![]() ![]() X-axes represent the ratio between the fluorescence emitted by β-arrestin2-mVenus and the luminescence emitted by D2R-WT or D2R-F189 5.38A-Rluc8. β-arrestin recruitment was assessed by BRET. Cells expressing a fixed amount of D2R-WT-Rluc8 or D2R-F189 5.38A-Rluc8 and increasing amounts of β-arrestin2-mVenus were incubated in the presence or absence of 10 μM quinpirole. ( E to F) Molecular proximity between D2R-WT or D2R-F189 5.38A and β-arrestin2 was detected with titration experiments performed in HEK293 cells. Average EC 50 and Emax values are shown in table S3. Dopamine-stimulated complementation of β-galactosidase was measured. ( D) D2R-WT or D2R-F189 5.38A were fused to a segment of β-galactosidase and expressed in CHO cells with β-arrestin2 fused to a complementing segment of β-galactosidase. Average EC 50 and Emax values are displayed in table S1. Dopamine-stimulated β-arrestin recruitment was assessed by BRET. ( C) The D2R-WT and F189 5.38A receptors were fused to Rluc8 and expressed in HEK293 cells with β-arrestin2-mVenus and GRK2. Average EC 50 and Emax values are displayed in table S3. ( B) HEK293 cells transiently expressing D2R-WT or D2R-F189 5.38A with the CAMYEL biosensor were assayed for inhibition of forskolin-stimulated cAMP production. ( A) HEK293 cells transiently expressing either D2R-WT or D2R-F189 5.38A with Goα1-Rluc8, β1, and γ2-mVenus were stimulated with dopamine and assayed for G protein activation by BRET. Average EC 50 and Emax values for functional assays are displayed in table S1. Data in (B) to (F) represent the mean ± SEM values of 3–5 independent experiments performed in technical triplicate. Functional data are expressed as a percentage of the maximum dopamine or MLS1547 responses for D2R-WT (% control). ( F) The D2R-WT and indicated mutant receptors were fused to Rluc8 and expressed with β-arrestin2-mVenus and GRK2 in HEK293 cells. ( E) HEK293 cells described in ( B) were stimulated with dopamine and assayed for G protein activation. ![]() Data are expressed as a percentage of the specific binding and fit using non-linear regression analyses (table S2). ( D) Membrane preparations from HEK293 cells expressing either D2R-WT or D2R-I184 EL2A, V190 5.39A or F189 5.38A were incubated with the indicated concentrations of dopamine and methylspiperone. ( C) HEK293 cells expressing D2R-F189 5.38A, Goα1-Rluc8, β1, and γ2-mVenus were incubated with 13 µM (EC 80) dopamine and the indicated concentrations of either sulpiride or MLS1547 and assayed for G protein activation by BRET. The cells were stimulated with MLS1547 and assayed for G protein activation by BRET. ( B) The D2R-WT or the indicated D2R mutants were expressed in HEK293 cells with Goα1-Rluc8, β1, and γ2-mVenus. ( A) Pharmacophore model for MLS1547 interactions with the D2R (modified from (27)). Our findings provide a structural basis for how ligand binding site alterations can allosterically affect GPCR-transducer interactions and result in biased signaling.Ĭopyright © 2020 The Authors, some rights reserved exclusive licensee American Association for the Advancement of Science. These analyses identified conformational rearrangements in β2R-Y199 5.38A that propagated from the extracellular ligand binding site to the intracellular surface, resulting in a modified orientation of the second intracellular loop in β2R-Y199 5.38A, which is predicted to affect its interactions with β-arrestin. To investigate the mechanism of this signaling bias, we used an active-state structure of the β 2-adrenergic receptor (β2R) to build β2R-WT and β2R-Y199 5.38A models in complex with the full β2R agonist BI-167107 for molecular dynamics simulations. This residue is relatively conserved among class A GPCRs, and analogous mutations within other GPCRs similarly impaired β-arrestin recruitment while maintaining G protein signaling. Here, we showed that residue Phe189 within this pocket (position 5.38 using Ballesteros-Weinstein numbering) functions as a microswitch for regulating receptor interactions with β-arrestin. This signaling bias was predicted to arise from unique interactions of the ligand with a hydrophobic pocket at the interface of the second extracellular loop and fifth transmembrane segment of the D2R. We previously identified a G protein-biased agonist of the D 2 dopamine receptor (D2R) that results in impaired β-arrestin recruitment. Signaling bias is the propensity for some agonists to preferentially stimulate G protein-coupled receptor (GPCR) signaling through one intracellular pathway versus another. ![]()
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