Extracellular mutations of protease-activated receptor-1 result in differential activation by thrombin and thrombin receptor agonist peptide

The protease-activated thrombin receptor-1 (PAR-1) can be activated by both the tethered ligand exposed by thrombin cleavage and a synthetic peptide h aving the tethered ligand sequence (thrombin receptor agonist peptide or TR AP). We conducted a mutational analysis of extracellular residues of the re ceptor potentially involved in interaction with both the tethered ligand an d the soluble peptide agonist. Agonist-stimulated calcium efflux in X. laev is oocytes or inositol phosphate accumulation in COS-7 cells was used to as sess receptor activation. We have also examined the binding of a radiolabel ed TRAP for the wild-type and mutant PAR-1 receptors. Our results indicated that most of the mutations strongly affected TRAP-induced responses withou t significantly altering thrombin-induced responses or TRAP binding. Severa l point mutations and deletion of extracellular domains (Delta EC3, Delta N H3) drastically altered the ability of mutant receptors to respond to TRAP, but not to thrombin, and did not affect the affinity for the radiolabeled TRAP by these mutant receptors. Only mutations that disrupted the putative disulfide bond or substitution of multiple acidic residues in the second ex tracellular loop by alanine had a significant effect on both ligand binding and thrombin activation. These results suggest that although both agonists can activate PAR-1, there are profound differences in the ability of throm bin and TRAP to activate PAR-1. In addition, we have found PAR-1 mutants wi th the ability to dissociate receptor-specific binding from functional acti vity.