Proteolytic cleavage of platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31) is regulated by a calmodulin‐binding motif

Abstract

Homophilic engagement of platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31) induces ‘outside‐in’ signal transduction that results in phosphorylation events and recruitment and activation of signalling molecules. The formation of signalling scaffolds with PECAM‐1 are important signalling events that modulate platelet secretion, aggregation and platelet thrombus formation. In this study, we describe a novel interaction between PECAM‐1 and cytosolic calmodulin (CaM) in platelets. Reciprocal co‐immunoprecipitation studies revealed that cytosolic CaM is constitutively associated with PECAM‐1 in resting, thrombin activated and aggregated human platelets. Our studies demonstrate that CaM directly interacts with a PECAM‐1 peptide (594–604) C595A containing the sequences ⁵⁹⁴KAFYLRKAKAK⁶⁰⁴. This CaM:PECAM‐1 interaction has a threefold higher affinity than CaM:GPVI interaction. It is potentiated by the addition of calcium ions, and dissociated by the CaM inhibitor, trifluoperazine. Treatment of platelets with CaM inhibitors triggers cleavage of PECAM‐1 in a time‐ and dose‐dependent manner. Furthermore, this membrane proximal portion of PECAM‐1 is conserved across mammalian species and the helical representation of basic/hydrophobic residues reveals a charge distribution analogous to other CaM‐binding motifs in other proteins. Taken together, these results suggest that this highly charged cluster of amino acids in the PECAM‐1 cytoplasmic domain directly interacts with CaM and this novel interaction appears to regulate cleavage of PECAM‐1.