title: Mapping transmembrane residues of proteinase activated receptor 2 (PAR2) that influence ligand-modulated calcium signaling.
abstract: Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor involved in metabolism, inflammation, and cancers. It is activated by proteolysis, which exposes a nascent N-terminal sequence that becomes a tethered agonist. Short synthetic peptides corresponding to this sequence also activate PAR2, while small organic molecules show promising PAR2antagonism. Developing PAR2ligands into pharmaceuticals is hindered by a lack of knowledge of how synthetic ligands interact with and differentially modulate PAR2. Guided by PAR2homology modeling and ligand docking based on bovine rhodopsin, followed by cross-checking with newer PAR2models based on ORL-1 and PAR1, site-directed mutagenesis of PAR2was used to investigate the pharmacology of three agonists (two synthetic agonists and trypsin-exposed tethered ligand) and one antagonist for modulation of PAR2signaling. Effects of 28 PAR2mutations were examined for PAR2-mediated calcium mobilization and key mutants were selected for measuring ligand binding. Nineteen of twenty-eight PAR2mutations reduced the potency of at least one ligand by >10-fold. Key residues mapped predominantly to a cluster in the transmembrane (TM) domains of PAR2, differentially influence intracellular Ca2+induced by synthetic agonists versus a native agonist, and highlight subtly different TM residues involved in receptor activation. This is the first evidence highlighting the importance of the PAR2TM regions for receptor activation by synthetic PAR2agonists and antagonists. The trypsin-cleaved N-terminus that activates PAR2was unaffected by residues that affected synthetic peptides, challenging the widespread practice of substituting peptides for proteases to characterize PAR2physiology.
authors.affiliation: Centre for Inflammation and Disease Research and ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia. Electronic address: j.suen@imb.uq.edu.au.
authors.forename: J Y
authors.initials: JY
authors.lastname: Suen
authors.affiliation: Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: M N
authors.initials: MN
authors.lastname: Adams
authors.affiliation: Centre for Inflammation and Disease Research and ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Lim
authors.affiliation: Centre for Inflammation and Disease Research and ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: P K
authors.initials: PK
authors.lastname: Madala
authors.affiliation: Centre for Inflammation and Disease Research and ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: W
authors.initials: W
authors.lastname: Xu
authors.affiliation: Centre for Inflammation and Disease Research and ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: A J
authors.initials: AJ
authors.lastname: Cotterell
authors.affiliation: Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: Y
authors.initials: Y
authors.lastname: He
authors.affiliation: Centre for Inflammation and Disease Research and ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia.
authors.forename: M K
authors.initials: MK
authors.lastname: Yau
authors.affiliation: Mater Research Institute-University of Queensland, Translational Research Institute, Woolloongabba, Qld 4102, Australia.
authors.forename: J D
authors.initials: JD
authors.lastname: Hooper
authors.affiliation: Centre for Inflammation and Disease Research and ARC Centre of Excellence in Advanced Molecular Imaging, Institute for Molecular Bioscience, The University of Queensland, Brisbane, Qld 4072, Australia. Electronic address: d.fairlie@imb.uq.edu.au.
authors.forename: D P
authors.initials: DP
authors.lastname: Fairlie
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002417:Cattle; D002460:Cell Line; D003412:Cricetulus; D006801:Humans; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009154:Mutation / Q000187:drug effects; D010455:Peptides / Q000494:pharmacology*; D000072417:Protein Domains / Q000502:physiology; D044464:Receptor, PAR-2 / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
chemical_list: D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D044464:Receptor, PAR-2; D014357:Trypsin; D002118:Calcium
keywords: 2f-LIGRLO-NH(2) (PubChem CID: 10395438); Agonist; Antagonist; GB110 (PubChem CID: 49843508); GB88 (PubChem CID: 73755230); Mutagenesis; PAR(2); Protease; Structure