates beta-catenin. Inhibition of PTP1B interrupted adhesion to E-cadherin-Fc of MDCK cells expressing WT beta-catenin but did not affect the adhesion of MDCK cells expressing Y489F or Y654F beta-catenin. Similarly, inhibition of PTP1B compromised the TER of MDCK cells expressing WT beta-catenin but did not affect the TER of MDCK cells expressing Y489F or Y654F beta-catenin. We conclude that phosphorylation of tyrosines 489 and 654 in beta-catenin is a necessary step in the process by which G protein-coupled H1 and PAR-2 receptors interrupt E-cadherin adhesion. We also conclude that activation of PAR-2 has no effect on the TER without first interrupting E-cadherin adhesion.
Winter
Michael C
MC
University of Iowa, Department of Internal Medicine, C33 GH, Iowa City, IA 52242, USA.
Shasby
Sandra
S
Shasby
D Michael
DM
D019943:Amino Acid Substitution; D000818:Animals; D015820:Cadherins / Q000502:physiology*; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D004285:Dogs; D017097:Electric Impedance; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D007739:L Cells; D051379:Mice; D009154:Mutation; D009842:Oligopeptides / Q000494:pharmacology; D010649:Phenylalanine / Q000502:physiology; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000819:agonists / Q000502:physiology*; D011969:Receptors, Histamine H1 / Q000502:physiology*; D014162:Transfection; D014443:Tyrosine / Q000235:genetics / Q000502:physiology*; D051176:beta Catenin / Q000235:genetics*
D015820:Cadherins; D009842:Oligopeptides; D044464:Receptor, PAR-2; D011969:Receptors, Histamine H1; D051176:beta Catenin; D014443:Tyrosine; D010649:Phenylalanine; D006632:Histamine; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1