title: Biased signaling of the angiotensin II type 1 receptor can be mediated through distinct mechanisms.
abstract: BACKGROUND
  Seven transmembrane receptors (7TMRs) can adopt different active conformations facilitating a selective activation of either G protein or β-arrestin-dependent signaling pathways. This represents an opportunity for development of novel therapeutics targeting selective biological effects of a given receptor. Several studies on pathway separation have been performed, many of these on the Angiotensin II type 1 receptor (AT1R). It has been shown that certain ligands or mutations facilitate internalization and/or recruitment of β-arrestins without activation of G proteins. However, the underlying molecular mechanisms remain largely unresolved. For instance, it is unclear whether such selective G protein-uncoupling is caused by a lack of ability to interact with G proteins or rather by an increased ability of the receptor to recruit β-arrestins. Since uncoupling of G proteins by increased ability to recruit β-arrestins could lead to different cellular or in vivo outcomes than lack of ability to interact with G proteins, it is essential to distinguish between these two mechanisms.
  
  
  RESULTS
  We studied five AT1R mutants previously published to display pathway separation: D74N, DRY/AAY, Y292F, N298A, and Y302F (Ballesteros-Weinstein numbering: 2.50, 3.49-3.51, 7.43, 7.49, and 7.53). We find that D74N, DRY/AAY, and N298A mutants are more prone to β-arrestin recruitment than WT. In contrast, receptor mutants Y292F and Y302F showed impaired ability to recruit β-arrestin in response to Sar1-Ile4-Ile8 (SII) Ang II, a ligand solely activating the β-arrestin pathway.
  
  
  CONCLUSIONS
  Our analysis reveals that the underlying conformations induced by these AT1R mutants most likely represent principally different mechanisms of uncoupling the G protein, which for some mutants may be due to their increased ability to recruit β-arrestin2. Hereby, these findings have important implications for drug discovery and 7TMR biology and illustrate the necessity of uncovering the exact molecular determinants for G protein-coupling and β-arrestin recruitment, respectively.
authors.affiliation: Laboratory for Molecular Cardiology, The Danish National Research Foundation Centre for Cardiac Arrhythmia, The Heart Centre, Copenhagen University Hospital, Rigshospitalet, and The Danish National Research Foundation Centre for Cardiac Arrhythmia, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Marie Mi
authors.initials: MM
authors.lastname: Bonde
authors.forename: Jonas Tind
authors.initials: JT
authors.lastname: Hansen
authors.forename: Samra Joke
authors.initials: SJ
authors.lastname: Sanni
authors.forename: Stig
authors.initials: S
authors.lastname: Haunsø
authors.forename: Steen
authors.initials: S
authors.lastname: Gammeltoft
authors.forename: Christina
authors.initials: C
authors.lastname: Lyngsø
authors.forename: Jakob Lerche
authors.initials: JL
authors.lastname: Hansen
mesh_terms: D000804:Angiotensin II / Q000378:metabolism; D000818:Animals; D019390:Arrestins / Q000235:genetics / Q000378:metabolism*; D001667:Binding, Competitive; D019556:COS Cells; D002522:Chlorocebus aethiops; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D009154:Mutation; D011485:Protein Binding; D044140:Receptor, Angiotensin, Type 1 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; D044140:Receptor, Angiotensin, Type 1; D011993:Recombinant Fusion Proteins; D000071557:beta-Arrestins; D000804:Angiotensin II; D049452:Green Fluorescent Proteins; D008156:Luciferases; D043802:GTP-Binding Protein alpha Subunits, Gq-G11