Spatio-temporal assessment of β-arrestin-centred “signalosomes” – the impact of effector binding on the functional selectivity of protease-activated receptor 2.
Structural Biology
Cell Biology
Final Report Abstract
The project was devised to investigate the functionality of β-arrestins in the context of pain signaling initiated by protease-activated receptor 2 (PAR2). As a G protein-coupled receptor (GPCR), PAR2 is part of the largest family of membrane proteins within the human body that influences many, if not all, physiological processes. Most of the 800 different GPCRs transmit their primary signals via approximately 30 distinct combinations of Gα and Gβγ subunits that make up the required G proteins to enable a certain degree of signal specificity. Yet only two ubiquitously expressed arrestin (β-arrestin1 and 2) and four GPCR kinase (GRK) isoforms (GRK2, 3, 5 and 6) have to mediate receptor desensitization, internalization, trafficking and specialized signaling responses for the entire GPCR superfamily. To investigate how arrestins and GRKs work together to enable targeted and diverse functions for so many different receptors and to specifically understand their involvement in the initiation of pain signalling via PAR2, new expression vectors were required for the assessment of GRK- specific β-arrestin recruitment to PAR2, which have now all been constructed. Utilizing two different activation mechanisms of the receptor, I was able to show that its main endogenous activator, trypsin, displays a distinctly different kinetic activation profile in comparison to the optimised peptide ligand 2f-LIGRLO-NH2 (2F). Leveraging a previously described assay system consisting of different GRK knockout cell lines, the results further show that all four ubiquitously expressed GRK isoforms are able to facilitate arrestin recruitment to PAR2 individually. Moreover, I present evidence that overexpression of any GRK isoform mediates a significant ligand-independent pre-association between the receptor and both β-arrestin isoforms. Finally, advanced biosensor assays have been established to investigate PAR2-induced β-arrestin conformational changes to explore whether there is a specific β-arrestin conformational state that supports the initiation of pain signalling. The resulting data show that β-arrestin2 undergoes significantly different conformational changes for coupling to PAR2 upon activation with trypsin and 2F. Ultimately, this DFG-funded project aimed to unveil the molecular basis of pain in acute and chronic inflammatory conditions. As one of the main mediators of pain originating from inflamed tissues, PAR2 is already established as a promising drug target, which is not therapeutically exploited yet. While the initial measurement systems to assess β-arrestin-mediated PAR2 functions have been established, this project will be continued through funding provided by the University of Nottingham to further focus on characterizing subcellular complex configurations between PAR2, β-arrestins and essential signaling effector proteins. The generated results already advanced our understanding of subcellular GPCR signaling and are anticipated to enable future drug discovery efforts to create clinical interventions that modulate GPCR signaling responses in specific subcellular compartments (spatial bias).
Publications
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Conformational flexibility of β‐arrestins – How these scaffolding proteins guide and transform the functionality of GPCRs. BioEssays, 45(8).
Haider, Raphael S.; Reichel, Mona; Matthees, Edda S. F. & Hoffmann, Carsten
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Arrestin‐centred interactions at the membrane and their conformational determinants. British Journal of Pharmacology, 182(14), 3135-3150.
Underwood, Owen; Haider, Raphael Silvanus; Sanchez, Julie & Canals, Meritxell
