Abstract 56: Thrombin Induces Protease-Activated Receptor 1 and PAR4 Heterodimers
Thrombin is a potent platelet agonist. Thrombin activates platelets and other cells of the cardiovascular system by cleaving its receptors, protease activated receptor 1 (PAR1), PAR4 or both. PARs are G-protein coupled receptors that activate cellular signaling through Gq and G12/13. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to specific residues in transmembrane helix 4 (TM4). We have also shown that coexpression of PAR1 with PAR4 lowers the threshold for PAR4 activation by thrombin ∼10-fold. The purpose of the current study is to examine the physical interaction between PAR1 and PAR4 and how these interactions influence PAR1’s ability to enhance PAR4 activation. The PAR1-PAR4 heterodimers were examined by bioluminescence resonance energy transfer (BRET). PAR1-PAR4 heterodimers were not detected under basal conditions. However, when the cells were stimulated with 10 nM thrombin, we were able to detect a strong interaction between PAR1 and PAR4. In contrast, stimulating PAR1, PAR4, or both with agonist peptides TFLLRN (100 microM) or AYPGKF (500 microM), respectively did not induce heterodimers. Further, point mutations that prevent PAR1 or PAR4 cleavage did not from heterodimers when stimulated with thrombin. Other proteases that activate PAR1 or PAR4 such as activated protein C (APC) and plasmin do not induce PAR1-PAR4 heterodimers. Finally, we have introduced point mutations in transmembrane helix 4 of PAR1 and PAR4 that prevent heterodimer formation when stimulated with thrombin. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation and have determined the PAR1-PAR4 heterodimer interface. Taken together, these data suggest that PAR1 and PAR4 have a dynamic interaction depending on the context of their expression. Since PAR1 is an attractive antiplatelet target, the molecular interactions of this receptor on the cells surface must be taken into account when developing and characterizing these antagonists.
- © 2012 by American Heart Association, Inc.