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. There is structural evidence that GPCRs, as a class, function as dimers and that dimerization can alter signaling specificity. Our previous studies have determined that PAR4 forms homodimers and have mapped the homodimer interface to 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) and bimolecular fluorescence complementation (BiFC). Similar to our previous studies with PAR4 homodimers, PAR1 homodimers were constitutive and did not require receptor activation. In contrast, 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. We next examined if PAR1-PAR4 heterodimers would be induced by stimulating PAR1 or PAR4 individually with their agonist peptides TFLLRN (100 μM) or AYPGKF (500 μM), respectively. The agonist peptides were unable to induce heterodimers when added to the cells individually or simultaneously. These data demonstrate that PAR1 and PAR4 require allosteric changes induced by receptor cleavage by thrombin to mediate heterodimer formation. To examine this further, we removed 37 amino acids from the C-terminus of PAR1, which disrupts the eighth helix. The truncated PAR1 was able to form constitutive heterodimers with PAR4 and these heterodimers were unaffected by thrombin. These data suggest that PAR1 is the allosteric modulator of the PAR1-PAR4 heterodimers. Finally, the stability of the constitutive PAR1 and PAR4 homodimers was unchanged in response to thrombin or the agonist peptides. 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.