title: Biased agonism and allosteric modulation of G protein-coupled receptor 183 - a 7TM receptor also known as Epstein-Barr virus-induced gene 2.
abstract: The GPCR Epstein-Barr virus-induced gene 2 (EBI2, also known as GPR183) is activated by oxysterols and plays a pivotal role in the regulation of B cell migration during immune responses. While the molecular basis of agonist binding has been addressed in several studies, the concept of biased agonism of the EBI2 receptor has not been explored.
  
  
  
  We investigated the effects of the EBI2 endogenous agonist 7α,25-dihydroxycholesterol (7α,25-OHC) on G protein-dependent and -independent pathways as well as sodium ion allosterism using site-directed mutagenesis and functional studies. Moreover, we generated a homology model of the EBI2 receptor to investigate the structural basis of the allosteric modulation by sodium.
  
  
  
  Residue N114, located in the middle of transmembrane-III at position III:11/3.35, was found to function as an efficacy switch. Thus, substituting N114 with an alanine (N114A) completely abolished heterotrimeric G protein subunit Gi α activation by 7α,25-OHC even though the specific binding of [3H]-7α,25-OHC increased. In contrast, the N114A mutant was still able to recruit β-arrestin and even had an enhanced potency (18.7-fold) compared with EBI2 wild type. Sodium had a negative allosteric effect on oxysterol binding that was mediated via N114, verifying the key role of N114. This was further supported by molecular modelling of the ion binding site based on a EBI2 receptor homology model.
  
  
  
  Collectively, our data point to N114 as a key residue for EBI2 signalling controlling the balance between G protein-dependent and -independent pathways and facilitating sodium binding.
authors.affiliation: Department of Biomedical Sciences, Laboratory for Molecular Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Viktorija
authors.identifier: 0000-0001-8786-348X
authors.initials: V
authors.lastname: Daugvilaite
authors.affiliation: Department of Biomedical Sciences, Laboratory for Molecular Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Christian Medom
authors.initials: CM
authors.lastname: Madsen
authors.affiliation: Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Michael
authors.initials: M
authors.lastname: Lückmann
authors.affiliation: Department of Biomedical Sciences, Laboratory for Molecular Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Clara Castello
authors.initials: CC
authors.lastname: Echeverria
authors.affiliation: Forum 1, Novartis Campus, CH-4056, Novartis Institutes for BioMedical Research, Basel, Switzerland.
authors.forename: Andreas Walter
authors.initials: AW
authors.lastname: Sailer
authors.affiliation: Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Thomas Michael
authors.initials: TM
authors.lastname: Frimurer
authors.affiliation: Department of Biomedical Sciences, Laboratory for Molecular Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Mette Marie
authors.initials: MM
authors.lastname: Rosenkilde
authors.affiliation: Department of Biomedical Sciences, Laboratory for Molecular Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Tau
authors.initials: T
authors.lastname: Benned-Jensen
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002478:Cells, Cultured; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D006888:Hydroxycholesterols / Q000494:pharmacology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D012964:Sodium / Q000494:pharmacology; D013329:Structure-Activity Relationship
chemical_list: C080105:GPR183 protein, human; D006888:Hydroxycholesterols; D043562:Receptors, G-Protein-Coupled; C057657:7,25-dihydroxycholesterol; D012964:Sodium