Identification of the regions of PECAM-1 involved in β- and γ-catenin associations

Abstract Platelet endothelial cell adhesion molecule-1 (PECAM-1) binds tyrosine-phosphorylated β-catenin and modulates β-catenin localization and sequestration. The biological significance of this interaction, while still unclear, it has been postulated to be involved in modulating adherens junction dynamics in response to perturbants [J. Clin. Invest. 109 (2002) 383]. Here we demonstrate that tyrosine-phosphorylated β-catenin, and to a lesser extent unphosphorylated β-catenin, interact with a portion of the cytoplasmic domain of PECAM-1 encoded by exon 15. Using RT-PCR, we obtained products representing alternatively spliced PECAM-1 isoforms from mouse kidney total mRNA and generated PECAM-1-GST constructs expressing full length and naturally occurring alternatively spliced PECAM-1 variants. Co-precipitation assays revealed that the protein sequence encoded by exon 15 is necessary for β-catenin binding. Transfections using deletion mutants confirmed the importance of the exon 15 sequence in this interaction. In contrast, γ-catenin–PECAM-1 interactions are thought to be modulated by an as yet undefined PECAM-1 serine phosphorylation and appear to mediate dynamic PECAM-1 intermediate filament cytoskeletal interactions [J. Biol. Chem. 275 (2000) 21435]. Here we demonstrate that the PECAM-1–γ-catenin interaction occurs via an exon 13-mediated process. GST-pull-down assays illustrated the importance of the exon 13 sequence in this interaction. Further, using site-directed mutagenesis of S 673 to C and D and S 669 and 670 to C, we confirmed the importance of S 673 and its phosphorylation state as a mediator of γ-catenin–PECAM-1 binding. Our studies define the exons of the PECAM-1 cytoplasmic domain that is involved in mediating these PECAM-1–catenin family member interactions and will allow investigators to better define the biological functions resulting from these interactions.