Dopamine D2 receptors form higher order oligomers at physiological expression levels

Abstract

G-protein-coupled receptors are generally thought to be organized as dimers; whether they form higher order oligomers is a topic of much controversy. We combined bioluminescence/fluorescence complementation and energy transfer to demonstrate that at least four dopamine D2 receptors are located in close molecular proximity in living mammalian cells, consistent with their organization as higher order oligomers at the plasma membrane. This implies the existence of multiple receptor interfaces. In addition to the symmetrical interface in the fourth transmembrane segment (TM4) we identified previously by cysteine (Cys) crosslinking, we now show that a patch of residues at the extracellular end of TM1 forms a second symmetrical interface. Crosslinking of D2 receptor with Cys substituted simultaneously into both TM1 and TM4 led to higher order species, consistent with our novel biophysical results. Remarkably, the rate and extent of crosslinking at both interfaces were unaltered over a 100-fold range of receptor expression. Thus, at physiological levels of expression, the receptor is organized in the plasma membrane into a higher order oligomeric structure.

Figure 1

D2R ‘dimerization' as assessed by protein complementation. HEK 293T cells transiently coexpressing D2R split Venus (A) or RLuc8 (C) or D2R and the corresponding TSHr splits (B, D) were harvested 48 h post-transfection, washed with PBS, centrifuged and resuspended in PBS. Fluorescence was recorded for 1 s using 500 nm excitation and 540 nm emission filters (Polarstar, BMG). Unfiltered luminescence was recorded for 1 s (Gain 3900). Background was determined with cells expressing only one of the receptor probes and the signal to background ratio was plotted against the FACS ratio (A–D). For the FACS ratio, cells transfected in parallel were labelled with primary and secondary antibodies (Abs) as described previously (Costagliola et al, 1998) and in the Methods. Relative staining for each receptor was determined independently in the same cells with the same secondary anti-mouse Ab to determine the FACS ratio. Representative results from at least three independent experiments are shown. Inset in (A) illustrates HTRF experiments performed in cells expressing identical amounts of D2–V1 and D2–V2 as compared with SF–D2 and SM–D2, respectively.

Figure 2

Biophysical evidence of higher order oligomerization. (A) Two-protomer BRET: increasing amounts of D2–Venus were coexpressed with constant amounts of either D2–RLuc8 or TSHr–RLuc8 in HEK 293T cells. At 48 h post-transfection, BRET was performed and the BRET signals were plotted against the relative expression levels of each tagged receptor. Results were analysed by nonlinear regression assuming a model with one site binding (GraphPad Prism 4.0) on a pooled data set from four independent experiments. (B) BRET was performed as described above in cells coexpressing constant amounts of D2–RLuc8 and increasing amounts of D2–Venus in the absence (▪ and solid line), or presence of three different class A GPCRs (D2R: □ and dashed line; TSHr: • and dotted line; CXCR4: × and dotted line). Cell surface expression of each untagged receptor used as a competitor was monitored by FACS (not shown). As above, pooled BRET signals from three independent experiments were plotted against relative expression ratios assuming a one-binding site model. (C) BRET assay was performed in cells coexpressing constant D2–RLuc8 and increasing D2–Venus with increasing amounts of untagged D2R as described above. The increasing level of surface expression of the competing D2R and surface expression of TSH and CXCR4 was confirmed by FACS (data not shown). A representative experiment performed three times is shown. (D and inset) Three-protomer BRET: cells coexpressing constant amounts of D2–L1 and D2–L2 as a BRET donor (complemented RLuc8) and increasing amounts of either D2–Venus or TSHr–Venus as the acceptors were treated and analysed identically as described in (A). Data from a pooled data set from four independent experiments are shown. (E) Three-protomer BRET: cells coexpressing increasing amounts of D2–V1 and D2–V2 as the BRET acceptor (complemented mVenus) and either D2–RLuc8 or TSHr–RLuc8 as donors were treated and analysed identically as described in (A). Data from a pooled data set from four independent experiments are shown. (F and inset) Four-protomer BRET: Cells coexpressing increasing amounts of D2–V1 and D2–V2 as the BRET acceptor (complemented mVenus) and constant amounts of either D2–L1 and D2–L2 or TSHr–L1 and TSHr–L2 as donors (complemented RLuc8) were treated and analysed identically as described in (A). Data from a pooled data set from four independent experiments are shown. Cartoons represent different receptor species: D2 depicted as a 7TM alone and TSHr as the 7TM with the large extracellular domain. mVenus and the splits are represented as elliptical and RLuc8 and the splits as rectangular. In the legends, + separates donor (first) from the acceptor (second) and _ indicates the complemented pairs.

Figure 3

TM1 forms a symmetrical ‘dimerization' interface in D2R. (A, B) Intact cells stably expressing substituted Cys mutants in TM1 from P32^1.30C to R61^1.59C (denoted using the indexing system described in Methods) (except for non-expressed N52^1.50C and the endogenous C56^1.54, which is present in the background construct and all the mutants) were treated with 1 mM CuP in a 1:2 molar ratio at 25°C for 10 min, washed with PBS buffer, treated with NEM and analysed by immunoblotting. (C–J) Crosslinking was performed as described above with increasing concentrations of CuP for the four Cys mutants of interest Y36^1.34C (C, D), Y37^1.35C (E, F), L40^1.38C (G, H) and L43^1.41C (I, J). The dimer fraction is plotted against CuP concentration to determine the apparent crosslinking rates and efficiency. (K, L). Intact cells were treated with 1 mM CuP for Y36^1.34C, L40^1.38C and L43^1.41C and 5 μM for Y37^1.35C in the absence or presence of 10 μM sulpiride or 10 μM quinpirole. Quantification of crosslinking fractions was performed as described in Methods. All experiments were repeated ⩾3 times, and one representative experiment is shown.

Figure 4

Molecular model of the TM1 interface and of a D2R oligomeric arrangement. Ribbon representations of (A) vertical, (B) extracellular and (C) cytoplasmic views of the proposed TM1–TM1 interacting regions of the D2R homodimer. The TM1 and H8 helices at the interface are highlighted by thicker traces. The Cβ atoms of residues that crosslink when mutated to Cys are shown in CPK representations in different colours. (D) Vertical and (E) extracellular views of the best fit between rhodopsin-based models of the D2R protomers (only TM1 helices are shown; orange and yellow colours) and the proposed B2AR-based model of the TM1–TM1 homodimer of D2R (cyan and grey colours). (F) Schematic representation of a possible D2R oligomeric organization based on inferences from our crosslinking studies (see Methods). Eight protomers are shown. The four protomers with red in the interior can be crosslinked in the ^1.35C/^4.58C construct. The four protomers contained in the yellow contour can be crosslinked in the ^1.35C/^5.41C construct (see Discussion).

Figure 5

TM1 and TM4 contribute to symmetrical interfaces in higher order D2R complexes. (A) Intact cells stably expressing a D2R TM1–TM4 double Cys mutant with both TM1 Y37^1.35C and the endogenous TM4 C168^4.58 were treated with increasing concentrations of CuP as described in Figure 3. A representative blot is shown with the different species as determined by the mass molecular weight standards. (B) Fractions of the different species at the indicated CuP concentration from three independent experiments (mean±s.d.). (C) Intact cells stably expressing F437^7.64C, L438^7.65C or K439^7.66C in H8 and C168^4.58 or C168^4.58S (as positive and negative control, respectively) were treated with 20 μM HgCl₂ at 25°C for 10 min and were prepared and immunoblotted as described in Figure 3. All the experiments were performed ⩾3 times, and a representative experiment is shown.

Figure 6

D2R crosslinking at physiologically relevant expression levels. HEK 293 T-rex cells were induced with 1 μg/ml tetracycline for the indicated times and Cys crosslinking was performed in cells expressing a substituted Cys at position 4.58 in TM4 (A–C), 1.35 in TM1 (D–F), or and both 1.35 and 4.58 (G–I). The experiments were performed as described for Figure 3. The fraction of immunoreactivity crosslinked to dimer (C, F) or to the various higher order species (I) was unchanged over the range of induction times, as shown by linear regression fitting. 4.58C (J–M) and 1.35C (N–Q) were induced for 4 h (K, O) or 21 h (L, P) and the cells were treated with increasing concentrations of CuP. Nonlinear regression analysis assuming a one-phase association (Prism 4.0) of the dimer fraction against [CuP] of three independent experiments (M, Q) is shown. Representative blots, in each case from three independent experiments, are shown.