RT Journal Article
SR Electronic
T1 The Physical Association of the P2Y12 Receptor with PAR4 Regulates Arrestin-mediated Akt Activation
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP mol.114.091595
DO 10.1124/mol.114.091595
A1 Aasma Khan
A1 Dongjun Li
A1 Salam Ibrahim
A1 Emer Smyth
A1 Donna S Woulfe
YR 2014
UL http://molpharm.aspetjournals.org/content/early/2014/04/10/mol.114.091595.abstract
AB It is now well-accepted that PAR1 and PAR4 have differential roles in platelet activation. PAR4, a low affinity thrombin receptor in human platelets, participates in sustained platelet activation in a P2Y12 dependent manner; however, the mechanisms are not defined. Our previous studies demonstrated that thrombin induces co-immunoprecipitation of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to co-regulate arrestin-2 signaling following PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4, but not PAR1 by Bioluminescent Resonance Energy Transfer (BRET) when the receptors were co-expressed in HEK 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. Using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant of PAR4: 'PAR4SFT', by replacing LGL194-196 at the base of transmembrane domain 4 with the corresponding aligned PAR1 residues SFT 220-222. PAR4SFT supported only 8.74% of PAR4 P2Y12 interaction, abolishing P2Y12-dependent arrestin recruitment to PAR4 and Akt activation. Nonetheless, PAR4SFT still supported homodimerization with PAR4. PAR4SFT failed to induce a calcium flux when expressed independently; however, co-expression of increasing concentrations of PAR4SFT together with PAR4 potentiated PAR4-mediated calcium flux, suggesting that PAR4 homodimers signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and also contributes to Gq-coupled calcium responses. PAR4-P2Y12 association supports arrestin-mediated sustained signaling to Akt. Hence PAR4-P2Y12 dimerization is likely to be important for the PAR4 and P2Y12-dependent stabilization of platelet thrombi.