Proteinase-Activated Receptor 4 (PAR4) Activation Triggers Cell Membrane Blebbing through RhoA and β-arrestin
Bibliographic record
Abstract
Abstract 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 serine proteinases such as thrombin, trypsin and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types including platelets, vascular smooth muscle cells and neuronal cells. In studying PAR4 signalling 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 signalling 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 Gα q/11 and Gα i signalling pathways and is dependent on signalling via the β-arrestin-1/-2 and RhoA signalling pathways. In order to gain a more comprehensive understanding of β-arrestin-mediated signalling downstream of PAR4 and to guide future studies, we undertook RNA-seq 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 relate back to known functions of PAR4 and 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.003 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".