Research ArticleGPCR SIGNALING

A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity

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Science Signaling  04 Feb 2020:
Vol. 13, Issue 617, eaaw5885
DOI: 10.1126/scisignal.aaw5885
  • Fig. 1 Investigation of structural elements supporting G protein–biased signaling by the D2R.

    (A) Pharmacophore model for MLS1547 interactions with the D2R [modified from (27)]. (B) The D2R-WT or the indicated D2R mutants were expressed in HEK293 cells with Goα1-Rluc8, β1, and γ2-mVenus. The cells were stimulated with MLS1547 and assayed for G protein activation by BRET. (C) HEK293 cells expressing D2R-F1895.38A, Goα1-Rluc8, β1, and γ2-mVenus were incubated with 13 μM (EC80) dopamine and the indicated concentrations of either sulpiride or MLS1547 and assayed for G protein activation by BRET. (D) Membrane preparations from HEK293 cells expressing either D2R-WT or D2R-I184EL2A, V1905.39A, or F1895.38A were incubated with the indicated concentrations of dopamine and [3H]methylspiperone. Data are expressed as a percentage of the specific binding and fit using nonlinear regression analyses (table S2). (E) HEK293 cells described in (B) were stimulated with dopamine and assayed for G protein activation. (F) The D2R-WT and indicated mutant receptors were fused to Rluc8 and expressed with β-arrestin2–mVenus and GRK2 in HEK293 cells. Dopamine-stimulated β-arrestin recruitment was assessed by BRET. Functional data are expressed as a percentage of the maximum dopamine or MLS1547 responses for D2R-WT (% control). Data in (B) to (F) represent the mean ± SEM values of three to five independent experiments performed in technical triplicate. Average EC50 and Emax values for functional assays are displayed in table S1.

  • Fig. 2 The F1895.38A mutation confers G protein signaling bias in the D2R.

    (A) HEK293 cells transiently expressing either D2R-WT or D2R-F1895.38A with Goα1-Rluc8, β1, and γ2-mVenus were stimulated with dopamine and assayed for G protein activation by BRET. Average EC50 and Emax values are displayed in table S1. (B) HEK293 cells transiently expressing D2R-WT or D2R-F1895.38A with the CAMYEL biosensor were assayed for inhibition of forskolin-stimulated cAMP production. Average EC50 and Emax values are displayed in table S3. (C) The D2R-WT and F1895.38A receptors were fused to Rluc8 and expressed in HEK293 cells with β-arrestin2–mVenus and GRK2. Dopamine-stimulated β-arrestin recruitment was assessed by BRET. Average EC50 and Emax values are displayed in table S1. (D) D2R-WT or D2R-F1895.38A were fused to a segment of β-galactosidase and expressed in CHO cells with β-arrestin2 fused to a complementing segment of β-galactosidase. Dopamine-stimulated complementation of β-galactosidase was measured. Average EC50 and Emax values are shown in table S3. (E and F) Molecular proximity between D2R-WT or D2R-F1895.38A and β-arrestin2 was detected with titration experiments performed in HEK293 cells. Cells expressing a fixed amount of D2R-WT–Rluc8 or D2R-F1895.38A-Rluc8 and increasing amounts of β-arrestin2–mVenus were incubated in the presence or absence of 10 μM quinpirole. β-Arrestin recruitment was assessed by BRET. X axes represent the ratio between the fluorescence emitted by β-arrestin2–mVenus and the luminescence emitted by D2R-WT or D2R-F1895.38A-Rluc8. Y axes represent the BRET ratio. (G and H) The D2R-WT and indicated mutant receptors fused to Rluc8 were expressed in HEK293 cells with β-arrestin2–mVenus and GRK2. (G) Dopamine- or (H) pramipexole-stimulated β-arrestin recruitment was assessed by BRET. Average EC50 and Emax values are displayed in table S5. All functional data are expressed as percentage of the maximum response observed for D2R-WT. Data points in (A) to (H) represent mean ± SEM of 3 to 14 independent experiments performed in technical triplicate.

  • Fig. 3 The G protein–biased D2R-F1895.38A exhibits impaired agonist-induced internalization.

    (A and B) HEK293 cells expressing either D2R-WT (A) or D2R-F1895.38A (B) were incubated for 1.5 hours with vehicle or 10 μM dopamine. Surface expression of the receptor was measured with an intact cell binding assay using [3H]sulpiride. Data are representative of three independent experiments. *P < 0.05, unpaired Student’s t test. (C) HEK293 cells transiently expressing either D2R-WT–Rluc8 or D2R-F1895.38A–Rluc8 with LYN-rGFP were treated with increasing concentrations of dopamine for 10 min. The interaction between D2R and LYN was measured by BRET. In the graph, the constitutive basal BRET is defined as 100% control, and maximum dopamine-induced decrease in BRET is defined as 0%. The EC50 for dopamine-induced internalization was 88 ± 19 nM. No measurable internalization was observed with the D2R-F1895.38A. Data in (A) to (C) are mean ± SEM of four independent experiments performed in technical triplicate.

  • Fig. 4 Mutation of 5.38 confers G protein signaling bias in multiple GPCRs.

    (A) The D3R-WT and D3R-F1885.38A were fused to Rluc8 and expressed in HEK293 cells with β-arrestin2–mVenus and GRK2. Dopamine-stimulated β-arrestin recruitment was assessed by BRET. (B) The D3R-WT and D3R-F1885.38A were expressed in HEK293 cells with Goα1-Rluc8, β1, and γ2-mVenus. The cells were stimulated with dopamine and assayed for G protein activation by BRET. (C) Dopamine-stimulated β-arrestin recruitment was assessed for the D4R-WT–Rluc8 and D4R-Y1925.38A-Rluc8 as described in (A). (D) Dopamine-stimulated Go activation was assessed for the D4R-WT and D4R-Y1925.38A as described in (B). (E) The β2R-WT and β2R-Y1995.38A were fused to Rluc8 and expressed in HEK293 cells with β-arrestin2–mVenus. Epinephrine-stimulated β-arrestin recruitment was assessed by BRET. (F) The β2R-WT and β2R-Y1995.38A were expressed in HEK293 cells with Gαs-RLuc8, β1, and γ2-mVenus. The cells were stimulated with epinephrine and assayed for G protein activation by BRET. (G) V2R-WT or the indicated V2R mutant was fused to Rluc8 and expressed in HEK293 cells with β-arrestin2–mVenus and assayed for AVP-stimulated β-arrestin recruitment by BRET. (H) HEK293 cells expressing either V2R-WT or the indicated V2R mutant were assayed for AVP-stimulated cAMP accumulation using the TR-FRET–based LANCE cAMP Detection kit. Data are expressed as a percentage of the maximum response for WT receptor. Average EC50 and Emax values are found in table S6. All data points represent the mean ± SEM of three to six independent experiments performed in technical triplicate.

  • Fig. 5 β2R-Y1995.38A changes the packing among TM4, TM5, and EL2.

    (A) The crystal structure of β2R–BI-167107 complex in active conformation (PDB ID 4LDE) (30). (B) Magnified view of the ligand binding site [the boxed region in (A)] in β2R-WT and β2R-Y1995.38A. In β2R-WT (brown structure), the side-chain ─OH of Tyr5.38 forms a hydrogen bond (dashed magenta line) with the backbone oxygen of Thr4.56. In the absence of this H-bond and the loss of the bulky side chain of Tyr5.38 in β2R-Y1995.38A (green structure), Tyr174EL2 bends down to interact with BI-167107. (C) The distance between Tyr5.38 and Thr4.56 (Cβ-Cβ) is larger in β2R-Y1995.38A, indicating a rearrangement of Thr4.56 away from the ligand binding site.

  • Fig. 6 Local disruption near position 5.38 propagates to the IL2 through the TM3-TM4-TM5 interface.

    (A) The β2R-WT (brown) and β2R-Y1995.38A (green). (B) The rhodopsin-Gi complex (PDB code 6CMO) (44) (gold) and the rhodopsin–β-arrestin complex (PDB code 4ZWJ) (45) (silver).

  • Table 1 Alignment of hydrophobic pocket residues for select GPCRs.

    The amino acid position in extracellular loop 2 (EL2) is delineated using the nomenclature developed by de Graaf et al. (76). Yellow indicates nonpolar amino acids with an aliphatic group (Ala, Val, Ile, Leu, Met, and Gly); light green indicates hydrophobic amino acids with an aromatic ring (Phe and Tyr); dark green indicates Trp; purple indicates polar amino acids with an uncharged side chain (Ser, Thr, Asn, and Gln); light gray indicates Pro; red indicates negatively charged amino acids (Glu and Asp); and blue indicates positively charged amino acids (Arg, His, and Lys).

    GPCRECL2-TM5 sequence*
    45.525.355.365.375.385.395.40
    D1RSerSerArgThrTyrAlaIle
    D2RIleAsnProAlaPheValVal
    D3RIleAsnProAspPheValIle
    D4RLeuAspArgAspTyrValVal
    D5RSerAsnArgThrTyrAlaIle
    5-HT1ARIleAspHisGlyTyrThrIle
    5-HT1BRValHisIleLysTyrThrVal
    5-HT2ARLeuAspAspAsnPheValLeu
    5-HT2BRLeuPheGlyAspPheMetLeu
    5-HT2CRLeuAspProAsnPheValLeu
    α1AIleGluProGlyTyrValLeu
    α1BValGluProPheTyrAlaLeu
    α1DIleGluAlaGlyTyrAlaVal
    α2AIleGlnLysTrpTyrValIle
    α2BLeuGluAlaTrpTyrIleLeu
    α2CLeuGluThrTrpTyrIleLeu
    β1RPheGlnArgAlaTyrAlaIle
    β2RPheGlnGlnAlaTyrAlaIle
    β3RPheGlnMetProTyrValLeu
    V2RAlaArgArgThrTyrValThr

    *Amino acid positions in transmembrane (TM) regions are delineated using the Ballesteros and Weinstein nomenclature (29).

    Supplementary Materials

    • stke.sciencemag.org/cgi/content/full/13/617/eaaw5885/DC1

      Fig. S1. D2R-WT and D2R mutants express to a similar extent using transient transfection.

      Fig. S2. Other D2R agonists exhibit G protein bias at the D2R-F1895.38A.

      Fig. S3. Compound CAB02-110 is a partial agonist at both G protein activation and β-arrestin recruitment.

      Fig. S4. Rotigotine- and apomorphine-stimulated internalization of the D2-WT and D2R-F1895.38A.

      Fig. S5. The β2R-Y1995.38A exhibits G protein bias with different agonists.

      Fig. S6. Partial agonist stimulation of β2R-mediated Gs activation and β-arrestin recruitment.

      Fig. S7. Distribution of distance between the oxygen of Tyr5.38 side-chain hydroxyl and the backbone oxygen atom of Thr4.56 in the β2R-WT.

      Fig. S8. IL2 moves downward in the β2R-Y1995.38A mutant.

      Table S1. β-Arrestin–BRET and Go BRET signaling by D2R-WT and D2R mutants.

      Table S2. Affinity constants (Ki values) for MLS1547 and dopamine binding to D2R-WT and D2R mutants.

      Table S3. β-Arrestin recruitment and regulation of cAMP signaling by D2R-WT and D2R-F1895.38A.

      Table S4. β-Arrestin and Go BRET signaling for D2R-WT and D2R-F1895.38A in response to full D2R agonists.

      Table S5. β-Arrestin–BRET recruitment for D2R-WT and various mutants at the Phe1895.38 position.

      Table S6. β-Arrestin– and G protein–mediated signaling by WT and 5.38 mutant D3R, D4R, β2R, and V2R.

      Table S7. Summary of MD simulations.

      Table S8. β2R–BI-167107 interacting residues.

    • This PDF file includes:

      • Fig. S1. D2R-WT and D2R mutants express to a similar extent using transient transfection.
      • Fig. S2. Other D2R agonists exhibit G protein bias at the D2R-F1895.38A.
      • Fig. S3. Compound CAB02-110 is a partial agonist at both G protein activation and β-arrestin recruitment.
      • Fig. S4. Rotigotine- and apomorphine-stimulated internalization of the D2-WT and D2R-F1895.38A.
      • Fig. S5. The β2R-Y1995.38A exhibits G protein bias with different agonists.
      • Fig. S6. Partial agonist stimulation of β2R-mediated Gs activation and β-arrestin recruitment.
      • Fig. S7. Distribution of distance between the oxygen of Tyr5.38 side-chain hydroxyl and the backbone oxygen atom of Thr4.56 in the β2R-WT.
      • Fig. S8. IL2 moves downward in the β2R-Y1995.38A mutant.
      • Table S1. β-Arrestin–BRET and Go BRET signaling by D2R-WT and D2R mutants.
      • Table S2. Affinity constants (Ki values) for MLS1547 and dopamine binding to D2R-WT and D2R mutants.
      • Table S3. β-Arrestin recruitment and regulation of cAMP signaling by D2R-WT and D2R-F1895.38A.
      • Table S4. β-Arrestin and Go BRET signaling for D2R-WT and D2R-F1895.38A in response to full D2R agonists.
      • Table S5. β-Arrestin–BRET recruitment for D2R-WT and various mutants at the Phe1895.38 position.
      • Table S6. β-Arrestin– and G protein–mediated signaling by WT and 5.38 mutant D3R, D4R, β2R, and V2R.
      • Table S7. Summary of MD simulations.
      • Table S8. β2R–BI-167107 interacting residues.

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