The carboxyl tail of protease-activated receptor-1 is required for chemotaxis - Correlation of signal termination and directional migration

The G protein-coupled thrombin receptor, protease-activated receptor 1 (PAR 1), mediates many of the actions of thrombin on cells including chemotaxis. In contrast to the reversible agonist binding that regulates signaling by most G protein-coupled receptors (GPCRs), PAR1 is activated by an irreversi ble proteolytic mechanism. Although activated PAR1 is phosphorylated, uncou pled, and internalized like typical GPCRs, signal termination is additional ly dependent on lysosomal degradation of cleaved and activated receptors. I n the present study we exploit two PAR1 mutants to examine the link between chemotaxis and receptor shutoff. One, a carboxyl tail deletion mutant (Y39 7Z), is defective in phosphorylation and internalization. The other, a carb oxyl tail chimeric receptor (P/S), is phosphorylated and internalized upon activation but recycles to the plasma membrane like reversibly activated GP CRs. Expression of these receptors in a hematopoietic cell line disrupted c ell migration along thrombin gradients. Thrombin activation of cells expres sing P/S or Y397Z resulted in persistent signaling independent of the conti nued presence of thrombin. Signaling in response to the soluble agonist pep tide SFLLRN was reversible for P/S but persisted for Y397Z. Strikingly, cel ls expressing P/S responded chemokinetically to thrombin but chemotacticall y to SFLLRN. In contrast, Y397Z-mediated migration was largely chemokinetic to both agonists. These studies suggest that termination of PAR1 signaling at the level of the receptor is necessary for gradient detection and direc tional migration.