Abstract
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.
1 Introduction
Dopamine is involved in the regulation of selective attention, memory, and learning, reward-driven behavior, and voluntary movement (; ). Dopamine receptors are G protein-coupled receptors (GPCRs) and come in five different subtypes, with D1-like receptors (D1R and D5R) coupling to stimulatory (at the level of adenylate cyclase modulation) Gαs/olf proteins and D2-like receptors (D2R, D3R, and D4R) coupling to inhibitory Gαi/o proteins. The D4R is encoded by the DRD4 gene, which is expressed most abundantly in the retina, with moderate expression in brain tissue (; ). Within the brain, notable D4R expression sites include GABAergic interneurons in the prefrontal cortex, as well as cortical glutamatergic pyramidal neurons and their corticostriatal terminals (; ; ). Using D4R knockout mice, the relative contribution of the D4R to total D2-like receptor expression has been estimated to 17% in the ventral striatum, 21% in the caudate-putamen and olfactory tubercle, and 40% in the hippocampus (). The D4R shares 41% and 39% sequence identity with the D2R and the D3R, respectively (Van Tol et al., 1991) and contains a region of variable number tandem repeats (VNTRs), giving rise to imperfect 16-residue repeats in its third intracellular loop (Van Tol et al., 1992; ). While a four-repeat variant is the most common in the global population, two- and seven-repeat variants are also common, with considerable geographical differences in the relative abundance of these variants (Van Tol et al., 1992; ). The seven-repeat variant has been associated with an increased risk of attention deficit hyperactivity disorder (ADHD) in several studies (; ), suggesting a role for this receptor in the regulation of selective attention. The D4R has also been proposed as a therapeutic target for substance use disorders and erectile dysfunction (; Wang et al., 2017). Moreover, the D4R is known to regulate autonomous nervous system activity () and has a similar affinity for both noradrenaline and dopamine (Sánchez-Soto et al., 2016).
Arrestins are proteins that interact with the intracellular loops and C-terminal regions of GPCRs. Arrestins typically compete with G proteins for receptor binding and sometimes initiate receptor internalization [16], leading to desensitization of G protein-dependent signaling. However, arrestins can also scaffold a host of other signaling proteins, thus setting off a distinct set of signaling events. Many GPCRs, including D2-like receptors, are known to signal via both the classical G protein-dependent pathways and the more recently described arrestin pathways (; Wingler and Lefkowitz, 2020). As their name implies, G protein-coupled receptor kinases (GRKs) are able to phosphorylate GPCRs, often agonist-dependently (). This phosphorylation has been found to increase the abilities of several GPCRs, including the D2R (Pack et al., 2018; ), to recruit arrestins. While there are several GRK isoforms, GRK2 is considered an important regulator of D2R signaling in the cerebral cortex (Urs et al., 2016).
Similarly to D2R and D3R, the coupling of D4R to inhibitory G proteins and consequent inhibition of adenylate cyclase and opening of G protein-coupled inward rectifier potassium (GIRK; also known as Kir3) channels have been repeatedly demonstrated (; ; Werner et al., 1996; ; ; Sahlholm et al., 2008). In contrast, while both D2R and D3R are known to recruit arrestins upon activation, there have been conflicting reports on the ability of D4R to interact with this class of signaling proteins. Indeed, some previous investigations could not detect significant agonist-induced arrestin recruitment to D4R, which was suggested to be “non-desensitizing” (Spooren et al., 2010; ; Zheng et al., 2020). Furthermore, little is known about the putative regulation of D4R signaling by GRKs. Here, we used a novel nanoluciferase complementation assay in HEK293T cells, as well as GIRK response desensitization in Xenopus oocytes, to investigate βarr2 recruitment to D4R and its modulation by GRK2 coexpression.
2 Materials and methods
2.1 Molecular biology
D4R-NP (‘native peptide’) and D2R-NP (a gift from Drs. Julien Hanson and Céline Laschet, University of Liège, Belgium) were in pcDNA3.1+ (Thermo Fisher Scientific, Waltham, MA). D4R-NP was designed to be analogous to D2R-NP, which was previously described by Laschet et al. (). Thus, in addition to the C-terminal linker (GNSGSSGGGGSGGGGSSG) and NP tag (GVTGWRLCERILA), D4R-NP contained an N-terminal cleavable influenza hemagglutinin signal peptide (KTIIALSYIFCLVFA) to promote surface expression, followed by a FLAG tag (DYKDDDDK). For oocyte experiments, D4R, RGS4, GIRK1 (Kir3.1), GIRK4 (Kir3.4), and GRK2 were in pXOOM (provided by Dr. Søren-Peter Olesen, University of Copenhagen, Denmark). Both D4R constructs were based on the four-repeat variant of human D4R (D4.4R) and were, along with GRK2-LgBiT (LgBiT fused directly C-terminally to human GRK2) and untagged human βarr2 and GRK2, synthesized by Genscript, Inc. (Piscataway, NJ) and cloned into pXOOM. LgBiT-βarr2 (rat βarr2 N-terminally fused to LgBiT.
VFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLITPDGSMLFRVTINS,
in pNBe3, Promega, Madison, WI) was also a gift from Drs. Julien Hanson and Céline Laschet. For oocyte experiments, the plasmids were linearized using the appropriate restriction enzymes (D4R, GIRK1, GIRK4, and RGS4; XhoI and βarr2 and GRK2; XbaI), followed by in vitro transcription using the T7 mMessage mMachine kit (Ambion, Austin, TX). The concentration and purity of cRNA were determined by spectrophotometry.
2.2 Luciferase complementation assay
We adapted a nanoluciferase assay described by to measure the interaction between D4R and downstream signaling proteins. HEK293T cells (a gift from Drs. Per Svenningsson and Xavier Morató Arús, Karolinska Institutet, Stockholm, Sweden) were incubated at 37 °C with 5% CO2 in 10 cm culture plates (VWR part of Avantor, Radnor, PA) containing Dulbecco’s modified eagle medium (DMEM; Thermo Fisher Scientific) supplemented with 0.01% penicillin/streptomycin (Thermo Fisher Scientific) and 10% FBS (Thermo Fisher Scientific). Cells were transfected with 1 µg/dish D4R-NP or D2R-NP and 1 µg/dish LgBiT-βarr2/GRK2-LgBiT along with 10 µg untagged GRK2, when indicated, using linear polyethylenimine (PEI; Polysciences Inc., Valley Road Warrington, PA (). The empty plasmid vector (pcDNA3.1+) was added to the transfection mixture to bring the total amount of transfected plasmid to 20 µg/dish. Subsequently, 24 h after transfection, cells were trypsinated and centrifugated for pellet recovery before resuspension in Hank’s balanced salt solution (HBSS; Corning, Tewksbury, MA). Cells were counted using a TC20 automated cell counter (Bio-Rad, Hercules, CA) and diluted in HBSS at 500,000 cells/ml. 100 µl cell solution was pipeted into each well of a flat-bottom white 96-well plate (Thermo Fisher Scientific). The nanoluciferase substrate furimazine was added to each well in the form of Nano-Glo live cell reagent (Promega) according to the manufacturer’s instructions. For concentration-response experiments, serial dilutions of dopamine (Sigma-Aldrich, St. Louis, MO) were added to the 96-well plate column-wise, with the first column receiving only vehicle (HBSS). The plate was incubated at room temperature for 5 min (or 3 min in experiments with GRK2-LgBiT) prior to luminescence measurement in a TriStar2 LB 942 multimode reader (Berthold Technologies, Bad Wildbad, Germany) with an integration time of 10 ms.
For time-resolved, repeated measurements, luminescence was recorded from each well each min for 1 h with an integration time of 10 ms. Following a baseline read of 8 min, 10 µL of dopamine dissolved in HBSS was injected into each well to result in a final concentration of 32 nM. After another 10 min, 10 µL of clozapine or raclopride (Tocris Bioscience, Bristol, UK), dissolved in DMSO and diluted in HBSS, were injected to yield a final concentration of 10 µM (clozapine) or 1 µM (raclopride). HBSS injection was used as control.
2.3 Oocyte preparation
The oocytes of the African clawed toad, Xenopus laevis, were surgically isolated as previously described (Sahlholm et al., 2011). The surgical procedures were approved by the Swedish National Board for Laboratory Animals and the Stockholm Ethical Committee (approval number 686–2021). After 1 day of incubation at 12°C, the oocytes were injected with 0.3 ng of D4R cRNA, 1.4 ng of RGS4 cRNA, 40 pg of each of GIRK1 and GIRK4 cRNA in a volume of 50 nL per oocyte using a Nanoject (Drummond Scientific, Broomall, PA). When applicable, 0.6–19 ng/oocyte of βarr2 and 0.3 ng/oocyte of GRK2 cRNA were also injected.
2.4 Electrophysiology methods
Following RNA injection and 6 days of incubation at 12 °C, electrophysiological experiments were performed on the oocytes using the parallel eight-channel, two-electrode voltage-clamp OpusXpress 6000A (Molecular Devices, San José, CA) (). Continuous perfusion, provided by Minipuls three peristaltic pumps (Gilson, Middleton, WI), was maintained at 3.5 mL/min. Data were acquired at a membrane potential of −80 mV and sampled at 156 Hz using OpusXpress 1.10.42 software (Molecular Devices). To increase the inward-rectifier potassium channel current at negative potentials, a high-potassium extracellular buffer was used (64 mM NaCl, 25 mM KCl, 0.8 mM MgCl2, 0.4 mM CaCl2, 15 mM HEPES, 1 mM ascorbic acid, adjusted to pH 7.4 with NaOH), yielding a K+ reversal potential of ∼−40 mV. Ascorbic acid was included to prevent spontaneous oxidation of DA.
2.5 Whole-cell enzyme-linked immunosorbent assay (ELISA)
Cell surface expression of D4R and D2R constructs was evaluated essentially as described by (). In brief, following 24 h incubation after transfection (as described above), cells were trypsinated, resuspended in DMEM (supplemented as above), counted and re-plated at 50,000 cells/well in a volume of 100 µL/well in transparent 96-well plates (Thermo Fischer Scientific) previously coated with poly-D-lysine (Sigma-Aldrich) and returned to the incubator for further growth and adhesion. After another 24 h incubation, DMEM was aspirated and wells were washed twice with 100 µL/well of chilled (4°C) phosphate-buffered saline (PBS; VWR part of Avantor) containing 0.5% (w/v) bovine serum albumin (BSA; Sigma-Aldrich). After washing, cells were incubated with 50 µL/well of horseradish peroxidase-linked mouse anti-FLAG M2 antibody (A8592; Sigma-Aldrich) diluted 1/20,000 with chilled PBS containing 1% BSA. Following 1 h incubation at 4°C, well contents were aspirated and washed four times with chilled PBS containing 0.5% BSA. Next, 50 µL/well of the horseradish peroxidase substrate 3,3′,5,5′-Tetramethylbenzidine (T0440; Sigma-Aldrich) was added and plates were incubated at 37°C for 20 min. Finally, 50 µL/well of 2 M HCl were added to each well and absorbance was measured at 450 nm in the Berthold TriStar2 LB 942 plate reader.
2.6 Data analysis
Concentration–response curves for nanoluciferase complementation data were calculated using the variable-slope sigmoidal functions in GraphPad Prism 8 (GraphPad Software, San Diego, CA).
The following equation was used for fitting:where Y is the response, Top is the maximal response as a fraction of the mean control (HBSS) luminescence and X is the logarithm of dopamine concentration. Data points are presented as mean ± SEM throughout. pEC50 values from individual experiments were compared using Student’s paired t-test, testing differences in pEC50s obtained from experiments performed in the presence or absence of exogenous GRK2. Experiments with and without GRK2 were carried out on the same day with cells of the same passage number.
Electrophysiology recordings were initially screened for inclusion in Clampfit 10.6 (Molecular Devices). Recordings in which holding currents at −40 mV were stable before and after the −80 mV step and in which the current (at −80 mV) after dopamine washout was equal to or smaller than before dopamine application were included in the analysis. Recordings were peak normalized and time-averaged using Matrix Laboratory 2018b (MathWorks, Natick, MA).
2.7 Statistical analysis
Statistical analysis was performed in GraphPad Prism 8 using the two-tailed, paried or unpaired Student’s t-test, one-way ANOVA with Dunnett’s test for multiple comparisons, or repeated measures ANOVA with Sidak’s test for multiple comparisons, as appropriate, for normally distributed data. Mann-Whitney test, Wilcoxon matched-pairs signed rank test, or Kruskal–Wallis test with Dunn’s correction for multiple comparisons were used when normal distribution of data could not be assumed. Normality was assessed using the Shapiro-Wilk test. The significance threshold was set to 0.05.
3 Results
First, we designed a nanoluciferase complementation assay to study βarr2 recruitment to D4R. To this end, we adapted a modified nanoBiT assay, which was recently demonstrated to work well with D2R (), for use with D4R. As the higher-affinity nanoluciferase fragment 'native peptide’ (NP) was reported to result in a higher signal-to-noise ratio compared to the lower-affinity SmBiT (), we created an analogous D4R construct, fusing NP to the C-terminus of D4R via a flexible linker (see Methods). The larger complementary fragment (LgBiT) was fused to the N-terminus of βarr2 (LgBiT-βarr2). In HEK293T cells transfected with D4R-NP and LgBiT-βarr2, a concentration-dependent increase in luminescence was observed when cells were stimulated with increasing dopamine in the presence of a constant concentration of the substrate furimazine (Figure 1A). This dopamine-dependent increase was not observed in cells transfected with only LgBiT-βarr2 or only D4R-NP (Figure 1A). Kinetic experiments revealed that the increase in luminescence after dopamine application was fully reversible upon the addition of 10 µM of the antipsychotic clozapine, which is a D4R antagonist (Van Tol et al., 1991; ) (Figure 1B). For comparison, βarr2 recruitment to D2R was also studied in a separate series of experiments. As previously described (), a concentration-dependent increase in luminescence was observed when cells coexpressing D2R-NP and LgBiT-βarr2, but not D2R-NP alone, were stimulated with increasing dopamine in the presence of furimazine (Figure 1C). In kinetic experiments, the increase in luminescence after dopamine application was fully reversible upon the addition of 1 µM of the D2R antagonist raclopride (Figure 1D).
FIGURE 1
Next, we wanted to assess the putative interaction between D4R and GRK2 itself. Dopamine application to cells co-expressing D4R-NP with LgBiT C-terminally fused to GRK2 (GRK2-LgBiT) revealed a dopamine concentration-dependent increase of luminescence (Figure 2A). Kinetic experiments revealed a transient dopamine-induced bioluminescence signal, which peaked rapidly following dopamine application (Figure 2B). No increase in luminescence was observed upon addition of dopamine to cells expressing GRK2-LgBiT without D4R-NP. The luminescence response in cells coexpressing D4R-NP and GRK2-LgBiT returned towards baseline (in the continued presence of dopamine) much faster than what was observed in cells co-expressing D4R-NP and LgBiT-βarr2 (c.f. Figure 1B; T1/2 2.9 ± 0.3 min vs 18.7 ± 3.5 min, n = 3 in each case; p = 0.011, Student’s unpaired t-test). For comparison, we also assessed the recruitment of GRK2 to the D2R. Similar to the findings with D4R-NP, dopamine concentration-dependently increased luminescence in cells transfected with GRK2-LgBiT and D2R-NP, although the fold-over-baseline increase in luminescence was greater than with the D4R-based construct (Figure 2C). Moreover, in kinetic experiments, the increase in luminescence was more sustained with D2R-NP (Figure 2D). Application of the D2R antagonist raclopride (1 µM) rapidly reversed the dopamine-induced luminescence increase (Figure 2D).
FIGURE 2
To evaluate the functional consequences of agonist-induced βarr2 recruitment to the D4R at the level of receptor desensitization, we studied the time course of GIRK currents evoked by the D4R in the absence or presence of βarr2. Xenopus oocytes are known not to express detectable levels of endogenous arrestins and GRKs () and therefore provide a suitable background on which to study the effect of exogenous βarr2 and GRK2. Additionally, after injection of the appropriate receptor and channel cRNAs, this system provides a facile readout of D4R-mediated GIRK activation (Wedemeyer et al., 2007; ; Sahlholm et al., 2008). Upon coexpression of D4R, GIRK1/4, and RGS4 with increasing amounts of βarr2 cRNA, a dosage-dependent increase in the rate of decay of dopamine-induced GIRK currents was observed. This effect reached significance for the largest amounts of co-injected βarr2 cRNA (Figures 3A, B), while there was no significant effect on the peak amplitudes of the evoked currents (Figure 3C).
FIGURE 3
Finally, we wanted to examine the effect of exogenous GRK2 coexpression on arrestin recruitment to the D4R. In the nanoluciferase complementation assay, cotransfection of D4R-NP and LgBiT-βarr2 with 10 µg/dish of GRK2-encoding plasmid increased the potency of dopamine by about 3-fold (Figures 4A, B). Observations were paired such that experiments with and without GRK2 were carried out on the same day with cells of the same passage number, transfected 24 h prior. There was a trend toward a decrease in the maximal dopamine-induced luminescence relative to baseline in the GRK2-coexpressing condition; however, this trend did not reach significance (Figure 4C). For comparison, the effect of GRK2 coexpression on βarr2 to the D2R was also studied. Cotransfection of D2R-NP and LgBiT-βarr2 with 10 µg/dish of the GRK2 plasmid increased the potency of dopamine by ca 10-fold (Figures 4D, E) and significantly reduced the maximal dopamine-induced luminescence increase from about 6-fold to 2-fold (Figure 4F). GRK2 coexpression was not accompanied by any significant change in cell surface FLAG immunoreactivity, neither in cells transfected with D4R-NP, nor with D2R-NP (Supplementary Figures S1A, B). In electrophysiology experiments in Xenopus oocytes, co-injection of GRK2 cRNA with D4R, GIRK1/4, RGS4, and βarr2 cRNA accelerated the decay rate of dopamine-induced GIRK currents (Figures 4G, H) as compared to oocytes expressing D4R, GIRK1/4, RGS4, and βarr2 in the absence of GRK2, while there was no significant effect on peak amplitudes of the elicited currents (Figure 4I). RGS4 is a GTPase accelerating protein which increases the rate of the G protein cycle and thus causes the state of receptor activity to have a more rapid impact on the GIRK current time course (). RGS4 was thus included to increase the measurable rate of desensitization of the GIRK response.
FIGURE 4
4 Discussion
In the present study, we report on the first nanoluciferase complementation assay capable of reporting on dopamine-induced arrestin recruitment to the D4R, as well as on the regulation of arrestin recruitment by GRK2. In agreement, the expression of βarr2 induced desensitization of GIRK current responses evoked via D4R in Xenopus oocytes and this desensitization was enhanced by exogenous GRK2. While both β-arrestin1 and β-arrestin2 (βarr2; also known as arrestin3) are expressed in the central nervous system and capable of interacting with D2-like receptors, βarr2 has been found to play the major role in D2R signaling and desensitization in vivo (; Skinbjerg et al., 2009). Here, we therefore chose to study βarr2 together with both D2R and the related D4R. Likewise, GRK2 has been found to have the strongest effect on D2R function of the five non-visual GRKs () and we thus focused our present efforts on this GRK isoform. Considering the prominent expression of both βarr2 and GRK2 in the prefrontal cortex (Urs et al., 2016) where D4R is also present (), it does not seem unlikely that these proteins may be native D4R interaction partners.
We note that some previous investigations that did not detect D4R-mediated arrestin recruitment used luciferase complementation () or fluorescence microscopy to assess redistribution of βarr2 to the plasma membrane (Spooren et al., 2010). On the other hand, some of the studies that did find evidence for such recruitment used PathHunter (; Pirzer et al., 2019) or bioluminescent resonance energy transfer (BRET) approaches to monitor βarr2-D4R interactions (Sánchez-Soto et al., 2016; ; Pavletić et al., 2022). Assay sensitivity would seem a likely explanation for these discrepancies. For example, using the brighter nanoluciferase () in the present investigation rather than the emerald luciferase employed in a previous complementation study () may have yielded a stronger signal, allowing an increase in luminescence upon βarr2 recruitment to be picked up. The smaller size of the nanoluciferase fragments and the flexible linker attaching the NP fragment to D4R could also mean that there is a greater likelihood of productive interaction (i.e.; enzyme complementation) and less likelihood of steric interference of the enzyme fragments with the interaction between the two tagged proteins.
The Xenopus oocyte GIRK assay is another highly sensitive assay that allows for kinetic, live-cell experiments. The dosage-dependent increase in GIRK response decay rate observed with increasing amounts of coinjected βarr2 cRNA suggests that βarr2 mediates desensitization of GIRK responses evoked by D4R, similar to what has been described for D2R in the corresponding assay (; ). In agreement with the results from the nanoluciferase complementation assay, coexpression of GRK2 further increased the rate of response decay.
Consistent with an interaction between D4R and GRK2, experiments in HEK cells transfected with D4R-NP and GRK2-LgBiT revealed an increase in luminescence upon the addition of dopamine. Compared to cells co-expressing D4R-NP and LgBiT-βarr2, this luminescence response peaked and decayed quite rapidly (c.f. Figures 1B, 2B), in the continued presence of dopamine. This behaviour may be interpreted as GRK2 competition for D4R binding with endogenous βarr2 (the affinity of which would increase after GRK2 phosphorylation, in line with the results discussed above), a higher affinity of GRK2 for unphosphorylated vs. phosphorylated D4R (with GRK2- D4R interaction decreasing once the receptor is phosphorylated), or both. Interestingly, the luminescence response in corresponding experiments with D2R-NP decayed considerably slower (c.f. Figures 2B, D), suggesting a more sustained interaction between GRK2 and the D2R.
The effect of GRK2 coexpression on LgBiT-βarr2 recruitment was more pronounced at the D2R compared to the D4R, both when considering the increase in dopamine potency and the decrease in maximal arrestin recruitment signal, which reached only a trend level at the D4R (Figures 4A–F). This signal reduction was not accompanied by a decrease in FLAG immunoreactivity on intact cells (Supplementary Figure S1), suggesting the cell surface expression of the D2R (and of the D4R in corresponding experiments) remained similar in the presence or absence of exogenous GRK2. Rather, we would speculate that the decrease in maximal dopamine-induced luminescence signal is due to competition between GRK2 and βarr2 for binding to the receptor, as was also suggested by (), who reported similar findings with regards to GRK2 and βarr2 recruitment to the D2R. The stronger effect of GRK2 coexpression on D2R signalling would seem consistent with the higher fold-over-baseline increase in luminescence and the slower response decay in experiments with GRK2-LgBiT and D2R-NP as compared to D4R-NP, as noted above.
5 Conclusion
In this study, we presented a nanoluciferase complementation assay able to report on dopamine-mediated βarr2 recruitment to the D4R. We also demonstrated that βarr2 recruitment is enhanced by GRK2 coexpression, although this effect is less pronounced than at the D2R. In addition, the potentiating action of GRK2 was observed at the level of βarr2-mediated desensitization of GIRK current responses evoked by the D4R. Finally, evidence was obtained for transient dopamine-induced recruitment of GRK2 to the D4R. The nanoluciferase complementation assay employed here may be useful for drug discovery efforts targeting the D4R. In addition, the new information regarding GRK2 regulation of D4R may prove relevant for understanding the biological functions of this relatively little-explored dopamine receptor.
Statements
Data availability statement
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.
Ethics statement
The animal study was reviewed and approved by the Swedish National Board for Laboratory Animals, Stockholm Ethical Committee (Stockholms djurförsöksetiska nämnd).
Author contributions
VB performed nanoluciferase complementation assays and drafted the manuscript together with KS. RÅ designed and performed electrophysiology experiments, NB performed nanoluciferase complementation assays, MV-L and EG-M performed experiments and helped establish assay conditions, and FC and KS supervised experimental work. KS conceived of and designed the study. All authors contributed to manuscript writing and approved the final version of the manuscript.
Funding
This study was funded by grants from the Lars Hierta Memorial Foundation, Åhlén foundation, and Magnus Bergvall Foundation. RÅ is supported by the Swedish Brain Foundation (PS2022-0040) and KS is supported by the Wallenberg Centre for Molecular Medicine.
Acknowledgments
VB, RÅ, NB, and KS participate in the European COST Action CA18133 (ERNEST).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar.2023.1087171/full#supplementary-material
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Summary
Keywords
luminescence measurements, luciferase, electrophysiology, HEK 293 cells, Xenopus laevis, G protein-coupled inwardly rectifying potassium channels
Citation
Burström V, Ågren R, Betari N, Valle-León M, Garro-Martínez E, Ciruela F and Sahlholm K (2023) Dopamine-induced arrestin recruitment and desensitization of the dopamine D4 receptor is regulated by G protein-coupled receptor kinase-2. Front. Pharmacol. 14:1087171. doi: 10.3389/fphar.2023.1087171
Received
02 November 2022
Accepted
18 January 2023
Published
27 January 2023
Volume
14 - 2023
Edited by
Stefano Espinoza, Istituto Italiano di Tecnologia, Italy
Reviewed by
Peter McCormick, Queen Mary University of London, United Kingdom
Marco Scarselli, University of Pisa, Italy
Updates
Copyright
© 2023 Burström, Ågren, Betari, Valle-León, Garro-Martínez, Ciruela and Sahlholm.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Kristoffer Sahlholm, kristoffer.sahlholm@umu.se
† These authors have contributed equally to this work
This article was submitted to Neuropharmacology, a section of the journal Frontiers in Pharmacology
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