Abstract
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.
Highlights
Thrombin-cleaved PAR1 reveals a tethered ligand that can transactivate PAR2
The ability of PAR1 to transactivate PAR2 would necessitate that the two receptors be in close proximity, likely in the form of a heterodimer
HeLa cells were cotransfected with FLAGPAR1 and HA-PAR2 and incubated with anti-FLAG and anti-HA antibodies for 1 h at 4 °C
Summary
Results: The thrombin-activated PAR1-PAR2 heterodimer displays unique trafficking behavior, recruitment of -arrestins to endosomes, and signaling responses compared with the receptor protomer. 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. We further demonstrate that thrombin activation of the PAR1-PAR2 heterodimer results in -arrestin recruitment through an interface that is different from that utilized by receptor protomers. -arrestins co-internalize with the thrombin-activated PAR1-PAR2 dimer and mediate ERK1/2 signaling in the cytosol while limiting nuclear ERK1/2 activation These results indicate that the PAR1-PAR2 dimer utilizes a unique -arrestin-binding interface and elicits signaling responses that are distinct from those induced by the PAR1 protomer
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