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Front Pharmacol
2023 Jan 01;14:1087171. doi: 10.3389/fphar.2023.1087171.
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Dopamine-induced arrestin recruitment and desensitization of the dopamine D4 receptor is regulated by G protein-coupled receptor kinase-2.
Burström V
,
Ågren R
,
Betari N
,
Valle-León M
,
Garro-Martínez E
,
Ciruela F
,
Sahlholm K
.
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The dopamine D4 receptor (D4R) is expressed in the retina, prefrontal cortex, and autonomic nervous system and has been implicated in attention deficit hyperactivity disorder (ADHD), substance use disorders, and erectile dysfunction. D4R has also been investigated as a target for antipsychotics due to its high affinity for clozapine. As opposed to the closely related dopamine D2 receptor (D2R), dopamine-induced arrestin recruitment and desensitization at the D4R have not been studied in detail. Indeed, some earlier investigations could not detect arrestin recruitment and desensitization of this receptor upon its activation by agonist. Here, we used a novel nanoluciferase complementation assay to study dopamine-induced recruitment of β-arrestin2 (βarr2; also known as arrestin3) and G protein-coupled receptor kinase-2 (GRK2) to the D4R in HEK293T cells. We also studied desensitization of D4R-evoked G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) current responses in Xenopus oocytes. Furthermore, the effect of coexpression of GRK2 on βarr2 recruitment and GIRK response desensitization was examined. The results suggest that coexpression of GRK2 enhanced the potency of dopamine to induce βarr2 recruitment to the D4R and accelerated the rate of desensitization of D4R-evoked GIRK responses. The present study reveals new details about the regulation of arrestin recruitment to the D4R and thus increases our understanding of the signaling and desensitization of this receptor.
FIGURE 1. Arrestin recruitment to the D4R as measured by a modified NanoBiT complementation bioluminescence assay. (A) A dopamine concentration-dependent increase in luminescence was observed in cells co-transfected with D4R-NP and LgBiT-βarr2, but not with either construct alone. (B) Kinetic experiments revealed a blockade of the dopamine-induced luminescence increase upon application of the D4R antagonist clozapine. (C) Dopamine concentration-response curve for the increase in luminescence in cells co-transfected with D2R-NP and LgBiT-βarr2, or with negative control (D2R-NP expressed alone). (D) Kinetic experiments revealed a blockade of the dopamine-induced luminescence increase upon application of the D2R antagonist raclopride. Asterisks and horizontal bars indicate intervals of statistically significant differences between HBSS control conditions and clozapine or raclopride. ***; p < 0.001, repeated measures ANOVA with Sidak’s multiple comparisons test. Luminescence data are expressed as fractions of the signal observed in vehicle-treated wells (A, C) or prior to dopamine application (B, D). Each data point in (A–D) represents mean ± SEM of eight replicate wells from one representative experiment performed three to five times. SEM bands are shown in (B, D). For some data points, SEM bars or bands are obscured by the symbols representing the means.
FIGURE 2. Assessment of GRK2 recruitment to the D4R and the D2R. (A) Concentration-response relationships for dopamine-induce luminescence increase in cells co-expressing D4R-NP with GRK2-LgBiT, or either construct expressed alone. Measurements were taken 3 min after the addition of dopamine. (B) Kinetic experiments revealed a transient increase in dopamine-induced luminescence in cells co-expressing D4R-NP with GRK2-LgBiT. (C) Concentration-response relationships for dopamine-induce luminescence increase in cells co-expressing D2R-NP with GRK2-LgBiT. Measurements were taken 3 min after the addition of dopamine. (D) Kinetic experiments showing the time course of the dopamine-induced luminescence increase in cells co-expressing D2R-NP with GRK2-LgBiT and the blockade of the response to dopamine upon application of the D2R antagonist raclopride. Asterisks and horizontal bar indicate an interval of statistically significant differences between the raclopride and HBSS control conditions. ***; p < 0.001, repeated measures ANOVA with Sidak’s multiple comparisons test. Luminescence data are expressed as fractions of the signal observed in vehicle-treated wells (A, C), or prior to dopamine application (B, D). Data points represent mean ± SEM from eight replicate wells from one representative example out of three independent experiments. SEM bands are shown in (B, D). For some data points, SEM bars or bands are obscured by the symbols representing the means.
FIGURE 3. βarr2-induced desensitization of D4R in Xenopus oocytes. Oocytes were injected with D4R cRNA together with RGS4 and GIRK1/4 channel subunit cRNAs, in addition to variable amounts of βarr2 cRNA, as indicated. (A) Peak-normalized and averaged traces of GIRK currents evoked by 1 μM dopamine in oocytes co-injected with varying amounts of βarr2 cRNA. (B) D4R desensitization rate expressed as residual GIRK response (fraction of initial peak response) after 415 s application of 1 µM dopamine. Data from the experiments shown in (A) Asterisks denote statistically significant effects of GRK2 coexpression vs control. ***; p < 0.001, **; p = 0.004, n.s.; not significant, one-way ANOVA with Dunnett’s test for multiple comparisons. (C) Peak amplitudes of GIRK current responses shown in (A) Data points represent mean ± SEM from three to nine oocytes, as indicated. Superimposed symbols in bar graphs represent individual data points. No statistically significant effects of GRK2 coexpression on current amplitude were observed (Kruskal–Wallis test with Dunn’s correction for multiple comparisons).
FIGURE 4. Impact of GRK2 co-expression on arrestin recruitment to the D4R and on desensitization of D4R-evoked GIRK responses. (A) Concentration-dependence of dopamine-induced luminescence increase in cells co-expressing LgBiT- βarr2 and D4R-NP, with and without exogenous GRK2 expression. Each data point represents mean ± SEM of data from eight replicate wells. Curves shown from one representative experiment out of a total of four. Data from all experiments are shown in (B, C). (B) Co-transfection of 10 µg of GRK2 significantly decreased the dopamine EC50 in the βarr2 recruitment assay by about 3-fold. *; p = 0.026, Student’s paired t-test. (C) Co-transfection of 10 µg of GRK2 did not significantly affect the Top parameter. ns; not significant, p = 0.125, Wilcoxon matched-pairs signed rank test. (D) Concentration-dependence of dopamine-induced luminescence increase in cells co-expressing LgBiT- βarr2 and D2R-NP, with and without exogenous GRK2 expression. Each data point represents mean ± SEM of data from eight replicate wells. Curves shown from one representative experiment out of a total of four. Data from all experiments are shown in (E, F). (E) Co-transfection of 10 µg of GRK2 significantly decreased the dopamine EC50 in the βarr2 recruitment assay by about 10-fold. **; p = 0.002, Student’s paired t-test. (F) Co-transfection of 10 µg of GRK2 significantly decreased the Top parameter. **; p = 0.003, Student’s paired t-test. (G) Peak-normalized and averaged traces of GIRK currents evoked by 1 μM dopamine in oocytes co-injected with D4R, RGS4, GIRK1/4, 1.9 ng βarr2, and 0 or 0.3 ng GRK2 cRNAs, as indicated. Data points represent mean ± SEM from six or seven oocytes. (H) Residual GIRK responses (fraction of initial peak response) after 415 s of dopamine application, representing the rate of desensitization under the conditions shown in (G). Co-expression of GRK2 significantly decreased the residual response amplitude. **; p = 0.005, Mann-Whitney test. (I) Peak currents for the conditions shown in (G). Superimposed symbols in bar graphs represent data from individual oocytes. No statistically significant effects of GRK2 coexpression on current amplitude were observed (ns; not significant, p = 0.090, Student’s unpaired t-test). For some data points in (A, D), SEM bars are obscured by the symbols representing the means.
Supplementary Figure 1. Surface expression, as determined by anti-FLAG whole-cell ELISA, of (A) D4R-NP and (B) D2R-NP in HEK 293T cells cotransfected with either receptor construct
and LgBit-βarr2 with or without exogenous GRK2. ns; not significant, Student’s paired t-test.
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