Helix-8 and Carboxyl Terminal Tail Regulation of Proteinase Activated Receptor-4 (PAR4) signalling

The 8th-helix (H8) and carboxyl terminal tail (CT) of GPCRs are crucial for interactions with intracellular signaling molecules and regulatory proteins. We investigated how H8 and CT residues influence signaling in PAR4, a tethered-ligand activated GPCR. Our analysis revealed that PAR4 activation by thrombin or AYPGKF-NH2 stimulates β-arrestin-1/-2 recruitment and activates multiple Gα proteins (Gαq, Gα11, Gαz, Gα15, Gα12, Gα13, Gαi1-3, GαoA/B). Mutation of the H8 sequence (R ³⁵² AGLFQRS ³⁵⁹ ) significantly reduced G protein activation and β-arrestin recruitment, with residues Leu ³⁵⁵ -Glu ³⁵⁷ being particularly important. Mutations of specific lysine residues in H8 and CT also impaired signaling. Additionally, we identified a crucial TM7-H8 interaction and found that CT phosphorylation sites regulate β-arrestin recruitment. Finally, using AlphaFold3 we predicted interactions between PAR4, β-arrestins, and G proteins, revealing novel receptor regulatory sites in the intracellular loops, transmembrane domains, H8 and CT.