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Proteinase-Activated Receptors (PARs) are a four-member family of G-protein coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by the serine proteinases such as thrombin, trypsin and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types including the platelets, vascular smooth muscle cells and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane with spherical membrane protrusions that resemble plasma membrane blebbing. Since non-apoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/Cas9 mediated gene editing approaches we establish that PAR4-dependent membrane blebbing occurs independently of the Gαq/11 and Gαi signaling pathways and is dependent on signaling via the β-arrestin-1/-2 pathway and RhoA signaling. In order to gain a more comprehensive understanding of β-arrestin mediated signaling downstream of PAR4 and to guide future studies, we undertook RNAseq analysis of PAR4 activated genes in control cells and in cells lacking β-arrestin-1/-2. A list of differentially expressed genes was generated followed by Gene Ontology (GO) and enrichment analysis revealing PAR4 regulation of genes involved in processes including blood coagulation and circulation, cell-cell adhesion, sensory perception and neuron-neuron synaptic transmission, terms that relates back to known function of PAR4 and that are consistent with our finding of membrane blebbing triggered by PAR4 activation. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA- and β-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4. Finally through CRISPR/Cas9-mediated targeting and RNA-seq analysis we catalogue here PAR4-dependent transcription that is dependent on β-arrestin.

Proteinase-Activated Receptor 4 Activation Triggers Cell Membrane Blebbing through RhoA andβ-Arrestin