The physical association of the P2Y12 receptor with PAR4 regulates arrestin-mediated Akt activation.
It is now well accepted that protease activated receptor (PAR) 1 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 the association of PAR4 with P2Y12, together with arrestin recruitment to the complex. Here we show that PAR4 and P2Y12 directly interact to coregulate Akt signaling after PAR4 activation. We observed direct and specific interaction of P2Y12 with PAR4 but not PAR1 by bioluminescent resonance energy transfer when the receptors were coexpressed in human embryonic kidney 293T cells. PAR4-P2Y12 dimerization was promoted by PAR4-AP and inhibited by P2Y12 antagonist. By using sequence comparison of the transmembrane domains of PAR1 and PAR4, we designed a mutant form 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, coexpression of increasing concentrations of PAR4SFT, together with PAR4 potentiated PAR4-mediated calcium flux, suggested that PAR4 act as homodimers to signal to Gq-coupled calcium responses. In conclusion, PAR4 LGL (194-196) governs agonist-dependent association of PAR4 with P2Y12 and 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-P2Y12 dependent stabilization of platelet thrombi.
Department of Biological Sciences, University of Delaware, Newark, Delaware (A.K., D.L., D.S.W.); and Institute for Translational Medicine and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (S.I., E.S.).
Aasma
A
Khan
Department of Biological Sciences, University of Delaware, Newark, Delaware (A.K., D.L., D.S.W.); and Institute for Translational Medicine and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (S.I., E.S.).
Dongjun
D
Li
Department of Biological Sciences, University of Delaware, Newark, Delaware (A.K., D.L., D.S.W.); and Institute for Translational Medicine and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (S.I., E.S.).
Salam
S
Ibrahim
Department of Biological Sciences, University of Delaware, Newark, Delaware (A.K., D.L., D.S.W.); and Institute for Translational Medicine and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (S.I., E.S.).
Emer
E
Smyth
Department of Biological Sciences, University of Delaware, Newark, Delaware (A.K., D.L., D.S.W.); and Institute for Translational Medicine and Pharmacology, University of Pennsylvania, Philadelphia, Pennsylvania (S.I., E.S.) dswoulfe@udel.edu.
Donna S
DS
Woulfe
D019390:Arrestins / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D057809:HEK293 Cells; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D015539:Platelet Activation / Q000502:physiology; D017434:Protein Structure, Tertiary; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12 / Q000378:metabolism*; D018179:Receptors, Thrombin / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013917:Thrombin / Q000378:metabolism
D019390:Arrestins; D008565:Membrane Proteins; C548576:P2RY12 protein, human; D058925:Receptors, Purinergic P2Y12; D018179:Receptors, Thrombin; D051057:Proto-Oncogene Proteins c-akt; D013917:Thrombin; C112908:protease-activated receptor 4; D002118:Calcium