Protease-activated receptor-1 can mediate responses to SFLLRN in thrombin-desensitized cells: Evidence for a novel mechanism for preventing or terminating signaling by PAR1's tethered ligand

The thrombin receptor PAR1 is activated when thrombin cleaves the receptor' s amino-terminal exodomain to reveal the new N-terminal sequence SFLLRN whi ch then acts as a tethered peptide ligand. Free SFLLRN activates PAR1 indep endent of receptor cleavage and has been used to probe PAR1 function in var ious cells and tissues. PAR1-expressing cells desensitized to thrombin reta in responsiveness to SFLLRN. Toward determining the mechanism of such respo nses, we utilized fibroblasts derived from a PAR1-deficient mouse. These ce lls were unresponsive to thrombin and SFLLRN and became sensitive to both l igands after transfection with human PAR1 cDNA. Moreover, PAR1-transfected cells responded to SFLLRN after thrombin-desensitization, indicating that s ignaling of thrombin-desensitized cells to SFLLRN was mediated by PAR1 itse lf. SFLLRN caused signaling in thrombin-desensitized cells when no uncleave d PARI was detectable on the cell surface; however, cleaved PARI was presen t. To determine whether the cleaved receptors could still signal, fibroblas ts were transfected with a PAR1 mutant containing a trypsin site/SFLLRN seq uence carboxyl terminal to the native thrombin site. These cells retained r esponsiveness to trypsin after thrombin-desensitization. Conversely, fibrob lasts expressing a PAR1 mutant with the trypsin site/SELLRN sequence amino terminal to the native thrombin site retained responsiveness to thrombin af ter trypsin-desensitization. This suggests that a population of thrombin-cl eaved PAR1 can respond both to exogenous SFLLRN and to a second tethered li gand. In this population, the tethered ligand unmasked by thrombin cleavage must not be functional, suggesting the possibility of a novel mechanism of receptor shutoff involving sequestration or modification of the tethered l igand to prevent or terminate its function.