Functional crosstalk and heteromerization of serotonin 5-HT2A and dopamine D2 receptors

Abstract

The serotonin 5-HT(2A) receptor (5-HT(2A)R) and dopamine D(2) receptor (D(2)R) are high-affinity G protein-coupled receptor targets for two different classes of antipsychotic drugs used to treat schizophrenia. Interestingly, the antipsychotic effects are not based on the regulation of same signaling mediators since activation of the 5-HT(2A)R and of the D(2)R regulate G(q/11) protein and G(i/o) protein, respectively. Here we use radioligand binding and second messenger production assays to provide evidence for a functional crosstalk between 5-HT(2A)R and D(2)R in brain and in HEK293 cells. D(2)R activation increases the hallucinogenic agonist affinity for 5-HT(2A)R and decreases the 5-HT(2A)R induced inositol phosphate production. In vivo, 5-HT(2A)R expression is necessary for the full effects of D(2)R antagonist on MK-801-induced locomotor activity. Co-immunoprecipitation studies show that the two receptors can physically interact in HEK293 cells and raise the possibility that a receptor heterocomplex mediates the crosstalk observed. The existence of this 5-HT(2A)R-D(2)R heteromer and crosstalk may have implications for diseases involving alterations of serotonin and dopamine systems and for the development of new classes of therapeutic drugs.

Fig. 1

5-HT_2AR radioligand competitive curves performed in HEK293 cells and mouse striatum. A, In HEK293 cells co-expressing 5-HT_2AR and D₂R, 5-HT_2AR agonist DOI affinity is higher in the presence of D₂R agonist quinpirole (10⁻⁵ M) (two-site model; K_i-high = 1.1 × 10⁻¹¹ M and K_i-low = 2.8 × 10⁻⁸ M) (○) from the control (one-site model; K_i = 1.7 × 10⁻⁸ M) (■). B, In HEK293 cells expressing only 5-HT_2AR, DOI affinity does not change in the presence of quinpirole (10⁻⁵ M) (two-site model; K_i-high = 2.9 × 10⁻¹⁰ M, K_i-low = 3.5 × 10⁻⁸ M) (○) from the control (two-site model; K_i-high = 5.8 × 10⁻¹⁰ M, K_i-low = 4.2 × 10⁻⁸ M) (■). C, In mouse striatum, DOI affinity is significantly higher in the presence of quinpirole (10⁻⁵ M) (two-site model; K_i-high = 1.5 × 10⁻¹¹ M and K_i-low = 4.7 × 10⁻⁷ M) (○) from the control (one-site model; K_i = 9.5 × 10⁻⁸ M) (■). D, In mouse striatum, the increased DOI affinity in the presence of quinpirole (10⁻⁵ M) (two-site model; K_i-high = 8.5 × 10⁻¹¹ M and K_i-low = 6.9 × 10⁻⁸ M) (○) from the control (one-site model; K_i = 3.3 × 10⁻⁶ M) (■) is decreased when D₂R antagonist raclopride is added (one-site model; K_i = 2.7 × 10⁻⁷ M) (●). Data are means ± SEM for each experiment performed in triplicate. The K_i values shown are for this single experiment. Averages of K_i values are indicated in Table 1.

Fig. 2

Inositol Phosphate (IP) production induced by 5-HT_2AR activation with natural agonist 5-HT or hallucinogenic drug DOI. A, Serotonin (5-HT)-stimulated 5-HT_2AR in HEK293 cells expressing 5-HT_2AR (△) and HEK293 cells co-expressing 5-HT_2AR and D₂R (■). D₂R expression does not affect 5-HT-induced 5-HT_2AR-IP₃ production (EC₅₀ = 5.5 × 10⁻⁷ M). B, DOI-stimulated 5-HT_2AR in HEK293 cells expressing 5-HT_2AR (△) (EC₅₀ = 3.8 × 10⁻⁶ M) and HEK293 cells co-expressing 5-HT_2AR and D₂R (■) (EC₅₀ = 6.7 × 10⁻⁸ M). D₂R expression increases DOI-induced 5-HT_2AR-IP₃ production. C, D₂R agonists (10⁻⁵ M), quinpirole (○) (EC₅₀ = 6.6 × 10⁻⁷ M) and ropinirole (◇) (EC₅₀ = 8 × 10⁻⁷ M), have no effect on 5-HT-induced 5-HT_2AR-IP₃ production (vehicle; EC₅₀ = 5.5 × 10⁻⁷ M). D, D₂R agonists (10⁻⁵ M), quinpirole (○) (EC₅₀ = 3.9 × 10⁻⁷ M) and ropinirole (◇) (EC₅₀ = 2.4 × 10⁻⁷ M) decrease DOI-induced 5-HT_2AR-IP₃ production (vehicle; EC₅₀ = 6.7 × 10⁻⁸ M). Data are means ± SEM for each experiment performed in triplicate. The EC₅₀ values shown are for this single experiment. Averages of EC₅₀ values over all experiments can be found in Table 2.

Fig. 3

5-HT_2AR is necessary to induce D₂R ligand dependent behavioral effects in mice. Expression of 5-HT_2AR is necessary for the inhibition of MK-801-induced locomotor activity by D₂R antagonist haloperidol. A, The time course of MK-801-induced locomotion is measured in 5 min blocks. Time of injections of MK-801 is indicated by arrows. Horizontal activity is measured in wild-type (WT) and 5-HT_2AR-KO mice. B, Corresponding bar graph of the total MK-801-induced locomotion as a summation of horizontal activity from t = 10 min to t = 120 min. Mice were administered haloperidol (1 mg/kg) or vehicle followed by MK-801 (0.5 mg/kg; i.p.) (n = 6; **p < 0.01; error bars shows SEM).

Fig. 4

5-HT_2AR and D₂R co-immunoprecipitate. A, Specific co-immunoprecipitation of 5-HT_2AR and D₂R in HEK293 cells. RLuc-tagged D₂R and GFP-tagged 5-HT_2AR were immunoprecipitated using anti-GFP or RLuc antibodies. Membranes were probed with either anti-RLuc antibody or anti-N-Cadherin antibody. The co-immunoprecipitated receptor complex is indicated by the box in the upper right panel. B, RLuc-tagged D₂R and GFP-tagged 5-HT_2AR, mGluR2 or mGluR3 were immunoprecipitated using anti-RLuc antibody and membranes were probed with anti-GFP antibody. A co-immunoprecipitated receptor is detected only for D₂R with 5-HT_2AR. C, RLuc-tagged D₂R and GFP-tagged D₂R, 5-HT_2AR, mGluR2 or mGluR3 were immunoprecipitated using anti-GFP antibody and membranes were probed with anti-RLuc antibody. Complex formation was observed only for D₂R homomers and D₂R-5-HT_2AR heteromers. Panels are representative of three independent experiments. WB, western blot. IP, immunoprecipitation.

Fig. 5

5-HT_2AR-D₂R heteromer crosstalk hypothesis: effects of D₂R expression and activation on 5-HT_2AR binding and signaling properties. On the left, serotonin 5-HT_2AR is illustrated as binding its agonist DOI and being coupled to G_q/11 protein. In the middle, dopamine D₂R expression might have an allosteric effect (illustrated with a white arrow) on 5-HT_2AR by decreasing DOI agonist affinity to 5-HT_2AR and increasing 5-HT_2AR coupling to G_q/11 protein. In contrast, on the right, D₂R activation (induced by its agonist quinpirole) leads to an increased affinity of 5-HT_2AR for DOI and a decreased G_q/11 protein coupling to 5-HT_2AR.