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. 2016 Sep 12:6:33233.
doi: 10.1038/srep33233.

Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

Alina Tabor  1 , Siegfried Weisenburger  2 , Ashutosh Banerjee  1 , Nirupam Purkayastha  1 , Jonas M Kaindl  1 , Harald Hübner  1 , Luxi Wei  2 , Teja W Grömer  3 , Johannes Kornhuber  3 , Nuska Tschammer  1 , Nigel J M Birdsall  4 , Gregory I Mashanov  4 , Vahid Sandoghdar  2 , Peter Gmeiner  1
Affiliations

Visualization and ligand-induced modulation of dopamine receptor dimerization at the single molecule level

Alina Tabor et al. Sci Rep. .

Abstract

G protein-coupled receptors (GPCRs), including dopamine receptors, represent a group of important pharmacological targets. An increased formation of dopamine receptor D2 homodimers has been suggested to be associated with the pathophysiology of schizophrenia. Selective labeling and ligand-induced modulation of dimerization may therefore allow the investigation of the pathophysiological role of these dimers. Using TIRF microscopy at the single molecule level, transient formation of homodimers of dopamine receptors in the membrane of stably transfected CHO cells has been observed. The equilibrium between dimers and monomers was modulated by the binding of ligands; whereas antagonists showed a ratio that was identical to that of unliganded receptors, agonist-bound D2 receptor-ligand complexes resulted in an increase in dimerization. Addition of bivalent D2 receptor ligands also resulted in a large increase in D2 receptor dimers. A physical interaction between the protomers was confirmed using high resolution cryogenic localization microscopy, with ca. 9 nm between the centers of mass.

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Figures

Figure 1
Figure 1. Visualization, tracking and analysis of the dimerization of single SNAP-tagged D2L receptors using SNAP-CD86 and SNAP-CD28 as monomeric and dimeric reference proteins.
(a,g,j) Schematic representation of the SNAP-tagged constructs. (b,h,k) Representative images of single CHO cells, stably transfected with the corresponding labeled protein and visualized by TIRF-M. Scale bar, 10 μm. The first 100 frames of the cell in b are shown in Supplementary Movie S1. Inserts correspond to higher magnification images of the areas in the white boxes. (c,i,l) Representative intensity distributions of fluorescent spots identified over the first 10-frame time window of TIRF illumination of CHO cells, stably transfected with the corresponding construct and labeled with Alexa546-BG. Number of identified particles, n = 5770 (c), 6252 (f) and 6458 (i). Data were fitted with a mixed Gaussian model. A mixed Gaussian fit after partial photobleaching (dotted line) was used to estimate the intensity of a single fluorescent molecules in each image sequence. (d) Individual trajectories of moving SNAP-D2L receptors were identified from the entire recording of the cell shown in (b). The insert shows a higher magnification that illustrates the random nature of the diffusive process. (e) Representative plot of the average mean square displacement (MSD) (mean ± s.d.) versus the time interval (δt) for the trajectories shown in (a). The plot is linear (r2 = 0.99 – linear fit (blue)), over a 3-s timescale, which is consistent with receptor movement following a random walk, and it shows no evidence for anomalous diffusive behavior. (f) Distribution of the diffusion coefficients of the receptor particles tracked in (d).
Figure 2
Figure 2. Dependence of the distribution of monomers and dimers on receptor density.
(a) Representative receptor density level: + 0.38 ± 0.016 spots μm−2 (mean ± s.d., 8 cells), ++ 0.52 ± 0.039 spots μm−2 (mean ± s.d., 10 cells), +++ 0.67 ± 0.031 spots μm−2 (mean ± s.d., 10 cells), ++++ 0.79 ± 0.046 spots μm2 (mean ± s.d., 10 cells). (b) The monomer/dimer ratios (mean ± s.d.) were calculated from the fitted fluorescence intensity distributions of fluorescent ligand receptor complexes using a mixed Gaussian model (compare to Fig. 1c). The statistical significance of the differences in monomer levels in the four groups was determined by an unpaired t-test (*p-value < 0.05, ***p-value < 0.010, ****p-value < 0.0001).
Figure 3
Figure 3. Transient dimer formation of SNAP-D2L receptors.
(a) 48 sequential frames of two Alexa546-labeled SNAP-D2L receptors showing transient dimer formation (frame rate of 19.32 fps) (also shown in Supplementary Movie S3). (b) Intensity profile (blue) of the marked fluorescent SNAP-D2L receptor shown in (a), compared to background intensity (grey). (c) A histogram of the lifetimes of 120 SNAP-D2L receptor dimers taken from trajectories similar to those in (a) and collected in 0.5 s bins. The solid line represents a one-phase exponential fit for a mean lifetime of 0.50 s (95% confidence interval: 0.44–0.60). (d) Effect of the size of SNAP-D2L receptors on their lateral diffusion. The diffusion coefficients (Dlat) of the analyzed receptor particles (n) are shown (monomers −0.104 ± 0.052 μm2 s−1, n = 412 from 3 cells and dimers −0.075 ± 0.027 μm2 s−1, n = 373 from 3 cells. Data represent mean ± s.d. The difference, determined by an unpaired t-test (****p-value < 0.0001) is significant and shows that the receptor mobility is negatively correlated with the size of the receptor complexes.
Figure 4
Figure 4. Visualization, diffusion and dimerization of Alexa546-labeled SNAP-D2L receptors and on membrane protrusions using HF-treated slides.
(a) Representative images of a single CHO cell stably expressing the SNAP-D2L receptor and seeded on HF-treated glass slides, labeled with Alexa546-BG and visualized by TIRF-M. The insert corresponds to higher magnification image of the area in the small white box. (b) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in a. The plot is linear (r2 = 0.99 – linear fit (blue)), over a 3-s timescale, which is consistent with receptor movement following a random walk. (c) The distribution of the diffusion coefficients of the analyzed receptor particles (n) are shown (n = 4409, 5 cells - HF-treated slide; n = 4409, 8 cells - non HF-treated slide) and revealed no evidence for anomalous diffusive behavior as a result of HF-treatment. (d) Representative intensity distribution of fluorescent spots identified over the first 10-frame time window of TIRF-illumination. Data were fitted with a mixed Gaussian model (sum of two Gaussian functions). (e) Representative TIRF-M images of CHO cells stably transfected with SNAP-D2L receptor, incubated in iso-osmotic (300 mOsm) (left) and hypo-osmotic PBS (108 mOsm) (right) for 2 h and labeled with Alexa546-BG. (f,g) Imaging of a region of membrane protrusions of CHO cells stably transfected with the SNAP-D2L receptor, incubated in hypo-osmotic (108 mOsm) PBS for 2 h and labeled with Alexa546-BG in epi-illumination (f) and TIRF-illumination (g). (h) Representative images of one membrane protrusion of a CHO cell stably expressing the labeled SNAP-D2L receptor and visualized by TIRF-M. (f) Plot of mean square displacement (MSD ± s.d.) versus the time interval (δt) of receptor particles that were tracked in (h). The plot is linear (r2 = 0.99 – linear fit (blue)), over a 2,5-s time scale and the calculation of the average diffusion coefficient Dlat of 0.077 ± 0.007 μm2 s−1 (mean ± s.d., 16 regions of membrane protrusions of 8 cells) revealed no evidence for anomalous diffusive behavior in the membrane protrusions. Scale bars, 10 μm.
Figure 5
Figure 5. Cryogenic localization microscopy of SNAP-D2L receptor dimers.
(a) Averaged wide-field dataset of a recording of a CHO cell stably expressing labeled SNAP-D2L. (b) Super-resolution reconstruction after 3B analysis of the area indicated by the white square in (a). (c) Histogram of the pairwise distances calculated from the emitter positions of the SNAP-D2L (blue) and SNAP-CD86 monomers (gray). The Gaussian fit determines the separation of the SNAP-D2L protomers to be μ = 9.1 ± 11.3 nm. (d) Schematic representation of an Alexa564-labeled SNAP-D2L receptor dimer detected by cryogenic localization microscopy.
Figure 6
Figure 6. Influence of monovalent (1a,b), bivalent (2a,b) and bivalent control (3a,b) dopamine D2 receptor antagonists on receptor dimerization.
(a) Chemical structures of monovalent ligands (1a–c), bivalent ligands (2a–c), and control ligands (3a,b). (b) Monomer/dimer ratios calculated from fitted fluorescence intensity distributions of Alexa546-labeled SNAP-D2L receptors incubated with monovalent (1a,b), bivalent (2a,b), control (3a,b) ligands using a mixed Gaussian model (Supplementary Table S2, Supplementary Fig. S6a). Data represent mean ± s.d. of n analysed cells (n = 16 for 1a, 8 for 1b, 8 for 2a, 16 for 2b, 6 for 3a and 8 for 3b. (c) Average diffusion coefficients (Dlat) of the corresponding ligand-SNAP-D2L receptor complexes of the same analyzed cells in (b). Data in (b,c) represent mean ± s.d., Statistical analysis was performed by an unpaired t-test (**p-value < 0.01, ****p-value < 0.0001) and showed that the receptor mobility is negatively correlated with the size of the receptor complexes. (d–g) Comparison of the apparent lifetimes of particle colocalization of the monomeric SNAP-CD86 and dimeric SNAP-CD28 control proteins proteins (e and f respectively) and the SNAP-D2L receptor in the absence and presence of the monovalent ligand 1a or bivalent ligand 2a (g, h, and f respectively). (d) Representative intensity profile of one trajectory which showed intensity doubling from the beginning of the particle tracking followed by one step intensity change which was used to calculate the colocalization time of two particles. (e–i) The apparent lifetime of particle colocalizations (τ; 95 confidence interval) was calculated by fitting colocalization time data with a one-phase exponential decay function. 120 trajectories like those shown in a were analyzed from 8 different cells in (e), 4 in (f), 6 in (g), 6 in (h) and 4 in (i), respectively.
Figure 7
Figure 7. Visualization, tracking and analysis of the dimerization of single SNAP-tagged D2L receptors and wild-type D2L receptors labeled with the fluorescent antagonist 1c.
(a,d) Schematic representation of the constructs. (b,e) Representative images of single CHO cells stably transfected with the two constructs, labeled and visualized by TIRF-M. Spot densities were 0.69 spots μm−2 (b) and 0.64 spots μm−2 (e). Inserts correspond to higher magnification images of the areas in the white boxes. Scale bar, 10 μm. (c,f) Representative intensity distributions of fluorescent spots identified over the first 10-frame time window of TIRF illumination of CHO cells, stably transfected with the corresponding constructs and labeled with the fluorescent ligand 1c (300 nM). Data were fitted with a mixed Gaussian model. A mixed Gaussian fit after partial photobleaching (dotted line) was used to estimate precisely the intensity of a single fluorescent spot in each image sequence.
Figure 8
Figure 8. Influence of antagonists (4a,b) and agonists (5a,b) on D2L, D2S and D3 receptor dimerization.
(a) Chemical structures of monovalent fluorescent antagonists (4a,b) and agonists (5a,b). (b) Representative images a single CHO cell stably expressing the D2L receptor, labeled with 4a (38 nM), visualized by TIRF-M. Scale bar, 10 μm. Insert correspond to a higher magnification image of the area in the white box. (c) Plot of average mean square displacement (MSD ± s.d.) versus the time interval (δt) of the receptor ligand complexes that were tracked (r2 = 0.99 – linear fit (blue)). (d) Intensity distribution of fluorescent spots identified over the first 10-frame time window of ligand receptor complex D2L-4a. (e,f) Monomer/dimer ratios of D2L, D2S and D3 receptors labeled with the fluorescent antagonists 4a,b and agonists 5a,b, calculated from fitted fluorescence intensity distributions of fluorescent ligand receptor complexes using a mixed Gaussian model (compare to (c) and Supplementary Table S5). (g) Average diffusion coefficients (Dlat) of the receptor complexes of the same cells analyzed in (e,f). Data in (e–g) represent mean ± s.d. n analysed cells (D2L: n = 18 for 4a, 19 for 5a, 14 for 4b and 17 for 5b, D2S: 13 for 4a, 12 for 5a, 19 for 4b and 12 for 5b, D3: 12 for 4a, 13 for 5a, 15 for 4b and 10 for 5b). (e) Representative effects of the size of D2L receptor-ligand complexes on their rates of lateral diffusion. The diffusion coefficient of D2L-4a monomers (0.116 ± 0.030 μm2 s−1, n = 838 from 3 cells) is greater than that of the dimers (0.087 ± 0.037 μm2 s−1, n = 185 from 3 cells). This is also found for D2L-5a monomers 0.134 ± 0.023 μm2 s−1 (n = 1396 from 3 cells) and dimers 0.106 ± 0.027 μm2 s−1 (n = 558 from 3 cells). Data represent means ± s.d. Statistical analysis was performed by an unpaired t-test (**p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001, n.s. - not significant).
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