Biomolecules & Therapeutics 2024; 32(1): 56-64
Biased Dopamine D2 Receptors Exhibit Distinct Intracellular Trafficking Properties and ERK Activation in Different Subcellular Domains
Shujie Wang, Lulu Peng and Kyeong-Man Kim*
Department of Pharmacology, College of Pharmacy, Chonnam National University, Gwangju 61186, Republic of Korea
Tel: +82-62-530-2936, Fax: +82-62-530-2949
The first two authors contributed equally to this work.
Received: February 19, 2023; Revised: June 18, 2023; Accepted: June 27, 2023; Published online: July 19, 2023.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Biased signaling or functional selectivity refers to the ability of an agonist or receptor to selectively activate a subset of transducers such as G protein and arrestin in the case of G protein-coupled receptors (GPCRs). Although signaling through arrestin has been reported from various GPCRs, only a few studies have examined side-by-side how it differs from signaling via G protein. In this study, two signaling pathways were compared using dopamine D2 receptor (D2R) mutants engineered via the evolutionary tracer method to selectively transduce signals through G protein or arrestin (D2G and D2Arr, respectively). D2G mediated the inhibition of cAMP production and ERK activation in the cytoplasm. D2Arr, in contrast, mediated receptor endocytosis accompanied by arrestin ubiquitination and ERK activation in the nucleus as well as in the cytoplasm. D2Arr-mediated ERK activation occurred in a manner dependent on arrestin3 but not arrestin2, accompanied by the nuclear translocation of arrestin3 via importin1. D2R-mediated ERK activation, which occurred in both the cytosol and nucleus, was limited to the cytosol when cellular arrestin3 was depleted. This finding supports the results obtained with D2Arr and D2G. Taken together, these observations indicate that biased signal transduction pathways activate distinct downstream mechanisms and that the subcellular regions in which they occur could be different when the same effectors are involved. These findings broaden our understanding on the relation between biased receptors and the corresponding downstream signaling, which is critical for elucidating the functional roles of biased pathways.
Keywords: Dopamine D2 receptor, Biased receptor, Endocytosis, Ubiquitination, ERK, Importin

G protein-coupled receptors (GPCRs) regulate various physiological processes through multiple signaling pathways, which can be classified into G protein-dependent and G protein-independent pathways (Wisler et al., 2018). G protein-independent pathways are primarily mediated by arrestin proteins previously recognized as mediators of agonist-induced receptor desensitization and endocytosis (Benovic et al., 1987; Lohse et al., 1990; Ferguson et al., 1996).

Unlike the traditional balanced agonists that affect both the G protein and arrestin pathways, some newly synthesized ligands have been reported to act more selectively on either one of the two (Wei et al., 2003; Urban et al., 2007; Violin et al., 2010). Although the specific mechanism of biased signaling remains unclear, recent studies suggest that biased agonists may stabilize GPCRs in distinct conformations (Reiter et al., 2012; Gurevich and Gurevich, 2020).

Currently, there is a great interest in the development of biased ligands because, compared with their traditional counterparts, they are believed to have the advantages of improved efficacy and reduced side effects in the treatment of certain diseases (Soergel et al., 2014; Violin et al., 2014). The main strategy for the design of biased ligands has been to utilize the insights gained through the microanalyses of GPCR structures (McCorvy et al., 2018; Sanchez-Soto et al., 2020).

Biased signaling can be induced by not only tailored ligands but also genetically modified GPCRs. For example, a mutation of an amino acid residue located in the hydrophobic pocket at the interface of the second extracellular loop and the fifth transmembrane segment of D2R (F5.38, F189) (Sanchez-Soto et al., 2020), a combined mutation of the Arg residue in the Asp-Arg-Tyr (DRY) motif (R3.50) and L3.41 (L123W) (Donthamsetti et al., 2020), and the alteration of four amino acid residues in the N-terminal region of the third intracellular loop (212IYIV215) (Lan et al., 2009) were reported to result in biased signaling through the G protein and/or arrestin pathways.

Some studies utilized a unique approach, evolutionary tracer (ET) method (Lichtarge et al., 1996), to identify D2R mutants that selectively signal through either the G protein or arrestin pathway (Peterson et al., 2015a, 2015b). In particular, these tailored D2R mutants were used in in vivo studies to elucidate the role of the arrestin pathway in amphetamine-induced locomotion (Peterson et al., 2015b).

In this study, the characteristics of ET-based D2R mutants were further examined to determine the differences between the G protein and arrestin signaling pathways. First, the pathway-selectivity of each mutant was determined, followed by the identification of the arrestin subtype involved and their relationships with receptor endocytosis and arrestin ubiquitination. In addition, we determined the differences in the subcellular regions of ERK activation upon agonistic activation of biased D2Rs. These findings were further validated by conducting experiments in cells where arrestin3 was depleted, ensuring the robustness of the results.



Dopamine (DA), (-)-quinpirole (Quin), leptomycin B (LMB), DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride), agarose beads coated with anti-FLAG antibodies (FLAG beads), and rabbit antibodies against actin or FLAG epitope were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). [3H]-Sulpiride (84 Ci/mmol) was purchased from PerkinElmer Life Sciences (Boston, MA, USA). Antibodies against arrestins, β-actin, and lamin B1 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against clathrin heavy chain (CHC) and caveolin1 (Cav1) were purchased from BD Biosciences (San Jose, CA, USA) and BD Transduction Laboratories (Franklin Lakes, NJ, USA), respectively. Pertussis toxin (PTX) was purchased from Calbiochem (Gibbs town, NJ, USA). Antibodies for importin β1, phospho-ERK1/2, and ERK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-mouse HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA).


Human embryonic kidney (HEK)-293 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in minimal essential medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere with 5% CO2. All knockdown (KD) cell lines were established by stable expression of short hairpin RNA (shRNA) plasmids. For arrestin-KD cells, HEK-293 cells stably transfected with pcDNA3.0 vector expressing a scrambled shRNA sequence (Con-KD cells) were used as negative control (Zhang et al., 2005). For the double knockdown of arrestin2 and arrestin3, the clones stably expressing the shRNA for arrestin2 were stably transfected with pcDNA3.1 expressing the shRNA for arrestin3. For other KD cells, HEK-293 cells stably expressing a scrambled shRNA sequence (PLKO.1) under puromycin selection were included as negative control.

Plasmid constructs

The mammalian expression constructs for wild-type human dopamine D2 receptor (hereafter, D2R) and arrestin3 have been described previously (Kim et al., 2001; Beom et al., 2004; Kim et al., 2011). The two D2R mutants with high degrees of functional separation, D2G (L125N, Y133L) and D2Arr (A135R, M140D) (Peterson et al., 2015b), were generated by site-directed mutagenesis.

ERK assay

Cells were starved overnight in serum-free culture medium containing 0.1% BSA and treated with 1 µM DA dissolved in 10 µM ascorbic acid. The medium was aspirated, and SDS sample buffer was added directly on the cells. Samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Nitrocellulose membranes were incubated for 1 h at room temperature in TBS-T (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5% Tween-20) containing 5% nonfat dry milk, followed by 1 h incubation with antibodies for phospho-ERK1/2 or ERK2 (1:1,000 dilution) and 1 h with secondary antibodies (1:5,000) in 2% nonfat dry milk. Protein bands were imaged and quantitated using a ChemiDoc MP imaging system (BioRad, Hercules, CA, USA).

Receptor endocytosis assay

Endocytosis of D2R was determined based on the hydrophilic properties of [3H]-sulpiride (Kim et al., 2001). HEK-293 cells expressing D2R were stimulated with 10 µM DA for 60 min and incubated with 250 µL of [3H]-sulpiride (2.2 nM, final concentration) at 4°C for 150 min in the presence or absence of an unlabeled competitive inhibitor (10 µM haloperidol). The cells were washed thrice with the same medium, and 1% SDS was added. Samples were mixed with 2 mL Lefkofluor scintillation fluid and counted on a Wallac 1450 MicroBeta TriLux Liquid Scintillation Counter (PerkinElmer Life Sciences).

Detection of arrestin3 ubiquitination

FLAG-arrestin3 and HA-Ub were used to co-transfect HEK-293 cells expressing the corresponding D2Rs. The cell lysates were solubilized in RIPA buffer (50 mM Tris, 150 mM NaCl, pH 8.0, 0.5% deoxycholate, 1% NP-40, 0.1% SDS) containing 1 mM sodium orthovanadate, 1 mM sodium fluoride, 10 mM N-ethylmaleimide, 5 μg/mL leupeptin, 5 μg/mL aprotinin, and 2 mM phenylmethylsulphonyl fluoride. Immunoprecipitation was performed with FLAG beads. The eluents were analyzed by immunoblotting.

Subcellular fractionation

Subcellular fractions were prepared as described previously (Min et al., 2019; Zhang et al., 2020). β-Actin and Lamin B1 were used as markers for the cytoplasmic and nuclear fractions, respectively.

Reporter gene assay

Cellular cAMP levels were measured by a reporter gene method as described previously (Cho et al., 2010; Zheng et al., 2011) using a reporter plasmid expressing firefly luciferase gene under the control of multiple cAMP response elements and a pRL-TK control vector. Cells were treated with 2 μM forskolin and quinpirole (10–12-10–8 M) for 4 h before harvesting. The relative luciferase expression was measured using a dual luciferase assay kit (Promega, Madison, WI, USA).


Cells were transfected with arrestin3-GFP along with D2Arr or D2G. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at 20°C. The cells were subsequently mounted on slides using Vectashield (Vector Laboratories; Burlingame, CA, USA) and imaged using a laser scanning confocal microscope (TCS SP5/AOBS/Tandem; Leica, Jena, Germany). The images were analyzed using the Fiji version of the image processing software ImageJ and colocalization was analyzed based on Pearson’s correlation coefficient (γ value) (Min et al., 2023).

Statistical analysis

Construction of dose-response curves and statistical analysis were performed on GraphPad Prism 5.0 (GraphPad Software Inc., La Jolla, CA, USA). Quantitated protein levels determined by immunoblotting were expressed as fold-control. Results were expressed as the mean ± standard deviation (SD). Differences between two groups were analyzed by Student’s t test (paired two-tailed), and those among multiple groups were analyzed by one-way ANOVA with Tukey’s post hoc test. A p-value lower than 0.05 was considered to indicate a significant difference.


Characterization of the biased signaling pathways of D2Rs

Two biased mutants of dopamine D2 receptors (D2Rs) that have been suggested to selectively signal through G protein (D2G) or through arrestin (D2Arr) pathways (Peterson et al., 2015a, 2015b) were further characterized to assess their suitability as a tool for developing biased ligands.

We first examined the G protein bias of D2G and D2Arr by determining the agonist-induced inhibition of cAMP production. As shown in Fig. 1A, WT-D2R and D2G showed similar levels of potency and efficacy for quinpirole in the inhibition of cAMP production. In contrast, D2Arr showed a significantly decreased potency and efficacy. IC50 values of quinpirole for the inhibition of cAMP production were 0.175 nM and 0.119 nM, for WT-D2R and D2G, respectively. In contrast, D2Arr could achieve about 50% inhibition of cAMP production upon treatment with 10 nM quinpirole.

Figure 1. Characterization of two representative biased signaling pathways of dopamine D2 receptors. Receptor expression levels were between 1.7-1.9 pmol/mg protein. Knockdown cells of arrestin2 (Arr2-KD), arrestins3 (Arr3-KD), and arrestin2 and arrestin3 (Arr2/3-KD) were described in our recent publication (Min et al., 2023). The knockdown efficiencies were about 90-95%. (A) HEK-293 cells were transfected with the cDNA of WT-D2R, D2G, or D2Arr, and reporter gene assay was conducted as described in the Materials and Methods. The CRE-luci values of the D2Arr group were significantly different from those of the other experimental groups at the concentration of quinpirole (Quin) between 10–10 M and 10–8 M (p<0.001, n=3). (B) HEK-293 cells expressing D2G or D2Arr were treated with 50 ng/ml pertussis toxin (PTX) overnight. Cells were treated with either vehicle or 1 μM DA for 5 min. ERK assay was conducted as described in the Materials and Methods. **p<0.01 compared to corresponding vehicle groups (n=3). (C) Con-KD or Arr2/3-KD cells were transfected with D2Arr or D2G. Cells were treated with either vehicle or 1 μM DA for 5 min. **p<0.01 compared to corresponding vehicle groups (n=3). #p<0.05, ##p<0.01 compared to Veh/Con-KD/D2Arr group (n=3). (D) Con-KD, Arr2-KD, or Arr3-KD cells were transfected with D2Arr. Cells were treated with either vehicle or 1 μM DA for 5 min. *p<0.05, **p<0.01 compared to corresponding vehicle groups (n=3). ###p<0.001 compared to Veh/Con-KD group (n=3).

Along with c-Src, ERK is a representative signaling effector known to be activated in an arrestin-dependent manner (Luttrell et al., 1999; Ahn et al., 2004). Treatment with pertussis toxin (PTX, 50 ng/mL, overnight) largely inhibited D2G-mediated ERK activation but did not affect that mediated by D2Arr (Fig. 1B). Conversely, D2Arr-mediated but not D2G-mediated ERK activation was inhibited in arrestin2/3-knockdown cells (Fig. 1C). We further observed that D2Arr-mediated ERK activation was blocked specifically by the knockdown of arrestin3 but not arrestin2 (Fig. 1D).

D2Arr, but not D2G, mediates arrestin ubiquitination involved in GPCR endocytosis

To determine whether the G protein-dependent or arrestin-dependent pathway is involved in the receptor endocytosis, we generated cells expressing WT-D2R, D2G, or D2Arr with either clathrin heavy chain (CHC) or caveolin1 (Cav1) knockdown. As shown in Fig. 2A, DA-induced endocytosis of D2R was significantly inhibited by the knockdown of CHC or Cav1, suggesting that both clathrin-mediated endocytosis (CME) and caveolar endocytosis are involved in the endocytosis of D2R. Similarly, the endocytosis of D2Arr was also inhibited by knockdown of CHC or Cav1. The levels of D2G endocytosis were significantly lower than those of WT-D2R or D2Arr endocytosis and not affected by knockdown of CHC or Cav1, indicating that non-CME/caveolar endocytosis was involved in D2G endocytosis. These results suggest that agonist-induced clathrin-mediated or caveolar endocytosis of D2R occurs through the arrestin-dependent pathway. In agreement with these results, the endocytosis of D2R still occurred in cells treated with PTX (Fig. 2B). PTX catalyzes the process of ADP-ribosylation specifically targeting the α subunits of the heterotrimeric Gi/o protein family. This modification serves to prevent the interaction between receptors and G-proteins (Burns, 1988).

Figure 2. Comparison of agonist-induced endocytosis and arrestin3 ubiquitination mediated by biased D2 receptors. Receptor expression levels were between 1.7-1.9 pmol/mg protein. (A) HEK-293 cells expressing pLKO.1-puro vector (Con-KD), or small hairpin RNA (shRNA) targeting clathrin heavy chain (CHC) or caveilin1 (Cav1) in pLKO.1, were transfected with the cDNA of WT-D2R, D2G, or D2Arr. Cells were treated with 10 μM DA for 1h, and the receptor endocytosis assay was conducted as described in the Materials and Methods. *p<0.05, **p<0.01, ***p<0.001 compared to corresponding Con-KD group. #p<0.05, ##p<0.01 compared to Con-KD/WT-D2R group (n=3). Cell lysates from Con-KD, Cav1-KD, and CHC-KD cells were immunoblotted with antibodies against Cav1/actin or CHC/actin. The knockdown efficiencies of Cav1 and CHC were about 90-95%. (B) HEK-293 cells were transfected with either 2 μg D2R-pCMV5 together with 2 μg pCMV5 or 2 μg D2R-pCMV5 together with arrestin3-pCMV5. Cells were treated with either vehicle or 50 ng/mL PTX overnight, followed by treatment with 1 μM DA for 1h. *p<0.05 compared to Veh/D2R group (n=3). (C) HEK-293 cells expressing D2Arr or D2G were transfected with hemagglutinin-tagged ubiquitin (HA-Ub) and FLAG-arrestin3. Cells were treated with 1 μM DA for 5 min, and ubiquitination assay was conducted as described in the Materials and Methods. **p<0.01 compared to other groups (n=3). (D) HEK-293 cells expressing D2Arr were treated with vehicle or 50 ng/mL PTX overnight. *p<0.05 compared to corresponding vehicle group (n=3).

Agonist-induced ubiquitination of arrestin3 in the nucleus (Zhang et al., 2020) is one of the critical events that mediate GPCR endocytosis (Shenoy et al., 2001). As shown in Fig. 2C, the ubiquitination of arrestin3 was induced by agonistic stimulation of D2Arr but not D2G. As in the case of D2R endocytosis (Fig. 2B), D2R-mediated ubiquitination of arrestin3 was insensitive to PTX treatment (Fig. 2D).

In summary, these results provide evidence that the endocytosis of D2R occurs in a manner dependent on arrestins, with the ubiquitination of arrestin3 playing a critical role. These findings are consistent with a previously published study showing that GPCRs can recruit arrestins in the absence of active G proteins (Grundmann et al., 2018).

Arrestin3 translocates to the nucleus and mediates ERK activation in response to agonist stimulation of D2Arr

ERK can be activated through both G protein-dependent and arrestin-dependent pathways. Interestingly, ERK activation through the two pathways is known to exhibit different characteristics. For example, studies conducted using the angiotensin II type 1A receptor have shown that G protein-dependent ERK activation is more rapid and transient compared to arrestin3-dependent activation (Ahn et al., 2004). In addition, rapid phase and late phase activation lead to nuclear and cytosolic accumulation of phosphorylated ERK, respectively. However, studies on other GPCRs such as free fatty acid receptor 4 have raised doubts on whether the time of activation is a factor that can specify ERK activation through two different pathways (Alvarez-Curto et al., 2016; Gurevich and Gurevich, 2020).

Thus, we investigated whether the activation of ERK in different subcellular locations could be a reliable marker for arrestin-dependent signaling pathway. As shown in Fig. 3A, agonist stimulation of D2G resulted in ERK activation in the cytoplasm but not in the nucleus. In contrast, ERK activation occurred both in the cytoplasm and in the nucleus in response to agonist stimulation of D2Arr.

Figure 3. Biased D2Rs mediate ERK activation in different subcellular regions. (A) HEK-293 cells expressing either D2Arr or D2G were treated with 1 μM DA for 5 min. Cell lysates were fractionated as described in the Materials and Methods, and were immunoblotted with antibodies against p-ERK1/2, ERK2, lamin b1 and β-actin which are nuclear and cytosolic markers, respectively. **p<0.01 compared to corresponding vehicle-treated groups (n=3). CF (cytoplasmic fraction) and NF (nuclear fraction) were used to denote the respective subcellular compartments. The expression levels of the receptors were measured to be approximately 1.2-1.5 pmol/mg protein. (B) Con-KD and arrestin3-KD HEK-293 cells expressing D2Arr were treated with 1 µM DA between 0-10 min. ***p<0.001 compared to 0 min/Con-KD group; ###p<0.001 compared to corresponding arrestin3-KD cell group (n=3). The expression levels of the receptors were measured to be approximately 1.3-1.5 pmol/mg protein.

To validate the findings regarding biased receptors, we conducted a comparison of D2R-mediated ERK activation between Con-KD and arrestin3-KD cells. According to the findings shown in Fig. 3B, when D2Arr was stimulated with an agonist, it led to the activation of ERK in both the cytosol and nucleus within a timeframe of 2 to 10 min. However, when cellular arrestin3 was specifically depleted through knockdown, it resulted in the selective inhibition of ERK activation in the nucleus.

The subcellular fractionation analysis (Fig. 4A) and confocal imaging experiments (Fig. 4B) revealed that upon agonist stimulation of D2Arr, arrestin3 translocated from the cytoplasm to the nucleus. This translocation was not observed in the case of D2G.

Figure 4. Nuclear translocation of arrestin3 in response to agonistic stimulation of arrestin-biased D2Rs. (A) HEK-293 cells expressing either D2Arr or D2G were treated with 1 μM DA for 5 min. Cell lysates were fractionated as described in the Materials and Methods. Co-immunoprecipitation was conducted from cytoplasmic or nuclear fractions, and the eluents were immunoblotted with antibodies against arrestin2/3. *p<0.05, **p<0.01 compared to corresponding vehicle-treated groups (n=3). #p<0.05 compared to DA/D2Arr group (n=3). (B) HEK-293 cells were transfected with arrestin3-GFP along with either D2Arr or D2G. Following a 7h treatment with 10 ng/mL leptomycin B (LMB), which inhibits exportin1, the major karyopherin receptor that mediates the nuclear export of a wide range of proteins, the cells were subsequently treated with 10 µM DA for 5 min. The colocalization between arrestin3 and DAPI, which was assessed and quantified using Pearson’s correlation coefficient (γ value), significantly increased upon DA treatment in cells expressing D2Arr, not D2G. Statistical differences among the experimental groups were verified by one-way ANOVA with Tukey’s post hoc. ***p<0.001 compared to other groups (n=5). The expression levels of the receptors were measured to be approximately 1.7-1.9 pmol/mg protein.

These results indicate that, concerning D2R, G protein-dependent ERK activation takes place in the cytosol, while arrestin-mediated activation occurs in both the nucleus and cytosol.

Importin-mediated nuclear entry of arrestin3 is responsible for the D2Arr-mediated ERK activation in the nucleus

MEK phosphorylates ERK in the threonine and tyrosine residues in the activation loop, inducing the conformational changes required for its full activation (Wortzel and Seger, 2011). The phosphorylation of the Ser-Pro-Ser (SPS) motif of ERK facilitates its binding to importin 7, which allows the nuclear entry of ERK (Maik-Rachline et al., 2019) and ERK-mediated activation of the transcription factors Elk-1 and CREB in the nucleus (Davis et al., 2000).

Because D2Arr-mediated ERK activation in the nucleus required arrestin3 expression, it is probable that the nuclear entry of arrestin3 is required for ERK activation. Considering that nuclear entry of arrestin3 is mediated by the importin complex (Zhang et al., 2020), it is likely that importin is needed for D2Arr-mediated ERK activation. Indeed, knockdown of importin 1 abolished D2Arr-mediated ERK activation and nuclear entry of arrestin3 (Fig. 5A, 5B).

Figure 5. Importin is involved in ERK activation mediated via arrestin-dependent signaling pathway of D2R that accompanies nuclear entry of arrestin3. (A) Con-KD or importin β1-KD (Impo-KD) HEK-293 cells were transfected with the cDNA of D2G or D2Arr. Cells were treated with 1 μM DA for 5 min, and ERK assay was conducted. **p<0.01, ***p<0.001 compared to the corresponding vehicle-treated groups (n=3). Cell lysates from Con-KD and importin β1-KD cells were immunoblotted with antibodies against importin β1 and actin. The knockdown efficiency of importin β1 was about 85%. (B) Con-KD and importin β1-KD HEK-293 cells were transfected with the cDNAs of D2Arr and FLAG-arrestin3. Cells were treated with 1 μM DA for 5 min, and the cell lysates were fractionated. Cytoplasmic and nuclear fractions were immunoblotted with antibodies against FLAG, lamin b1, and β-actin. **p<0.01 compared to corresponding vehicle-treated cells (n=3).

ERK activation via D2R signaling and arrestin3 dependence initiates within the nucleus, followed by the subsequent translocation of activated ERK to the cytosol

Our results showed that the D2Arr activates the ERK both in the cytosol and nucleus. We hypothesized that the nuclear entry of arrestin3 is required for D2Arr-mediated ERK activation in the nucleus. Therefore, we expected that knocking down importin β1, which mediates the nuclear entry of arrestin3, would partially block D2Arr-mediated ERK activation. However, our results showed that knocking down either arrestin3 or importin β1 almost completely abolished D2Arr-mediated ERK activation.

Therefore, our conjecture was that arrestin3 facilitates ERK activation within the nucleus, and subsequently, the phosphorylated ERK relocates to the cytosol (Adachi et al., 2000). As illustrated in Fig. 6, the treatment leptomycin B (LMB), a substance that hinders the nuclear export of multiple proteins, impeded the activation of ERK in the cytosol. This observation implies that the phosphorylated ERK originating from the nucleus thereafter undergoes translocation to the cytosol.

Figure 6. D2R- and arrestin3-mediated ERK activation initiates within the nucleus, followed by the subsequent translocation of activated ERK to the cytosol. HEK-293 cells expressing D2Arr were treated with vehicle or 10 ng/mL LMB for 7h. Following this, the cells were treated with 1 μM DA for a time period ranging from 0 to 10 min. Subsequently, the cell lysates were divided into cytoplasmic and nuclear fractions, and immunoblotting was performed using antibodies specific to p-ERK, ERK2, lamin b1, and β-actin. ***p<0.001 compared to 0 min/Veh group; ##p<0.01 compared to corresponding LMB-pretreated group (n=3). The expression levels of the receptors were measured to be approximately 1.4-1.5 pmol/mg protein.

Fig. 7 shows a summary of the current study. D2R signals through G protein- and arrestin-dependent pathways. The G protein pathway mediates the inhibition of adenylyl cyclase, whereas the arrestin pathway mediates arrestin ubiquitination and receptor endocytosis. Both pathways mediate ERK activation, but the subcellular regions in which ERK activation occurs are different. The G protein pathway mediates ERK activation in the cytosol, but the arrestin pathway, which also mediates ERK activation in the nucleus, accompanies the translocation of arrestin to the nucleus. Following its activation in the nucleus, ERK undergoes subsequent migration to the cytosol.

Figure 7. Diagram showing the molecular cascades involved in the biased signaling pathways of dopamine D2 receptor. The G protein pathway is responsible for D2R-mediated adenylyl cyclase inhibition, while the arrestin pathway is involved in arrestin ubiquitination and receptor endocytosis. In terms of regulating ERK, the G protein pathway induces ERK activation in the cytosol. In contrast, the arrestin pathway facilitates ERK activation within the nucleus, followed by the translocation of phosphorylated ERK to the cytosol. Gi/o, inhibitory/other heterotrimeric guanine nucleotide-binding proteins; AC, adenylyl cyclase; impo, importin α/β; ERK-p, phospho-ERK.

Signaling of the GPCRs through arrestin-mediated pathway is usually measured through receptor endocytosis or translocation of arrestin from the cytosol to the plasma membrane. Given that arrestin ubiquitination is needed for the translocation of arrestins to the plasma membrane and receptor endocytosis (Shenoy et al., 2001, 2007), the initial cellular event that determines the arrestin-dependent pathway of GPCRs is likely to be Mdm2-mediated arrestin ubiquitination. In a previous study, it was reported that a key factor determining Mdm2-mediated arrestin ubiquitination is arrestin nuclear entry through importin; and that Gβγ and clathrin are involved in the nuclear entry of arrestin (Zhang et al., 2020). Therefore, it would be important to elucidate how Gβγ and clathrin affect arrestin nuclear entry via importin to understand the mechanistic aspects of the arrestin-dependent pathway of GPCRs.

Figs. 3 and 6 show that the G protein-dependent signaling pathway of D2R mediates ERK activation in the cytosol. In contrast, the arrestin-dependent pathway facilitates the activation of ERK in the nucleus, followed by the translocation of the activated ERK to the cytosol. These results differ from those obtained previously utilizing angiotensin II type 1 receptor (Tohgo et al., 2002; Ahn et al., 2004) as well as the chimeric receptor between β2 adrenergic receptor and the vasopressin type 2 receptor (Tohgo et al., 2003). These studies have shown that the rapid and late phases of ERK activation, which are presumably G protein-dependent and arrestin-dependent, respectively; the former leading to the accumulation of activated ERK in the nucleus and the latter in the cytosol.

It is not currently clear why such differences are observed between experiments, but possible explanations could be proposed. First, some of the experimental results were obtained through mutants or chimeric receptors between different GPCRs, and these modified receptors may induce changes that are more diverse than desired. Second, it may not be informative to compare D2R and angiotensin II type 1 receptor because they show a marked difference in their binding ability to arrestin (Oakley et al., 2000). A more definitive conclusion seems to require further data obtained for the same class of GPCRs and under more native experimental conditions.

It has been observed in a previous study that the knockdown of arrestins does not affect D2R-mediated ERK activation (Quan et al., 2008), which contradicts with more recent reports of biased mutant D2Rs and ligands (Lan et al., 2009; Peterson et al., 2015b; Chen et al., 2016; McCorvy et al., 2018; Bonifazi et al., 2019; Donthamsetti et al., 2020; Sanchez-Soto et al., 2020).

Several plausible explanations could be considered for this discrepancy. For example, if the G protein and arrestin pathways are complementary for ERK activation, either of them would be sufficient for ERK activation when the other is blocked. Another reason may be the dual function of arrestins; while they mediate the arrestin pathway for ERK activation, arrestins might concurrently desensitize the G protein pathway. Therefore, when endogenous arrestin is removed, G protein-dependent ERK activation might be enhanced as the arrestin-mediated D2R desensitization is disabled. Another explanation is from a technical point of view; shRNA-mediated knockdown does not completely eliminate the endogenous target protein. If a small amount of arrestin is sufficient to support its own signaling pathway, there may not be any appreciable change in ERK activation even when the levels of arrestin are largely reduced.

Overall, this study investigated the two biased signaling of D2R and identified interesting differences between the two. The G protein pathway mediated the inhibition of cAMP production and ERK activation in the cytosol, while the arrestin pathway governed the ERK activation in the nucleus along with functions related to the intracellular trafficking of GPCRs. These results will be of significant help in understanding which functions of GPCRs are regulated through biased signaling pathways and, further, in explaining the in vivo therapeutic effects of newly developed biased ligands.


This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (KRF-2020R1I1A3062151); Korea Drug Development Fund funded by Ministry of Science and ICT, Ministry of Trade, Industry, and Energy, and Ministry of Health and Welfare (HN21C1076).


The authors have no conflicts of interest to declare.

  1. Adachi, M., Fukuda, M. and Nishida, E. (2000) Nuclear export of MAP kinase (ERK) involves a MAP kinase kinase (MEK)-dependent active transport mechanism. J. Cell Biol. 148, 849-856.
    Pubmed KoreaMed CrossRef
  2. Ahn, S., Shenoy, S. K., Wei, H. and Lefkowitz, R. J. (2004) Differential kinetic and spatial patterns of beta-arrestin and G protein-mediated ERK activation by the angiotensin II receptor. J. Biol. Chem. 279, 35518-35525.
    Pubmed CrossRef
  3. Alvarez-Curto, E., Inoue, A., Jenkins, L., Raihan, S. Z., Prihandoko, R., Tobin, A. B. and Milligan, G. (2016) Targeted elimination of G proteins and arrestins defines their specific contributions to both intensity and duration of G protein-coupled receptor signaling. J. Biol. Chem. 291, 27147-27159.
    Pubmed KoreaMed CrossRef
  4. Benovic, J. L., Kuhn, H., Weyand, I., Codina, J., Caron, M. G. and Lefkowitz, R. J. (1987) Functional desensitization of the isolated beta-adrenergic receptor by the beta-adrenergic receptor kinase: potential role of an analog of the retinal protein arrestin (48-kDa protein). Proc. Natl. Acad. Sci. U. S. A. 84, 8879-8882.
    Pubmed KoreaMed CrossRef
  5. Beom, S., Cheong, D., Torres, G., Caron, M. G. and Kim, K. M. (2004) Comparative studies of molecular mechanisms of dopamine D2 and D3 receptors for the activation of extracellular signal-regulated kinase. J. Biol. Chem. 279, 28304-28314.
    Pubmed CrossRef
  6. Bonifazi, A., Yano, H., Guerrero, A. M., Kumar, V., Hoffman, A. F., Lupica, C. R., Shi, L. and Newman, A. H. (2019) Novel and potent dopamine D(2) receptor Go-protein biased agonists. ACS Pharmacol. Transl. Sci. 2, 52-65.
    Pubmed KoreaMed CrossRef
  7. Burns, D. L. (1988) Subunit structure and enzymic activity of pertussis toxin. Microbiol. Sci. 5, 285-287.
  8. Chen, X., Mccorvy, J. D., Fischer, M. G., Butler, K. V., Shen, Y., Roth, B. L. and Jin, J. (2016) Discovery of G protein-biased D2 dopamine receptor partial agonists. J. Med. Chem. 59, 10601-10618.
    Pubmed KoreaMed CrossRef
  9. Cho, D., Zheng, M., Min, C., Ma, L., Kurose, H., Park, J. H. and Kim, K. M. (2010) Agonist-induced endocytosis and receptor phosphorylation mediate resensitization of dopamine D(2) receptors. Mol. Endocrinol. 24, 574-586.
    Pubmed KoreaMed CrossRef
  10. Davis, S., Vanhoutte, P., Pages, C., Caboche, J. and Laroche, S. (2000) The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J. Neurosci. 20, 4563-4572.
    Pubmed KoreaMed CrossRef
  11. Donthamsetti, P., Gallo, E. F., Buck, D. C., Stahl, E. L., Zhu, Y., Lane, J. R., Bohn, L. M., Neve, K. A., Kellendonk, C. and Javitch, J. A. (2020) Arrestin recruitment to dopamine D2 receptor mediates locomotion but not incentive motivation. Mol. Psychiatry 25, 2086-2100.
    Pubmed KoreaMed CrossRef
  12. Ferguson, S. S., Downey, W. E., Colapietro, A. M., Barak, L. S., Menard, L. and Caron, M. G. (1996) Role of beta-arrestin in mediating agonist-promoted G protein-coupled receptor internalization. Science 271, 363-366.
    Pubmed CrossRef
  13. Grundmann, M., Merten, N., Malfacini, D., Inoue, A., Preis, P., Simon, K., Ruttiger, N., Ziegler, N., Benkel, T., Schmitt, N. K., Ishida, S., Muller, I., Reher, R., Kawakami, K., Inoue, A., Rick, U., Kuhl, T., Imhof, D., Aoki, J., Konig, G. M., Hoffmann, C., Gomeza, J., Wess, J. and Kostenis, E. (2018) Lack of beta-arrestin signaling in the absence of active G proteins. Nat. Commun. 9, 341.
    Pubmed KoreaMed CrossRef
  14. Gurevich, V. V. and Gurevich, E. V. (2020) Biased GPCR signaling: possible mechanisms and inherent limitations. Pharmacol. Ther. 211, 107540.
    Pubmed KoreaMed CrossRef
  15. Kim, K. M., Valenzano, K. J., Robinson, S. R., Yao, W. D., Barak, L. S. and Caron, M. G. (2001) Differential regulation of the dopamine D2 and D3 receptors by G protein-coupled receptor kinases and beta-arrestins. J. Biol. Chem. 276, 37409-37414.
    Pubmed CrossRef
  16. Kim, M. L., Sorg, I. and Arrieumerlou, C. (2011) Endocytosis-independent function of clathrin heavy chain in the control of basal NF-kappaB activation. PLoS One 6, e17158.
    Pubmed KoreaMed CrossRef
  17. Lan, H., Liu, Y., Bell, M. I., Gurevich, V. V. and Neve, K. A. (2009) A dopamine D2 receptor mutant capable of G protein-mediated signaling but deficient in arrestin binding. Mol. Pharmacol. 75, 113-123.
    Pubmed KoreaMed CrossRef
  18. Lichtarge, O., Bourne, H. R. and Cohen, F. E. (1996) An evolutionary trace method defines binding surfaces common to protein families. J. Mol. Biol. 257, 342-358.
    Pubmed CrossRef
  19. Lohse, M. J., Benovic, J. L., Codina, J., Caron, M. G. and Lefkowitz, R. J. (1990) beta-Arrestin: a protein that regulates beta-adrenergic receptor function. Science 248, 1547-1550.
    Pubmed CrossRef
  20. Luttrell, L. M., Ferguson, S. S., Daaka, Y., Miller, W. E., Maudsley, S., Della Rocca, G. J., Lin, F., Kawakatsu, H., Owada, K., Luttrell, D. K., Caron, M. G. and Lefkowitz, R. J. (1999) Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science 283, 655-661.
    Pubmed CrossRef
  21. Maik-Rachline, G., Hacohen-Lev-Ran, A. and Seger, R. (2019) Nuclear ERK: mechanism of translocation, substrates, and role in cancer. Int. J. Mol. Sci. 20, 1194.
    Pubmed KoreaMed CrossRef
  22. Mccorvy, J. D., Butler, K. V., Kelly, B., Rechsteiner, K., Karpiak, J., Betz, R. M., Kormos, B. L., Shoichet, B. K., Dror, R. O., Jin, J. and Roth, B. L. (2018) Structure-inspired design of beta-arrestin-biased ligands for aminergic GPCRs. Nat. Chem. Biol. 14, 126-134.
    Pubmed KoreaMed CrossRef
  23. Min, X., Sun, N., Wang, S., Zhang, X. and Kim, K. M. (2023) Sequestration of Gbetagamma by deubiquitinated arrestins into the nucleus as a novel desensitization mechanism of G protein-coupled receptors. Cell Commun. Signal. 21, 11.
    Pubmed KoreaMed CrossRef
  24. Min, X., Zhang, X., Sun, N., Acharya, S. and Kim, K. M. (2019) Mdm2-mediated ubiquitination of PKCbetaII in the nucleus mediates clathrin-mediated endocytic activity. Biochem. Pharmacol. 170, 113675.
    Pubmed CrossRef
  25. Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G. and Barak, L. S. (2000) Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J. Biol. Chem. 275, 17201-17210.
    Pubmed CrossRef
  26. Peterson, S. M., Pack, T. F. and Caron, M. G. (2015a) Receptor, ligand and transducer contributions to dopamine D2 receptor functional selectivity. PLoS One 10, e0141637.
    Pubmed KoreaMed CrossRef
  27. Peterson, S. M., Pack, T. F., Wilkins, A. D., Urs, N. M., Urban, D. J., Bass, C. E., Lichtarge, O. and Caron, M. G. (2015b) Elucidation of G-protein and beta-arrestin functional selectivity at the dopamine D2 receptor. Proc. Natl. Acad. Sci. U. S. A. 112, 7097-7102.
    Pubmed KoreaMed CrossRef
  28. Quan, W., Kim, J. H., Albert, P. R., Choi, H. and Kim, K. M. (2008) Roles of G protein and beta-arrestin in dopamine D2 receptor-mediated ERK activation. Biochem. Biophys. Res. Commun. 377, 705-709.
    Pubmed CrossRef
  29. Reiter, E., Ahn, S., Shukla, A. K. and Lefkowitz, R. J. (2012) Molecular mechanism of beta-arrestin-biased agonism at seven-transmembrane receptors. Annu. Rev. Pharmacol. Toxicol. 52, 179-197.
    Pubmed KoreaMed CrossRef
  30. Sanchez-Soto, M., Verma, R. K., Willette, B. K. A., Gonye, E. C., Moore, A. M., Moritz, A. E., Boateng, C. A., Yano, H., Free, R. B., Shi, L. and Sibley, D. R. (2020) A structural basis for how ligand binding site changes can allosterically regulate GPCR signaling and engender functional selectivity. Sci. Signal. 13, eaaw5885.
    Pubmed KoreaMed CrossRef
  31. Shenoy, S. K., Barak, L. S., Xiao, K., Ahn, S., Berthouze, M., Shukla, A. K., Luttrell, L. M. and Lefkowitz, R. J. (2007) Ubiquitination of beta-arrestin links seven-transmembrane receptor endocytosis and ERK activation. J. Biol. Chem. 282, 29549-29562.
    Pubmed KoreaMed CrossRef
  32. Shenoy, S. K., Mcdonald, P. H., Kohout, T. A. and Lefkowitz, R. J. (2001) Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin. Science 294, 1307-1313.
    Pubmed CrossRef
  33. Soergel, D. G., Subach, R. A., Burnham, N., Lark, M. W., James, I. E., Sadler, B. M., Skobieranda, F., Violin, J. D. and Webster, L. R. (2014) Biased agonism of the mu-opioid receptor by TRV130 increases analgesia and reduces on-target adverse effects versus morphine: a randomized, double-blind, placebo-controlled, crossover study in healthy volunteers. Pain 155, 1829-1835.
    Pubmed CrossRef
  34. Tohgo, A., Choy, E. W., Gesty-Palmer, D., Pierce, K. L., Laporte, S., Oakley, R. H., Caron, M. G., Lefkowitz, R. J. and Luttrell, L. M. (2003) The stability of the G protein-coupled receptor-beta-arrestin interaction determines the mechanism and functional consequence of ERK activation. J. Biol. Chem. 278, 6258-6267.
    Pubmed CrossRef
  35. Tohgo, A., Pierce, K. L., Choy, E. W., Lefkowitz, R. J. and Luttrell, L. M. (2002) beta-Arrestin scaffolding of the ERK cascade enhances cytosolic ERK activity but inhibits ERK-mediated transcription following angiotensin AT1a receptor stimulation. J. Biol. Chem. 277, 9429-9436.
    Pubmed CrossRef
  36. Urban, J. D., Clarke, W. P., Von Zastrow, M., Nichols, D. E., Kobilka, B., Weinstein, H., Javitch, J. A., Roth, B. L., Christopoulos, A., Sexton, P. M., Miller, K. J., Spedding, M. and Mailman, R. B. (2007) Functional selectivity and classical concepts of quantitative pharmacology. J. Pharmacol. Exp. Ther. 320, 1-13.
    Pubmed CrossRef
  37. Violin, J. D., Crombie, A. L., Soergel, D. G. and Lark, M. W. (2014) Biased ligands at G-protein-coupled receptors: promise and progress. Trends Pharmacol. Sci. 35, 308-316.
    Pubmed CrossRef
  38. Violin, J. D., Dewire, S. M., Yamashita, D., Rominger, D. H., Nguyen, L., Schiller, K., Whalen, E. J., Gowen, M. and Lark, M. W. (2010) Selectively engaging beta-arrestins at the angiotensin II type 1 receptor reduces blood pressure and increases cardiac performance. J. Pharmacol. Exp. Ther. 335, 572-579.
    Pubmed CrossRef
  39. Wei, H., Ahn, S., Shenoy, S. K., Karnik, S. S., Hunyady, L., Luttrell, L. M. and Lefkowitz, R. J. (2003) Independent beta-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2. Proc. Natl. Acad. Sci. U. S. A. 100, 10782-10787.
    Pubmed KoreaMed CrossRef
  40. Wisler, J. W., Rockman, H. A. and Lefkowitz, R. J. (2018) Biased G protein-coupled receptor signaling: changing the paradigm of drug discovery. Circulation 137, 2315-2317.
    Pubmed KoreaMed CrossRef
  41. Wortzel, I. and Seger, R. (2011) The ERK cascade: distinct functions within various subcellular organelles. Genes Cancer 2, 195-209.
    Pubmed KoreaMed CrossRef
  42. Zhang, X., Min, X., Wang, S., Sun, N. and Kim, K. M. (2020) Mdm2-mediated ubiquitination of beta-arrestin2 in the nucleus occurs in a Gbetagamma- and clathrin-dependent manner. Biochem. Pharmacol. 178, 114049.
    Pubmed CrossRef
  43. Zhang, X., Wang, F., Chen, X., Li, J., Xiang, B., Zhang, Y. Q., Li, B. M. and Ma, L. (2005) Beta-arrestin1 and beta-arrestin2 are differentially required for phosphorylation-dependent and -independent internalization of delta-opioid receptors. J. Neurochem. 95, 169-178.
    Pubmed CrossRef
  44. Zheng, M., Cheong, S. Y., Min, C., Jin, M., Cho, D. I. and Kim, K. M. (2011) beta-arrestin2 plays permissive roles in the inhibitory activities of RGS9-2 on G protein-coupled receptors by maintaining RGS9-2 in the open conformation. Mol. Cell. Biol. 31, 4887-4901.
    Pubmed KoreaMed CrossRef

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