Research ArticleGPCR SIGNALING

Single-molecule diffusion-based estimation of ligand effects on G protein–coupled receptors

See allHide authors and affiliations

Science Signaling  18 Sep 2018:
Vol. 11, Issue 548, eaao1917
DOI: 10.1126/scisignal.aao1917
  • Fig. 1 Activation model, TIRFM image, MSD-Δt plots, and comparison of diffusion, ligand occupancy, and G protein activation of mGluR3.

    (A) Activation model of mGluR. The crystal structures of the ECDs of mGluR1 in the inactive [blue; Protein Data Bank (PDB), 1EWT] and active (red; PDB, 1EWK) states were constructed with PyMOL (www.pymol.org/). The crystal structure of the TMD (blue and red; PDB, 4OR2) is also shown. (B) Representative TIRFM image of a human embryonic kidney (HEK) 293 cell expressing TMR-labeled mGluR3 (left, whole-cell image; right, magnified view of the area within the blue dashed square). Right: The trajectories of mGluR3 molecules are shown as yellow lines. (C to E) MSD-Δt plots of the trajectories of mGluR3 under the indicated ligand conditions. (C) Inverse agonist (LY341495) dependency. (D and E) Agonist (LY379268) dependencies at 100 nM LY314195 without (D) and with (E) 1 μM MNI137. Data are means ± SEM of 20 cells. *P < 0.01 [one-way analysis of variance (ANOVA)] when comparing the MSDs among the five ligand conditions at each Δt. (F and G) Dose-dependent changes in DAv. (F) LY341495 dependency (blue squares; EC50 = 28.2 ± 0.9 nM) and LY379268 dependency (red circles; EC50 = 1.19 ± 0.02 μM) in the absence of other ligands. (G) LY379268 dependencies at 100 nM LY341495 without (red circles; EC50 = 1.03 ± 0.08 μM) and with (green squares; EC50 = 0.34 ± 0.003 μM) 1 μM MNI137. Data are means ± SEM of 20 cells. *P < 0.01 (two-tailed t test) when compared with the leftmost point in each curve (F). **P < 0.01 (two-tailed t test) when comparing with and without 1 μM MNI137 in (G). (H and I) Dose-dependent changes in specific [3H]LY341495 binding. [3H]LY341495 saturation binding [H, blue squares; dissociation constant (Kd) = 47.4 ± 1.7 nM]. Replacement of 100 nM [3H]LY341495 with LY379268 in the absence (H and I, red circles; IC50 = 0.55 ± 0.08 μM) and presence (I, green squares; IC50 = 0.60 ± 0.03 μM) of 1 μM MNI137. The amount of nonspecifically bound 100 nM [3H]LY341495 was 50 ± 11 fmol. Data are means ± SEM of three independent experiments. The same membrane preparation from mGluR3-expressing cells was analyzed within the same panel. *P < 0.01 (two-tailed t test) when compared with the leftmost point in each curve in (H). No statistically significant difference was detected with and without 1 μM MNI137 in (I). P > 0.05 by two-tailed t test. (J and K) Dose-dependent changes in the efficiency of G protein activation in membranes from mGluR3-expressing cells. LY341495 dependency (blue closed squares; IC50 = 2.11 ± 0.18 nM) and LY379268 dependency (red closed circles; EC50 = 0.025 ± 0.0029 μM) without other ligands are shown in (J). LY379268 dependencies at 100 nM LY341495 without (red closed circles; EC50 = 1.77 ± 0.39 μM) and with (green closed squares; EC50 = 9.34 ± 4.44 μM) 1 μM MNI137 are shown in (K). Open circles and squares indicate the G protein activation efficiency of mock-transfected cell membranes under the same ligand conditions as for the closed circles and squares, respectively. Data are means ± SEM of three to five independent experiments. *P < 0.01, #P < 0.03 (two-tailed t test) when compared with the leftmost point in each curve in (J). **P < 0.01 (two-tailed t test) with and without 1 μM MNI137 in (K).

  • Fig. 2 VB-HMM analysis of the trajectories of mGluR3 molecules.

    (A) Every step in the trajectories shown in Fig. 1B was categorized into four diffusion states. The immobile, slow, medium, and fast states are shown in blue, yellow, green, and red, respectively. (B) Histogram of the displacement during 30.5 ms of all of the trajectories (open black bars; 28,092 steps from 573 trajectories) on a cell divided into four single-step distributions of random walks (see Eq. 6 in Materials and Methods). The immobile, slow, medium, and fast states are shown in blue, yellow, green, and red, respectively. (C and D) Dose-dependent changes in the fractions of the diffusion states. (C) LY341495 dependency. (D) LY379268 dependencies in the presence of 100 nM LY314195. The immobile, slow, medium, and fast states are shown in blue, yellow, green, and red, respectively. All data are means ± SEM of 20 cells. *P < 0.01 (two-tailed t test) when compared with the leftmost point in each curve. (E) Four state transition diagrams of mGluR3 under inactive (1 μM LY314195) and active (100 μM LY379268 with 100 nM LY341495) ligand conditions. The diffusion coefficient and fraction of each state are shown next to the circles, the size of which reflects the size of the fraction. The SEM is indicated in parentheses (n = 20 cells). The arrows between states reflect the rate constants of the state transition estimated from fig. S4. The statistically significant changes in rate constants between the two conditions in fig. S4 are shown as colored arrows (blue, inactive; red, active). The asterisk indicates statistically significant differences in the fractions or in the diffusion coefficients compared with the inactive ligand conditions (*P < 0.01 by two-tailed t test).

  • Fig. 3 Effects of the PTX on the molecular behavior of mGluR3.

    (A) Schematic model of the effect of PTX on the GPCR–Gi/o protein interaction. Left: In the absence of PTX, a certain amount of GPCR is precoupled with Gi/o protein in the inactive state. The Gi/o protein is released from the GPCR upon activation after the guanosine diphosphate (GDP)/guanosine 5′-triphosphate (GTP) exchange reaction. A GPCR in the active state continuously turns over the G proteins, during which time the transient binding and release of Gi/o proteins occur repeatedly. Right: In contrast, the precoupling and turnover of Gi/o protein are inhibited by the ADP ribosylation caused by PTX. The crystal structures (PDB IDs: 4OR2, 1GP2, 1GIA, and 1PRT) were drawn with PyMOL to be representative of a GPCR, a heterotrimeric G protein, an activated Gα, and PTX, respectively. (B to D) Comparison of the MSD-Δt plots of mGluR3 trajectories in the presence or absence of PTX. The MSD-Δt plots for the basal (vehicle), inactive (100 nM LY314195), and active (100 μM LY379268) ligand conditions are shown in top panels of (B) to (D), respectively. Middle: Similar comparisons of MSD-Δt plots with or without the PTX B oligomer for the inactive and active ligand conditions. The asterisk indicates a statistically significant difference when compared with the vehicle control (P < 0.01 by two-tailed t test). Bottom: Comparison of the fractions of the diffusion states estimated from VB-HMM analysis. Results are from the same experiments as those depicted in (B) to (D) and are means ± SEM of 20 cells. *P < 0.01, #P < 0.05 (two-tailed t test) when compared with the vehicle control.

  • Fig. 4 Colocalization analysis of mGluR3 and Go protein.

    (A and B) Representative images of colocalization between TMR-labeled mGluR3 (red) and SiR-labeled Go protein (green). The mGluR3 and Go proteins formed transient complexes in both the inactive (A; 1 μM LY341495) and active (B; 100 μM LY379268) conditions. Scale bars, 1 μm. Images are representative of 20 cells. (C and D) Comparison of the MSD-Δt plots of the trajectories of mGluR3 in dual-color TIRFM movies with and without PTX. The MSD-Δt plots for the inactive (1 μM LY314195) and active (100 μM LY379268) ligand conditions are shown in (C) and (D), respectively. (E) The fraction of mGluR3 molecules that colocalized with Go proteins was estimated from the total trajectories under the inactive (blue) and active (red) ligand conditions with and without PTX. (F) Cumulative probability histograms of the colocalization duration under the inactive (blue) and active (red) ligand conditions without (filled markers) and with (empty markers) PTX. Curves were fitted using a two-component exponential function (see Eq. 8 in Materials and Methods). (G and H) Comparison of the fractions of the diffusion states estimated from VB-HMM analysis without (G) and with (H) PTX. The fractions estimated from the trajectories of total mGluR3 molecules under the inactive and active ligand conditions are indicated by the blue and red shaded bars, respectively. The fractions estimated from the trajectories of mGluR3 colocalized with Go protein under the inactive and active ligand conditions are indicated by blue and red solid bars, respectively. Data in (C) to (H) are means ± SEM of 20 cells. *P < 0.01 and #P < 0.05 by two-tailed t test.

  • Fig. 5 Colocalization analysis of mGluR3 and CLC.

    (A) Schematic of the clustering of mGluR3 in a CCP, which is followed by internalization. (B) Representative images of the colocalization of TMR-labeled mGluR3 (red) and GFP-labeled CLC (green). The mGluR3 forms a cluster during the colocalization with CLC after 0.34 s and disappears after 4.5 s. Scale bars, 1 μm. Images are representative of 18 cells. (C) Intensity changes of the particles shown in (B) are indicated by red and green arrows. A rapid increase in the TMR-labeled mGluR3 intensity coincided with its colocalization with GFP-labeled CLC. TMR and GFP intensities decreased simultaneously after 4.5 s. a.u., arbitrary units. (D) Comparison of the fractions of the diffusion states estimated from VB-HMM analysis. The fractions estimated from the trajectories of total mGluR3 under the inactive (100 nM LY314195) and active (100 μM LY379268) conditions are indicated by blue and red shaded bars, respectively. The fractions estimated from the trajectories of mGluR3 colocalized with CLC under the inactive and active conditions are indicated by blue and red solid bars, respectively. (E) The fraction of mGluR3 molecules that colocalized with CLC was estimated from the trajectories of total mGluR3 under the inactive (blue) and active (red) conditions. (F and G) Cumulative probability histograms of the colocalization duration under the inactive (blue) and active (red) ligand conditions. The curves in (F) were fitted with a two-component exponential function (see Eq. 8 in Materials and Methods) to show the time constants and fraction (inset) of colocalization in (G). Data in (D) to (G) are means ± SEM of 17 cells for the inactive ligand conditions and of 18 cells for the active ligand conditions. *P < 0.01 and #P < 0.05 by two-tailed t test.

  • Fig. 6 Effects of the RNAi-mediated knockdown of CLC on the molecular behavior of mGluR3.

    (A) Western blotting analysis of CLC abundance in HEK 293 cells transfected with mock siRNA (Lipofectamine RNAiMax transfection reagent was used without any siRNA) or CLC-specific siRNA. Top left: The bands observed in the 25- to 30-kDa range in the upper panel represent CLC. Bottom left: Western blotting analysis of β-actin, used as a loading control, in the same lysates. Western blots are representative of four experiments. Right: Comparison of normalized density of Western blots. Data are means ± SEM of four experiments. (B to D) Comparison of the MSD-Δt plots of the trajectories of mGluR3 in cells transfected with mock siRNA or CLC-specific siRNA. MSD-Δt plots are shown for the basal (vehicle) (B), inactive (1 μM LY314195) (C), and active (100 μM LY379268) (D) ligand conditions. (E to G) Comparisons of the fractions of the diffusion states estimated from VB-HMM analysis of the data shown in (B) to (D). Data are means ± SEM of 20 cells per condition. *P < 0.01, #P < 0.05 when compared to Mock as assessed by two-tailed t test.

  • Fig. 7 Schematic of mGluR3 diffusion in the plasma membrane.

    The equilibrium among the four diffusion states of mGluR3 (immobile, slow, medium, and fast) is altered upon ligand stimulation because of differences in the accessibility of mGluR3 to plasma membrane domains that depend on functional states, including G protein binding or clathrin-binding domains.

  • Table 1 Comparison of the DAv values of nine GPCRs in various phylogenetic positions with or without ligands.

    The class, group, and cluster of GPCRs are listed according to previous reports (1, 2). DAv was calculated from the MSD in fig. S8 based on Eqs. 1 and 2 in Materials and Methods. All data are means ± SEM of 20 cells. P values indicating the statistically significant difference between the vehicle and ligand conditions were calculated on the basis of Welch’s two-tailed t test. Under ligand conditions, GPCR-expressing HEK 293 cells were stimulated by the compound listed in the rightmost column. MECA is initials of GPCRs in this branch: the melanocortin receptors (MCRs), endothelial differentiation G-protein coupled receptors (EDGRs), cannabinoid receptors (CNRs), and adenosin binding receptors (ADORAs); DHA, docosahexaenoic acid; NECA, 5′-N-ethylcarboxamidoadenosine; TRAP-6, thrombin receptor activator peptide 6.

    GPCRClassGroupBranchEndogenous
    ligand
    G protein
    selectivity
    DAv (μm2/s)t test
    P value
    Compounds
    tested
    VehicleLigand
    ADRB2AαAmineAdrenalineGs0.078 ± 0.00240.051 ± 0.00231.2 × 10−9Isoproterenol (10 μM)
    HTR2AAαAmineSerotoninGq/Gi0.079 ± 0.00220.065 ± 0.0342.1 × 10−3Serotonin (10 μM)
    HRH1AαAmineHistamineGq0.064 ± 0.00230.045 ± 0.00251.1 × 10−6Histamine (1 μM)
    ADORA2AAαMECAAdenosineGs0.66 ± 0.00220.058 ± 0.00168.0 × 10−3NECA (10 μM)
    FFAR4AαMelatoninFree fatty acidGq0.083 ± 0.00250.048 ± 0.00733.5 × 10−13DHA (100 nM)
    CXCR4AγChemokineChemokineGi0.087 ± 0.00340.068 ± 0.00171.7 × 10−5CXCL12 (20 nM)
    F2RAδPurineThrombinGq/Gi/G120.084 ± 0.00410.060 ± 0.00171.4 × 10−5TRAP-6 (10 μM)
    GCGRBSecretinPeptideGlucagonGs/Gq0.075 ± 0.00280.042 ± 0.00258.7 × 10−11Glucagon (1 μM)
    mGluR3CAmino acidGlutamateGi0.47 ± 0.00220.039 ± 0.00189.1 × 10−3LY379268 (100 μM)
    0.047 ± 0.00150.064 ± 0.00181.6 × 10−8LY341495 (1 μM)

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/548/eaao1917/DC1

    Fig. S1. Evaluation of the effect of the HaloTag fusion to mGluR3.

    Fig. S2. Comparison of HaloTag and SNAP-tag ligands by single-molecule imaging.

    Fig. S3. Example of the VB-HMM analysis of mGluR3 trajectories.

    Fig. S4. Dose-dependent change in the time constants of the state transition.

    Fig. S5. Dose-dependent change in the diffusion coefficient of each diffusion state.

    Fig. S6. Correlation between mean oligomer size and receptor density under various ligand conditions.

    Fig. S7. Correlation between DAv and receptor density under various ligand conditions.

    Fig. S8. MSD-Δt plots of the trajectories of GPCRs with or without agonist.

    Fig. S9. Histograms showing displacement during 33 ms of the trajectories for mGluR3, Go protein, and CLC molecules, categorized into four diffusion states, using VB-HMM analysis.

    Movie S1. TIRFM of TMR-labeled mGluR3 molecules on a HEK 293 cell.

    Movie S2. Dual-color TIRFM of TMR-labeled mGluR3 and SiR-labeled Go protein.

    Movie S3. Accumulation of mGluR3 molecules followed by disappearance with rapid movement.

    Movie S4. Dual-color TIRFM of TMR-labeled mGluR3 and GFP-labeled CLC.

    Movie S5. TIRFM of various GPCR molecules on a HEK 293 cell with and without agonist.

  • The PDF file includes:

    • Fig. S1. Evaluation of the effect of the HaloTag fusion to mGluR3.
    • Fig. S2. Comparison of HaloTag and SNAP-tag ligands by single-molecule imaging.
    • Fig. S3. Example of the VB-HMM analysis of mGluR3 trajectories.
    • Fig. S4. Dose-dependent change in the time constants of the state transition.
    • Fig. S5. Dose-dependent change in the diffusion coefficient of each diffusion state.
    • Fig. S6. Correlation between mean oligomer size and receptor density under various ligand conditions.
    • Fig. S7. Correlation between DAv and receptor density under various ligand conditions.
    • Fig. S8. MSD-Δt plots of the trajectories of GPCRs with or without agonist.
    • Fig. S9. Histograms showing displacement during 33 ms of the trajectories for mGluR3, Go protein, and CLC molecules, categorized into four diffusion states, using VB-HMM analysis.
    • Legends for Movies S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). TIRFM of TMR-labeled mGluR3 molecules on a HEK 293 cell.
    • Movie S2 (.mp4 format). Dual-color TIRFM of TMR-labeled mGluR3 and SiR-labeled Go protein.
    • Movie S3 (.mp4 format). Accumulation of mGluR3 molecules followed by disappearance with rapid movement.
    • Movie S4 (.mp4 format). Dual-color TIRFM of TMR-labeled mGluR3 and GFP-labeled CLC.
    • Movie S5 (.mp4 format). TIRFM of various GPCR molecules on a HEK 293 cell with and without agonist.

Navigate This Article