ENDOTHELIAL-CELL THROMBIN RECEPTORS AND PAR-2 - 2 PROTEASE-ACTIVATED RECEPTORS LOCATED IN A SINGLE CELLULAR ENVIRONMENT

Human endothelial cells express thrombin receptors and PAR-2, the two known members of the family of protease-activated G protein-coupled re ceptors. Because previous studies have shown that the biology of the h uman thrombin receptor varies according to the cell in which it is exp ressed, we have taken advantage of the presence of both receptors in e ndothelial cells to examine the enabling and disabling interactions wi th candidate proteases likely to be encountered in and around the vasc ular space to compare the responses elicited by the two receptors when they are present in the same cell and to compare the mechanisms of th rombin receptor and PAR-2 clearance and replacement in a common cellul ar environment. Of the proteases that were tested, only trypsin activa ted both receptors. Cathepsin G, which disables thrombin receptors, ha d no effect on PAR-2, while urokinase, kallikrein, and coagulation fac tors IXa, Xa, XIa, and XIIa neither substantially activated nor notice ably disabled either receptor. Like thrombin receptors, activation of PAR-2 caused pertussis toxin sensitive phospholipase C activation as w ell as activation of phospholipase A(2), leading to the release of PGI (2). Concurrent activation of both receptors caused a greater response than activation of either alone. It also abolished a subsequent respo nse to the PAR-2 agonist peptide, SLIGRL, while only partially inhibit ing the response to the agonist peptide, SFLLRN, which activates both receptors. After proteolytic or nonproteolytic activation, PAR-2, like thrombin receptors, was cleared from the endothelial cell surface and then rapidly replaced with new receptors by a process that does not r equire protein synthesis. Selective activation of either receptor had no effect on the clearance of the other. These results suggest that th e expression of both thrombin receptors and PAR-2 on endothelial cells serves more to extend the range of proteases to which the cells can r espond than it does to extend the range of potential responses. The re sults also show that proteases that can disable these receptors can di stinguish between them, just as do most of the proteases that activate them. Finally, the residual response to SFLLRN after activation of th rombin receptors and PAR-2 raises the possibility that a third, as yet unidentified member of this family is expressed on endothelial cells, one that is activated by neither thrombin nor trypsin.