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A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3
Published on: January 24, 2016
Activated protein C drives β-arrestin-2- and c-Src-dependent phosphorylation of Cav1 and modulates Cav1 association
Huaping Qin1, Lennis B Orduña-Castillo1, Olivia Molinar-Inglis1
1Department of Pharmacology, School of Medicine, University of California, San Diego, La Jolla, California, USA.
Abstract:
G-protein-coupled receptors (GPCRs) display bias toward either G proteins or GPCR kinase (GRK)-mediated β-arrestin (βarr) signaling depending on the agonist-stabilized receptor conformation. The cellular context and subcellular location of GPCRs can also influence biased signaling through mechanisms that are not well understood. The protease-activated receptor-1 (PAR1) exhibits signaling bias in response to thrombin and activated protein C (APC). APC-induced βarr2-biased signaling requires PAR1 compartmentalization in caveolae, a subtype of lipid rafts, whereas thrombin-activated PAR1 G protein signaling does not. Caveolin-1 (Cav1) is the principal structural component of caveolae and regulates protein-protein interactions. The mechanisms by which Cav1 contributes to APC-PAR1-induced βarr2-biased signaling are not known. Here, we report that a substantial population of endogenous PAR1 colocalizes with Cav1 in endothelial cells and is modulated by APC, assessed by single-molecule super-resolution stochastic optical reconstruction microscopy imaging. APC activation of PAR1 also induces Cav1 tyrosine-14 phosphorylation through a βarr2- and c-Src-dependent pathway, which disrupts PAR1-Cav1 coassociation. A smaller population of endogenous GRK5 was also found to colocalize with Cav1 in endothelial cells and was modestly altered by APC activation of PAR1. Moreover, GRK5 was found to interact with Cav1 in intact cells through an N-terminal aromatic-rich Cav1 binding motif. Mutation of this motif disrupts GRK5-Cav1 binding, shifts GRK5 predominantly to the cytoplasm rather than the plasma membrane, and perturbs GRK5-mediated βarr2 recruitment to APC-activated PAR1. Thus, beyond its structural function, Cav1 participates in protein-protein interactions with PAR1 and GRK5, two key effectors that enable APC-induced βarr2 signaling.
Insights
Caveolin-1 (Cav1) is crucial for biased signaling of protease-activated receptor-1 (PAR1) in endothelial cells. Cav1 interacts with PAR1 and GRK5, enabling activated protein C (APC)-induced β-arrestin-2 (βarr2) recruitment.
Area of Science:
- Cellular Biology
- Molecular Pharmacology
- Biochemistry
Background:
- G protein-coupled receptors (GPCRs) exhibit biased signaling, directing agonists towards specific pathways like G protein or β-arrestin signaling.
- The protease-activated receptor-1 (PAR1) shows differential signaling bias depending on the agonist, with activated protein C (APC) favoring β-arrestin-2 (βarr2) signaling.
- Caveolin-1 (Cav1), a structural protein in caveolae, is implicated in regulating GPCR localization and signaling, but its precise role in PAR1 bias is unclear.
Purpose of the Study:
- To elucidate the mechanisms by which Caveolin-1 (Cav1) mediates APC-induced β-arrestin-2 (βarr2) biased signaling through PAR1.
- To investigate the interaction between Cav1, PAR1, and GRK5 in endothelial cells during APC activation.
Main Methods:
- Single molecule super-resolution stochastic optical reconstruction microscopy (STORM) imaging to assess PAR1 and Cav1 colocalization.
- Analysis of Cav1 tyrosine-14 phosphorylation following APC activation.
- Co-immunoprecipitation assays to study protein-protein interactions between Cav1, GRK5, and PAR1.
- Site-directed mutagenesis of the Cav1 binding motif in GRK5.
Main Results:
- A significant population of endogenous PAR1 colocalizes with Cav1 in endothelial cells, modulated by APC.
- APC activation induces Cav1 tyrosine-14 phosphorylation via a βarr2- and c-Src-dependent pathway, disrupting PAR1-Cav1 association.
- GRK5 interacts with Cav1 through an N-terminal motif, and disruption of this interaction impairs GRK5 localization and βarr2 recruitment to APC-activated PAR1.
Conclusions:
- Caveolin-1 (Cav1) plays a critical role beyond structure, actively participating in protein-protein interactions essential for APC/PAR1-induced βarr2 biased signaling.
- Cav1 facilitates the interaction between GRK5 and PAR1, which is necessary for efficient βarr2 recruitment and signaling bias.
- These findings reveal a novel mechanism for Cav1 in regulating GPCR signaling specificity.
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