Structural analysis of β-arrestin signalling at Protease-activated receptor 2 (PAR2)

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dc.contributor.authorJogensjö, Jonathan
dc.contributor.departmentChalmers tekniska högskola / Institutionen för biologi och biotekniksv
dc.contributor.departmentChalmers University of Technology / Department of Biology and Biological Engineeringen
dc.date.accessioned2019-07-03T14:52:13Z
dc.date.available2019-07-03T14:52:13Z
dc.date.issued2018
dc.description.abstractG protein-coupled receptors (GPCRs) are the most common receptor class for drug targeting. They are key targets for more than 35% of clinically used drugs as they are important in controlling a diversity of processes within pathophysiology. GPCR signalling was first thought to only be G protein-mediated. However, it has recently been shown that β-arrestins can act as an adaptor protein creating their own G protein-independent signalling. One GPCR, the protease-activated receptor 2 (PAR2), has been shown to be involved in inflammation and pain; metabolic, cardiovascular and neurological systems but also cancers which makes it an interesting drug target. Recently, the crystal structure of PAR2 together with two antagonists was solved indicating a suggested orthogonal binding site for agonists. The aim of this project was to evaluate possible important amino acid residues of PAR2 inducing β-arrestin recruitment for the agonists SLIGKV, SLIGRL, GB110 and 2f-LIGRLO. This was done by using single point mutated receptors based on the solved crystal structure using DiscoverX PathHunter β-Arrestin Assay for GPCR Cell Lines. The β-arrestin recruitment assay is based on an enzyme fragment complementation (EFC) technology where PAR2 is tagged with an Enzyme Donor (PK) and β-arrestin with an Enzyme Acceptor (EA). Optimization of the β-arrestin recruitment assay and construction of PAR2-PK plasmids needed for transient transfections of 1321N1 β-arrestin-EA cells were carried out. The optimized conditions for β-arrestin recruitment assay were found to be 4000 cells/well, 90 minutes of agonist incubation time and 60 minutes of detection time for the U2OS cell line stably expressing PAR2-PK and β-arrestin-EA. Constructed PAR-PK plasmids were shown to have the correct fragment sizes and sequences after ScaI restriction enzyme digestion and Sanger sequencing respectively. 1321N1 β-arrestin-EA cells transiently transfected with nine PAR2-PK plasmids were shown to have PAR2 expressed at the membrane surface for all constructed plasmids. Data suggests the importance of residues E232, Y323, Y326 and D228 of PAR2 for β-arrestin-mediated signalling induced by agonists SLIGKV, SLIGRL, GB110 (except E232) and 2f-LIGRLO. Data also suggest that the proposed orthogonal site in the published crystal structure is the orthogonal binding site in β-arrestin recruitment for agonist SLIGKV, SLIGRL, GB110 and 2f-LIGLRO.
dc.identifier.urihttps://hdl.handle.net/20.500.12380/255855
dc.language.isoeng
dc.setspec.uppsokLifeEarthScience
dc.subjectLivsvetenskaper
dc.subjectBiokemi och molekylärbiologi
dc.subjectLäkemedelskemi
dc.subjectMedicinsk bioteknologi (med inriktning mot cellbiologi)
dc.subjectLife Science
dc.subjectBiochemistry and Molecular Biology
dc.subjectMedicinal Chemistry
dc.subjectMedical Biotechnology (with a focus on Cell Biology)
dc.titleStructural analysis of β-arrestin signalling at Protease-activated receptor 2 (PAR2)
dc.type.degreeExamensarbete för masterexamensv
dc.type.degreeMaster Thesisen
dc.type.uppsokH
local.programmeBiotechnology (MPBIO), MSc
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