Transactivation of the PAR1-PAR2 heterodimer by thrombin elicits β-arrestin-mediated endosomal signaling.

Thrombin cleaves the N terminus of PAR1, generating a new N-terminal domain that functions as a tethered ligand that binds intermolecularly to activate PAR2 in trans. The mechanisms that regulate PAR1-PAR2 heterodimer signaling and trafficking are not known. We now report that PAR1 and PAR2 form a heterodimer that exhibits unique trafficking and signaling behaviors compared with receptor protomers. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, co-immunoprecipitation, and cells expressing receptors exogenously and endogenously, we show that PAR1 and PAR2 specifically interact and form stable dimers. Intriguingly, the PAR1-PAR2 heterodimer displays constitutive internalization that is driven by PAR1 C-terminal tail sorting motifs and is a process that enhances dimer formation. Upon thrombin activation, PAR1-PAR2 dimers co-internalize and recruit β-arrestins to endosomes. Remarkably, PAR1-PAR2 heterodimers appear to utilize a distinct interface for β-arrestin interaction compared with receptor protomers. Moreover, thrombin-activated PAR1-PAR2 heterodimers enhance β-arrestin-mediated ERK1/2 activation in the cytoplasm, whereas activated ERK1/2 induced by the thrombin-activated PAR1 protomer redistributes to the nucleus. Thus, the formation of PAR1-PAR2 heterodimers provides additional modes of thrombin-stimulated signaling responses that appear to be distinctly regulated compared with the receptor protomer.