Biased signaling agonist of dopamine D3 receptor induces receptor internalization independent of β-arrestin recruitment

Pharmacological Research 143 (2019) 48–57

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Biased signaling agonist of dopamine D3 receptor induces receptor internalization independent of β-arrestin recruitment Wei Xua, Maarten E.A. Reithc, Lee-Yuan Liu-Chend, Sandhya Kortagerea,b,

T

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a

Department of Microbiology and Immunology, Drexel University College of Medicine, PA 19129, United States Department of Pharmacology and Physiology, Drexel University College of Medicine, PA 19102, United States c Department of Psychiatry, Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, NY 10016, United States d Department of Pharmacology, Temple University School of Medicine, Philadelphia, PA 19140, United States b

A R T I C LE I N FO

A B S T R A C T

Keywords: β-Arrestin 1 β-Arrestin 2 Biased agonism Dopamine D3 receptor (D3R) Clathrin Caveolin Dynamins G protein-coupled receptor kinase 2 (GRK2) GRK-interacting protein 1 (GIT1) Internalization

Agonist-induced internalization of G protein-coupled receptors (GPCRs) is a significant step in receptor kinetics and is known to be involved in receptor down-regulation. However, the dopamine D3 receptor (D3R) has been an exception wherein agonist induces D3Rs to undergo desensitization followed by pharmacological sequestration – which is defined as the sequestration of cell surface receptors into a more hydrophobic fraction within the plasma membrane without undergoing the process of receptor internalization. Pharmacological sequestration renders the receptor in an inactive state on the membrane. In our previous study we demonstrated that a novel class of D3R agonists exemplified by SK608 have biased signaling properties via the G-protein dependent pathway and do not induce D3R desensitization. In this study, using radioligand binding assay, immunoblot or immunocytochemistry methods, we observed that SK608 induced internalization of human D3R stably expressed in CHO, HEK and SH-SY5Y cells which are derived from neuroblastoma cells, suggesting that it is not a cell-type specific event. Further, we have evaluated the potential mechanism of D3R internalization induced by these biased signaling agonists. SK608-induced D3R internalization was time- and concentration-dependent. In comparison, dopamine induced D3R upregulation and pharmacological sequestration in the same assays. GRK2 and clathrin/dynamin I/II are the key molecular players in the SK608-induced D3R internalization process, while β-arrestin 1/2 and GRK-interacting protein 1(GIT1) are not involved. These results suggest that SK608promoted D3R internalization is similar to the type II internalization observed among peptide binding GPCRs.

1. Introduction Agonists bind to G-protein coupled receptors (GPCRs) at the plasma membrane and initiate conformational changes resulting in activation of one or more downstream signaling pathways. Most GPCRs then undergo three temporally distinct regulatory processes: desensitization (seconds to hours), internalization (minutes to hours), and downregulation (hours to days). Desensitization is referred to as attenuation of responsiveness to agonists following prolonged or repeated activation, which is a multistep process involving GRK-mediated receptor phosphorylation and β-arrestin recruitment [1]. Internalization or endocytosis is a process of rapid agonist-induced movement of the phosphorylated GPCR into intracellular compartments from the plasma membrane, where it is able to bind hydrophobic ligands, but not hydrophilic ligands [2–5]. The internalized GPCRs can propagate signals through β-arrestin-dependent novel transduction pathways.

Subsequently, the receptors are sorted either to recycling pathways returning to the plasma membranes or degraded or downregulated through lysosomal or proteasomal pathways [6–8]. Although D2R and D3Rs share 46% overall amino acid homology and 78% identity in the seven transmembrane domains and they both recruit of the inhibitory G-protein (Gαi) [9–11], D2R and D3R have distinct regulatory or signaling mechanisms [12,13]. While D2R undergoes dopamine (DA)-induced receptor phosphorylation and β-arrestin mediated translocation and internalization, D3R has not been documented to undergo significant phosphorylation, translocation and internalization, but instead has stronger constitutive interaction with βarrestin than D2R [12,14,15]. Several studies have also demonstrated that DA does not induce D2R desensitization under normal conditions [16–18], but does so only under prolonged treatment of up to 24 h [19,20]. However, DA or other agonists readily induce desensitization of D3R [17,21,22]. In addition, agonist-activated D3Rs are also

⁎ Corresponding author at: Department of Microbiology and Immunology, Drexel University College of Medicine, 2900, Queen Lane, Philadelphia, PA 19129, United States. E-mail address: [email protected] (S. Kortagere).

https://doi.org/10.1016/j.phrs.2019.03.003 Received 4 January 2019; Received in revised form 1 March 2019; Accepted 1 March 2019 Available online 04 March 2019 1043-6618/ © 2019 Elsevier Ltd. All rights reserved.

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demonstrated a time-dependent increase in the number of internalized receptors, but DA-induced D3R upregulation was not time-dependent (Fig. 1B). PKC activator phorbol-12-myristate-13-acetate (PMA), a positive control, significantly induced D3R internalization following 60 min exposure, but not at 24 h (Fig. 1B), a finding that is in agreement with previous reports on PMA-induced heterologous D3R internalization in HEK293-D3R cells [24]. Assessment of GPCR internalization with radiolabeled ligand binding of hydrophobic and hydrophilic ligands has been demonstrated to be an effective and accurate method for quantifying surface receptor internalization [23,31]. A requirement for successful application of this method is that no unwashed agonist remains to interfere with subsequent radioligand binding. We found that DA or SK608 and its analogs bound to the surface receptors could be easily washed out with cold PBS buffer unlike other hydrophobic ligands and also had no further effect on the D3R binding affinity and Bmax of [3H]methylspiperone (Supplementary Figure S1) (Min, Zheng et al. 2013). The results on DA-induced D3R pharmacological sequestration and D2R internalization in the present study using the radioligand binding method are in agreement with previous reports [12,22,32–35]. In order to determine whether SK608-induced D3R internalization and DA-induced surface D3R upregulation are cell type specific events, we assessed the effects of DA, PMA or SK608 on surface expression of D3R in HEK-D3R cells or SH-SY5Y-D3R cells. As shown in Supplementary Figure S2, pretreatment with 3 μM SK608 or 100 nM PMA for 60 min significantly induced HEK-D3R internalization, however, DA did not upregulate the surface D3R expression in HEK-D3R cells. Interestingly, in SH-SY5Ycells SK608 induced D3R internalization, DA or PMA did not have any effects. D2R and D3R were reported to be endogenously expressed in differentiated SH-SY5Y cells [36,37], but we found that D2R-like binding with [3H]methylspiperone was not detectable with blank undifferentiated SH-SY5Y cells (105 cells/each reaction tube), thus excluding the potential influence of endogenous D2R or D3R expression. Although internalization of certain GPCRs is described to be cell type-dependent which is attributed to disparity in cellular contents of essential components involved in internalization [38,39], SK608-induced D3R internalization was observed in all the three cell types. In contrast, DA-induced surface D3R upregulation was only observed in CHO-D3R cells and PMA-induced internalization was observed in CHO-D3R and HEK-D3R cells but not SH-SY5Y-D3R cells. These results suggest that only SK608 could internalize D3R irrespective of the cell line. D3R internalization was confirmed with immunoblotting in addition to radioligand binding method. HEK-FLAG-D3R cell membranes were resolved with SDS–PAGE and the receptor was detected with immunoblotting with M1 anti-FLAG monoclonal antibody. FLAG-D3R was observed as a broad and diffuse 45–55 kDa band and a less diffuse 35–40 kDa band. In non-transfected HEK cells, no protein bands were detected (data not shown) at the reported molecular weights suggesting that the 45–55 kDa band is the glycosylated form of D3R and the 35–40 kDa band may be the-partially glycosylated form of D3R [35]. As shown in Fig. 2A-B, pretreatment with 0.1 μM PMA or 3 μM SK608 for 30 min significantly reduced the glycosylated form and increased the partially glycosylated form, but treatment with 1 μM DA did not. This decrease in glycosylated form may occur when GPCRs undergo internalization or during down regulation when receptors get degraded. In analyzing our results from the binding assay with [3H]methylspiperine, we observed a reduction in only the surface D3R receptors but the total number of D3Rs remain unchanged, suggesting that PMA and SK608 induced receptor internalization, but not downregulation. Our results also showed that SK608 or PMA increased D3R partially glycosylated forms suggesting that SK608 or PMA may enhance partially glycosylated D3R export. D3R internalization was also validated by immunocytochemistry. HEK FLAG-D3R cells were incubated with M1 mouse anti-FLAG antibody for 30 min at 37C and treated with vehicle or 0.1 μM PMA or 3 μM

evidenced to undergo pharmacological sequestration, which is defined as the sequestration of cell surface receptors into a more hydrophobic fraction within the plasma membrane without undergoing the process of receptor internalization into intracellular regions [22,23]. An integral part of this process is a receptor conformational change rendering the relevant binding site inaccessible to hydrophilic ligands. Pharmacological sequestration of D3Rs has been shown to be a Gβγ- and βarrestin-dependent mechanism [22]. Like most class A GPCRs, D3Rs are known to undergo PKC-mediated heterologous desensitization and internalization involving the clathrin and dynamin dependent pathway [24]. In contrast, none of the commonly known D3R agonists promote significant D3R internalization. We have recently designed and developed novel compounds SK609 and its analogs that are highly selective D3R agonists and have atypical signaling properties [25]. SK609 has shown efficacy in reversing akinesia and reducing L-dopa induced dyskinesia in hemiparkinsonian rats [26]. Moreover, SK609 and its analogs do not induce D3R desensitization in vitro and exhibit biased signaling properties that are mediated through the G-protein dependent pathways [27]. In this study, we characterized the agonist-mediated internalization of these biased signaling agonists using human D3R stably expressed in CHO cells (CHOD3R), HEK293 cells (HEK-D3R) and neuronal-like SH-SY5Y cells (SHSY5Y-D3R). We also investigated the potential mechanism and molecular players involved in the process of D3R internalization induced by SK608. 2. Results and discussion We have recently designed and tested a novel series of phenylethylamine molecules with high selectivity for D3R and have agonists activity. SK609 a leading member of this series had a binding affinity of 283 nM, SK608 a para chloro analog had an affinity of 103 nM and SK213, a ortho chloro analog had an affinity of 3.7 μM at the high affinity site of D3R as assessed by radioligand binding assay. In the same assay, none of the molecules had measurable affinity for D2R [27]. All three molecules had functional selectivity for D3R and elicited no functional response at D2R. 2.1. DA induced D2R internalization but upregulated surface D3R in CHO cells We first assessed and compared whether exposure to DA induced internalization of the human D2R or D3R. CHO-D2R or CHO-D3R cells were treated with 10 μM DA or vehicle at 37 °C for 60 min and internalized receptors were determined by [3H]methylspiperone binding. Consistent with previous studies [12,22,24,28–30], DA (10 μM, 60 min) promoted significant internalization of CHO-D2R, with about 35% of surface receptors being translocated to the cell interior (Fig. 1A). However, DA did not induce CHO-D3R internalization, a finding in agreement with previous reports on HEK293-D3R cells [12,22,24]. Moreover, DA increased the surface expression of D3R by about 20% of the control cells, i.e. causing upregulation. 2.2. Novel D3R selective agonist SK608 significantly induced D3R internalization Our previous studies demonstrated that DA induced D3R desensitization but D3R selective agonists SK609 and its analogs did not induce desensitization in HEK-D3R cells [27]. In the present study, we determined whether SK609 and its analogs (SK608, SK213) promoted CHO-D3R internalization. As shown in Fig. 1B, while 10 μM DA significantly increased surface D3R, 10 μM of SK609, SK608 or SK213 induced D3R internalization following a 60-min or 24-h exposure. Further, SK608-induced internalization or DA-induced upregulation was also dose-dependent with an EC50 value of 0.794 ± 0.02 μM or 47 ± 4 nM, respectively (Fig. 1C). SK608 at these doses also 49

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Fig. 1. (A) Comparison of DA-induced changes in cell surface D2R and D3R. CHO-D2R cells or CHO-D3R cells were pretreated with vehicle, 10 μM DA for 60 min. (B) Comparison of the effect of DA, SK609, SK213, SK608, and PMA on internalization of D3R. CHO-D3R cells were pretreated with vehicle, DA, SK609, SK213, SK608 and of phorbol 12-myristate 13-acetate (PMA) at indicated concentrations for 60 min or 24 h. (C) Dose response curves of DA and SK608 in inducing D3R upregulation or internalization and pharmacological sequestration. CHO-D3R cells were pretreated with vehicle, indicated concentrations of DA for 10 min for pharmacological sequestration and 60 min for upregulation or SK608 for 60 min for internalization and pharmacological sequestration, respectively. Post treatment in each case cells were then chilled and washed and [3H]methylspiperone (about 1 nM) binding was carried out on intact cells in Kreb’s buffer at 4 °C for 2 h as described in Material and Methods. For total and cell-surface receptors, nonspecific binding was defined as the binding in the presence of 10 μM haloperidol and 10 μM (-) sulpiride, respectively. Data were normalized against vehicle-treated cells (100%). The data represent the mean ± s.e.m of six independent experiments performed in duplicate. (A) **, P < 0.01, compared with vehicle control using student t-test. B,C - *, P < 0.05, **, P < 0.01 and ***, P < 0.001, compared with vehicle control by one-way ANOVA followed by Dunnett’s multiple comparison test.

Fig. 2. Comparison of the effect of SK608, PMA and DA on the internalization of D3R stably expressed in HEK293 cells using western blot and immunohistochemistry. (A–B) HEK-FLAGD3R cells were incubated with serum-free DMEM for 30 min and then treated with vehicle, 1 μM DA, 0.1 μM PMA or 3 μM SK608 for 30 min. Cell membranes were made and subjected to SDS − PAGE. Immunoblotting was performed using M1 mouse anti-FLAG antibody as described in Material and Methods. Plasma membrane marker GAPDH was also determined with rabbit anti- GAPDH polyclonal antibody after stripping the blots of the same PVDF membrane. Glycosylated form band (A) and partially glycosylated form band (B) were quantified by densitometry and normalized to GAPDH, respectively. The graphs represent mean ± s.e.m. of five independent experiments as % of vehicle control (100%). *P < 0.05 compared with vehicle control by one-way ANOVA followed by Dunnett’s multiple-comparison test. (C-F) HEK-FLAG-D3R cells grown on culture slides for 48 h were washed and incubated with serum-free DMEM for 30 min. Cells were incubated with M1 mouse anti-FLAG antibody for 30 min and then treated with vehicle, 1 μM DA, 0.1 μM PMA or 3 μM SK608 for 30 min. Cells were fixed with paraformaldehyde, permeated, stained with goat anti-mouse IgG conjugated with Alexa-Fluor 488. Images were acquired using 40x objective.

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SK608 for 30 min and then fixed and permeabilized and were imaged using goat anti-mouse IgG conjugated with Alexa Fluor 488. As shown as Fig. 2C and D, vehicle or DA treatment did not lead to any changes in FLAG-D3R localization with the majority of D3Rs being localized to the cell surface. In contrast, in 0.1 μM PMA or 3 μM SK608-treated cells, most of the FLAG-D3Rs were localized to the intracellular region of the cells and were observed as punctate aggregates (Fig. 2E and F). These observations of D3R distribution in response to DA, PMA and SK608 are in complete agreement with our radioligand binding assay and immunoblot experiment results. All three results confirm that SK608 induces D3R internalization. Differences in GPCR density may induce variations in the distribution of the receptors in plasma membranes and internal membranes which could influence the kinetics of the receptor. Therefore, it is critical to evaluate the influence of receptor expression on receptor internalization. In our studies, the expression level of D3R in CHO-D3R, HEK-D3R and SH-SY5Y-D3R was found to be 9.1, 12.2 and 1.2 pmol/ mg protein, respectively. Our results demonstrated that SK608-induced D3R internalization was similar in all the three cell lines which have varying levels of D3R expression (supplementary Figure S2 and Figs. 1 and 2), indicating that SK608-induced D3R internalization is independent of receptor density. To the best of our knowledge, these results represent the first report that a selective D3R agonist induces significant D3R homologous internalization and hence we sought to understand the molecular players involved in D3R internalization and trafficking in CHO-D3R cells.

Treatment of CHO-D3R cells with 3 μM SK608 or 1 μM DA resulted in a time-dependent increase in internalization or upregulation of CHOD3R respectively (Fig. 3A). DA-induced D3R upregulation was initially rapid with a half-maximal increase at about 2–5 min and then reached a plateau at about 10 min. SK608-induced D3R internalization, however, gradually increased without approaching a plateau until 60 min. These results indicate that SK608-induced D3R internalization and DA-induced upregulation are different regulatory processes with different dynamic properties. While extensive studies on GPCR internalization and underlying mechanisms have been performed, the transport processes from endoplasmic reticulum (ER) to the cell surface and the regulatory mechanisms of export trafficking of GPCRs are not well understood [7]. Although DA-induced surface D3R upregulation in CHO-D3R cells is a cell type-dependent phenomenon, it is a good cellular model to investigate the underlying mechanisms of D3R export or upregulation and to compare it with the process of internalization.

HEK293-D3R cells [22]. We assessed dose dependence and time course of DA or SK608 induced pharmacological sequestration in CHO-D3R cells. Cells were pretreated with vehicle, indicated doses of DA for 10 min or SK608 for 60 min at 37 °C and then washed with cold PBS buffer at 4 °C. Surface D3R binding was determined by [3H]sulpiride, which is a hydrophilic compound and binds only to the cell surface receptors on intact cells. As shown by previous studies [22], [3H]sulpiride can only bind available surface D3Rs; however, when an agonist sequesters receptors, their binding sites are buried in a more hydrophobic portion of the plasma membrane, inaccessible to [3H]sulpiride but accessible to [3H]methylspiperone as this molecule can traverse the membrane due to its hydrophobic nature. In the [3H]sulpiride binding assay, SK608 (10−9 to 10-5 M) had no effect on surface D3R, but DA dose-dependently reduced surface D3R binding with an EC50 value of 35 ± 2 nM (Fig. 1C). For time course experiments, cells were treated with or without 1 μM DA or 3 μM SK608 at 37C for the indicated intervals and surface D3R binding was determined by [3H]sulpiride. As shown in Fig. 3B, DA rapidly reduced surface D3R binding of [3H]sulpiride, indicating pharmacological sequestration occurring as early as 2 min; a plateau was reached at about 5–10 min. However, under the same treatment conditions the number of receptors on the surface was not reduced, but in fact increased as determined by [3H]methylspiperone (Fig. 3A). These results are in complete agreement with previous studies on HEK293 cells [22]. In contrast, SK608 did not affect the levels of surface receptor binding of [3H]sulpiride, although it reduced by about 20% the number of surface D3Rs as measured with [3H]methylspiperone (Fig. 1). Surface receptor binding to a hydrophilic ligand depends on several factors including a) affinity of the ligand to the receptor b) number of surface receptors and c) accessibility of the binding sites of surface receptor to the ligands. Although DA increased the surface D3R expression as determined by hydrophobic [3H]methylspiperone ligand binding (Figs. 1 and 3A), according to the published model [22] these surface receptors are pharmacologically sequestered on the membrane with less accessibility to hydrophilic ligands such as [3H]sulpiride. In contrast, D3R maintained normal surface receptor binding in the SK608-pretreated CHO-D3R cells. The mechanism involved is not clear. One possibility is that SK608-induced D3R conformational changes result in both internalization and increase of the binding site accessibility of surface D3R to sulpiride so that surface binding is equilibrated. These results suggest that activation of D3R by DA or SK608 may initiate distinct conformational changes at the plasma membrane and thus triggers D3R pharmacological sequestration or internalization respectively. The DA-induced D3R pharmacological sequestration results in the present study using the radioligand binding method, are in agreement with previous published reports [12,22,32–35].

2.4. DA, but not, SK608 induces D3R pharmacological sequestration

2.5. Molecular players involved in SK608 induced D3R internalization

Previous studies have shown that DA did not induce D3R internalization but resulted in significant pharmacological sequestration in

To assess the molecular players involved in SK608-induced internalization of D3R, CHO-D3R cells were used and the effect of Gα,

2.3. SK608-induced D3R internalization or DA-induced D3R upregulation has different kinetics

Fig. 3. Time course of (A) SK608-induced internalization and DA-induced upregulation of D3R and (B) DA or SK608-mediated pharmacological sequestration of the D3R. CHO-D3R cells were pretreated with vehicle, 1 μM DA and 3 μM SK608 for the indicated periods of time. Cells were then chilled and washed and [3H]methylspiperone (about 1 nM) binding (A) or [3H]sulpiride (about 2 nM) binding (B) to total and cell-surface receptors was carried out on intact cells as described in Fig. 1 legend. The data represent the mean ± s.e.m. of five to six independent experiments performed in duplicate.

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Fig. 4. Effect of various molecular players on SK608 induced D3R internalization or DA induced pharmacological sequestration. CHOD3R cells were treated with vehicle, 1 μM DA and 3 μM SK608 at 37 °C for 1 h after pretreatment with PTX (100 ng/ml) for 18 h, or 24 h later with transient transfection of expression constructs for GRK2-K220R and GIT1 and β-arrestin 1-V53D and β-arrestin 2-V54D or vector, respectively. D3R internalization (A) was determined by [3H]methylspiperone binding assay and pharmacological sequestration (B) by [3H]sulpiride binding assay as described in Material and Methods. The data represent the mean ± s.e.m. of five to six independent experiments performed in duplicate as % change of the control **, P < 0.01, when compared with the control; ##, P < 0.01, compared with DA or SK608; by one way ANOVA followed by Dunnett’s multiplecomparison test.

GRK2, GIT1, β-arrestin 1 and 2 were evaluated. DA-induced D3R upregulation and pharmacological sequestration were examined for comparison and contrast. The internalization of some GPCRs involves G proteins, but others do not [31,40,41]. In order to clarify whether G protein coupling is required for D3R internalization, upregulation and pharmacological sequestration, CHO-D3R cells were pretreated with pertussis toxin (PTX) (100 ng/ml) for 18 h to block pertussis toxinsensitive Gi/o proteins [42,43], followed by 1 μM DA or 3 μM SK608 treatment for 60 min. PTX, had no effect on the basal or SK608-induced internalization or DA-induced D3R upregulation and pharmacological sequestration (Fig. 4). These results demonstrate that activation/coupling of Gi/o is not necessary for D3R internalization/upregulation and pharmacological sequestration. Our finding that Gi/o had no effect on D3R internalization is similar to other GPCRs including delta or kappa opioid receptors and CCR1 receptor [31,40,41]. G protein-coupled receptor kinases (GRKs) play important roles in GPCR desensitization, internalization and trafficking in both phosphorylation dependent and independent processes [44]. Among the seven isoforms of GRKs in mammals, GRK2, GRK3, GRK5 and GRK6 are found to be expressed throughout the brain [45,46], and GRK2, GRK3 and GRK6 have critical roles in regulating both the D1R-like and the D2R-like signaling in vitro and in vivo [47]. Numerous studies have demonstrated that GRK2 play important roles in regulating the signaling and trafficking of multiple DA receptors including D2R and D3R [15,28,48,49]. In this study, we tested the roles of GRK2 in SK608induced D3R internalization and DA-induced D3R upregulation and pharmacological sequestration. CHO-D3R cells were transiently transfected with expression constructs for dominant negative mutant GRK2K220R or vector, followed by the treatment with vehicle, 1 μM DA or 3 μM SK608 for 60 min, and D3R internalization and pharmacological sequestration were determined. As a first step, the overexpression of dominant negative mutant GRK2-K220R was confirmed using western blot techniques (Supplementary Figure S3). The same construct was also used in previous studies to test agonist-induced phosphorylation and internalization of GPCRs [31,50,51]. Our results showed that GRK2-K220R completely blocked SK608-induced D3R internalization. In contrast, it had no effect on DA-induced D3R upregulation (Fig. 4A) and pharmacological sequestration (Fig. 4B). It significantly increased surface receptor, suggesting reduction of basal internalization resulting in an increase in surface D3R and (Fig. 4A). Although we have no direct evidence that SK609 induces D3R phosphorylation in vitro, our present results suggest that GRK2 is required for the basal and SK608-induced D3R internalization in CHO cells. CHO and HEK293 cells which express relatively high levels of GRK2 and β-arrestins, thus using dominant negative mutants of GRK2 and β-

arrestin isoforms rather than the overexpression of the wild types have been demonstrated to be more appropriate for understanding GPCR internalization and trafficking [31,52–54]. The effects of GRK2 on DAinduced D3R pharmacological sequestration observed in our study is similar to the previous reports that demonstrated that GRK2 knockdown using the shRNA method had no effect on this process [22]. Similarly, reports on D2R that siRNA knockdown of endogenous GRK2 significantly increased cell surface expression of the D2R and also impaired agonist-promoted D2R internalization in HEK293 T cells [55,56]. These results provide supporting evidence for the role of GRK2 in promoting both basal and agonist-induced internalization of D2R and D3R. However, overexpression of GRK2 K220R had no effect on PMAinduced D3R internalization, indicating that GRK2 is not involved in D3R heterologous internalization promoted by PMA [24]. GPCRs have different requirements for GRK-mediated phosphorylation for their internalization. It is reported that the agonist-induced desensitization, β-arrestin recruitment, or internalization of the D2R is receptor phosphorylation-independent, but GRK-mediated phosphorylation of the D2R regulates the intracellular trafficking or sorting of D2R after internalization [55,56]. These features of D2R phosphorylation are different from other GPCRs such as the β2-AR and m2 muscarinic cholinergic receptors (m2 AchR) wherein co-expression of GRK2 dominant negative mutant was shown to attenuate agonist-promoted phosphorylation and internalization of the receptors [50,57]. DA has been reported to promote weak or no phosphorylation of D3R even with overexpression of GRK2 and GRK3 and does not induce D3R internalization in HEK293 cells [12]. Interestingly, GRK4α or GRK4γ overexpression in CHO cells was found to significantly phosphorylate D3R when activated by a D2/D3 agonist PD128907 [58], although GRK4 is not known to be abundantly expressed in the brain. The GRK-interacting protein GIT1 was reported to slow internalization of β2-AR and numerous other GPCRs that are internalized through the clathrin-coated pit pathway in a β-arrestin- and dynaminsensitive manner [59,60]. We next assessed whether overexpression of GIT1 affects SK608-induced D3R internalization or DA-induced pharmacological sequestration. CHO-D3R cells were transiently transfected with expression constructs for GIT1 which was validated by western blot (Supplementary Figure S3), or vector, followed by the treatment with vehicle, 1 μM DA or 3 μM SK608 for 60 min, and D3R internalization and pharmacological sequestration were determined. The results demonstrate that overexpression of GIT1 had no effect on basal D3R internalization, SK608-induced D3R internalization (Fig. 4A), DAinduced pharmacological sequestration or upregulation (Fig. 4B). Although GRK2 plays an important role in SK608-induced D3R internalization, these results suggest that GIT1 is not involved in these 52

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processes. β-arrestins play important roles in internalization and signal transduction of most GPCRs [61,62]. In our previous studies, however, we found that while D2R/D3R agonist PD128907 potently recruits β-arrestin-2 for signaling at D3R, the D3R selective agonists SK609 and its analogs do not recruit β-arrestin-2 [27]. To examine the role of β-arrestins in SK608-induced D3R internalization or DA-induced D3R upregulation and pharmacological sequestration, CHO-D3R cells were transiently transfected with expression constructs of dominant negative mutants, namely β-arrestin1-V53D, β-arrestin2-V54D, or vector, followed by the treatment with vehicle, 1 μM DA or 3 μM SK608 for 60 min, and D3R internalization/upregulation and pharmacological sequestration were determined. Although a truncated version of β-arrestin (319–418) was reported to be more effective than the full length dominant negative mutant, V53D mutant was demonstrated to block arrestin-associated GPCR internalization and agonist-induced trafficking [54], and the full length β-arrestin1-V53D was used to study arrestin associated GPCR internalization and signaling [51,52,63]. In this study, we validated the overexpression of β-arrestin-1 V53D by western blot (Supplementary Figure S3) and demonstrated that it had no effect on basal and SK608-induced D3R internalization or DA-induced upregulation and pharmacological sequestration (Fig. 4), indicating that β-arrestin-1 was not involved in these processes. Overexpression of dominant negative mutant β-arrestin-2 V54D (Supplementary Figure S3), however, significantly reduced DA-induced pharmacological sequestration (Fig. 4B). Our results are in agreement with previous studies [22] that demonstrated that co-expression of βarrestin-2 increased the D3R pharmacological sequestration and knockdown of β-arrestin-1/2 inhibited pharmacological sequestration [22]. The basal and SK608-induced internalization or DA-induced upregulation were unaffected by these dominant negative mutants of βarrestin1 or 2 (Fig. 4). These results are similar to certain GPCRs such as the leukotriene B(4) (LTB(4)) receptor (BLT1) and protease-activated receptor-1 (PAR1) wherein β-arrestin1/2 had no effect on basal (constitutive) and agonist-induced internalization [39,64]. It has been noted that participation of GIT1 appears to be correlated with the recruitment of β-arrestin. GPCRs that internalize in a β-arrestin-dependent pathway are also found to be responsive to GIT1 [60]. Our findings that SK608-induced D3R internalization is both β-arrestins- and GIT1- independent support the notion that this novel compound does not activate or signal through the β-arrestin mediated pathways. Among most Class-A GPCRs, β-arrestins bind to phosphorylated GPCRs which leads to the recruitment of clathrin and the adaptor protein complex-2 (AP-2), components of the endocytic machinery, to induce receptor internalization [61,62,65,66]. A number of studies have indicated that DA-induced internalization of D2R in HEK293 cells is regulated by β-arrestins [18,56]. However, our results demonstrate that both the dominant negative mutants β-arrestin-1 (V53D) and βarrestin- 2 (V54D) (Fig. 4A) failed to reduce SK608-induced D3R internalization suggesting that SK608-induced D3R internalization in CHO cells is β-arrestin-independent and is different from D2R. This finding is in agreement with our previous report that SK609 and its analogues do not significantly recruit β-arrestin-2 and hence do not induce desensitization [27] and with other published studies that PMAinduced heterologous D3R internalization is also β-arrestin-2-independent [24]. GRK2 is found to interact with a number of protein partners which may play distinct roles in GPCR internalization and may be specific to a GPCR. GRK2-dependent and β-arrestin1/2-independent internalization of D3R induced by SK608 is an interesting observation that seems to deviate from well-known relationship between GRK2 and β-arrestin1/2 in receptor trafficking. However, there are instances of GPCR internalization that involve GRK2 without the involvement of β-arrestin 1/2 [44]. In the case of β-adrenergic receptor, GRK2 has been shown to bind to phosphoinositide 3-kinase (PI3K) and recruit it to the cell surface

following ligand stimulation, and this interaction was key to receptor internalization, likely by enhanced recruitment of AP2 to the receptor [67,68]. It was also reported that the C terminus of GRK2 directly binds to clathrin, which can facilitate internalization of certain GPCRs [69,70]. Arrestin-independent internalization has been reported for several members of the GPCR superfamily, including angiotensin type 1 A receptor (AT1 AR), 5-hydroxytryptamine 2 A receptor, m2 muscarinic cholinergic receptors (M2R), gonadotropin releasing hormone receptor, secretin receptor (SR), N-formyl peptide receptor, leukotriene B4 receptor 1(BLT1), prokineticin receptor 2 (PKR2), protease-activated receptor-1 (PAR1) and protease-activated receptor-4 (PAR4) [39,51,53,62,64,71–74]. Among these GPCRs, AT1 AR, SR, and M2R were shown to internalize independently of the clathrin-coated pit pathway [53,60,72]. Hence, we predict that SK608-induced D3R internalization may involve GRK2-dependent, but β-arrestin-independent, mechanisms with the involvement of other proteins such as clathrin or calveolin. GPCRs are demonstrated to undergo internalization by two main endocytic pathways which are either clathrin- or caveolin-dependent [75,76]. It is reported that D2R internalization is known to be GRK/βarrestin-dependent via both clathrin- and caveolae-mediated endocytic pathways [12,77,78] and PKC-mediated D3R heterologous internalization is caveolin-independent [24]. In order to determine whether SK608-induced D3R internalization involved the clathrin or caveolin pathways or both, CHO-D3R cells were pretreated with: (1) 0.45 M sucrose for 20 min, which was recently demonstrated to block both clathrin and caveolin endocytic pathways [79], (2) 30 μM pitstop 2 for 15 min which competitively inhibits clathrin terminal domain to inhibit clathrin-mediated internalization [80], (3) 80 μM dynasore for 30 min, which is an inhibitor of dynamin1/2 [42], (4) 3 mM MβCD for 30 min, which selectively inhibits caveolin endocytic routes without affecting clathrin-mediated internalization [34,79], and each of the 4 conditions followed by the treatment with vehicle, 1 μM DA or 3 μM SK608 for 60 min, and D3R internalization/upregulation and pharmacological sequestration were determined. Pretreatment of sucrose completely inhibited SK608-induced D3R internalization but had no effect on DA-induced D3R upregulation or pharmacological sequestration (Fig. 5A and B). Interestingly, sucrose itself did not affect surface D3R binding to hydrophilic ligand [3H] sulpiride, but significantly increased D3R binding to [3H]sulpirdie in SK608-pretreated CHO-D3R cells and this effect may result from increasing the accessibility of D3R to a hydrophilic ligand. Pretreatment with pitstop 2 (30 μM, 15 min) upregulated surface D3R and completely blocked SK608-induced D3R internalization (Fig. 5A), indicating involvement of clathrin in basal and SK608-induced D3R internalization. Our result that pitstop 2 pretreatment reduced basal D3R internalization is different from previous studies by Guo et al, which showed that Pitstop2 did not affect the surface expression of D3R in HEK293 cells [79]. These discrepancies may be associated with distinct cell types (CHO cells vs HEK293 cells) and methods used (radioactive binding assay in the present study vs antibody-based and enzyme-linked immunosorbent assay in Guo’s study). Although pitstop 2 alone did not affect surface D3R binding to [3H] sulpiride, it significantly increased surface D3R binding in SK608-pretreated CHO-D3R cells (Fig. 5B), which may result from increasing surface D3R numbers and/or accessibility to hydrophilic ligand. Pitstop2 greatly reduced DA-induced pharmacological sequestration, but had no effect on DA-induced upregulation, suggesting that the or DAinduced pharmacological sequestration may be clathrin-dependent (Fig. 5A and B). Interestingly, a previous study by Min et al. reported that DA-induced D3R pharmacological sequestration was not affected by the knockdown of the clathrin heavy chain in HEK-D3R cells [22], suggesting that clathrin is not involved in the process. However, this is in contrast with our results that suggest that DA-induced pharmacological sequestration is clathrin-dependent, which could be attributed to the cell type differences (HEK vs. CHO cells). 53

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Fig. 5. Effect of hypertonic sucrose, pitstop2, dynasore, and MβCD on D3R internalization or pharmacological sequestration. CHO-D3R cells were treated with vehicle, 1 μM DA or 3 μM SK608 at 37 °C for 1 h after pretreatment with sucrose (0.45 M, 20 min), pitstop2 (30 μM, 15 min), dynasore (80 μM, 30 min), and MβCD (3 mM, 30 min), respectively. D3R internalization (A) was determined by [3H]methylspiperone binding assay and pharmacological sequestration (B) by [3H]sulpiride binding assay as described in Material and Methods. The data represent the mean ± s.e.m. of five to six independent experiments performed in duplicate as % change of the control. **, P < 0.01, when compared with the control; # P < 0.05 and ##, P < 0.01, compared with DA or SK608 by one way ANOVA followed by a Dunnett’s multiplecomparison test.

Dynamin I/II are GTPases that are required for the detachment of newly formed vesicles from the plasma membrane and regulate both clathrin- and caveolin-dependent internalization of GPCRs [12,79,81–83]. Pretreatment with dynamin I/II inhibitor dynasore significantly upregulated surface D3R (Fig. 5A), indicating reduced basal internalization of D3R and completely abolished SK608-induced D3R internalization (Fig. 5B), indicating that the basal and SK608-induced D3R internalization are dynamin I/II-dependent. These results are similar to reports that dynamins are involved in agonist-induced internalization of several GPCRs using dynasore or the dominant negative dynamin mutant [31,42]. PMA-induced D3R internalization was found to be also dynamin-dependent as co-expression of the dominant negative dynamin I mutant inhibited PMA-induced D3R internalization [24]. Dynasore greatly enhanced DA-induced D3R upregulation but had no effect on DA-induced pharmacological sequestration (Fig. 5A and B), demonstrating involvement of dynamins in DA-induced D3R upregulation. Pitstop2 was recently reported to show only marginal selectivity toward clathrin- vs. caveolin-mediated internalization of β2AR in HEK293 cells [79]. It was reported that 3 mM MβCD for 30 min can be used to specifically inhibit caveolin-dependent internalization of β2 AR and D2R in HEK293 or COS-7 cells [79]. Pretreatment of 3 mM MβCD for 30 min had no effect on the basal and SK608-induced D3R internalization or DA-induced upregulation and pharmacological sequestration in CHO-D3R cells (Fig. 5A and B), strongly suggesting that all the processes are caveolin-independent. Altogether, our results demonstrate that SK608-induced D3R internalization is clathrin/dynamin-dependent and caveolin-independent. It is noteworthy that the GRK2 dominant negative mutant, clathrin and dynamin I/II inhibitors effectively reduced the basal level of D3R internalization (Figs. 4A, 5 A) resulting in a lower steady state level of internalized D3R. In addition, SK608 effectively enhanced this process, but DA had an opposite effect. It is well known that GPCR expression levels at the cell surface are a balance of export, internalization, and degradation [7]. Our results imply that the D3R stably expressed in CHO cells may undergo constitutive internalization, externalization, and recycling in the absence of an agonist via regulatory and dynamic processes. These processes result in a balance of a steady-state level of exported receptor and internalized receptor and are in agreement with similar reports on constitutive agonist-independent internalization and recycling of D2R [84]. It is also noteworthy that DA-induced D3R pharmacological sequestration is correlated with D3R desensitization in a Gβγ- and β-arrestin-dependent manner [22]. In conclusion, the results summarized in Table 1 demonstrate, for the first time, that agonist-induced D3R internalization is a GRK2-, clathrin-, and dynamin I/II- dependent, but Gi/o, β-arrestins, GIT1, and

Table 1 Summary of molecular players involved in SK608-induced D3R internalization and DA-induced D3R pharmacological sequestration.

Gi/o proteins GRK2 GIT1 β-arrestin-1 β-arrestin-2 Clathrin Caveolin DynaminI/II

SK608-induced internalization

DA-induced Pharmacological sequestration

No Yes No No No Yes No Yes

No No No No Yes Yes No No

caveolae independent complex process. Both SK608 and PMA induced D3R internalization involve clathrin and dynamin but are independent of β-arrestins and caveolin. Significantly, SK608-induced D3R “homologous” internalization is GRK2-dependent and cell type-independent, which may be triggered by GRKs in a phosphorylation-dependent manner. In comparison, DA-induced pharmacological sequestration involves β-arrestin 2 and clathrin, which positively contribute to the process. DA-induced surface D3R upregulation is cell type-dependent and involves dynamin1/2 which negatively regulate this process. 3. Materials and methods 3.1. Materials [3H]methylspiperone (85.5 Ci/mmol) and [3H] sulpiride (84 Ci/ mmol) were purchased from PerkinElmer (Boston, MA). Tetracycline, hygromycin and blasticidin, DA, (+) butaclamol, haloperidol, (-) sulpiride, sucrose, methyl-β-cyclodextrin (MβCD) and ascorbic acid were obtained from Sigma-Aldrich (St. Louis, MO), G418 from Gemini BioProducts (West Sacramento, CA), cell culture reagents and Lipofectamine 3000 transfection kit from Invitrogen (Carlsbad, CA). GRK2 dominant negative mutant (GRK2-K220R) was a gift from Dr. Robert Lefkowitz (Addgene plasmid # 35403), Flag-ECFP-GIT1 from Dr. Rick Horwitz (Addgene plasmid # 15223), β-arrestin-1 dominant negative mutant-GFP (V53D) and β-arrestin-2 dominant negative mutant-GFP (V54D) from Dr. Marc Caron. SK609 and its analogs namely SK608 and SK213 were synthesized in-house and characterized using NMR and Mass spectrometry and their chemical structures, binding affinity and functional selectivity to dopamine receptors [27]. 3.2. Cell Culture and transfection CHO cells stably expressing human D2 receptor or human D3 54

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presence of 10 μM haloperidol and 10 μM (-) sulpiride, respectively. While methylspiperone, which is a hydrophobic antagonist ligand, can bind to both cell surface and intracellular receptors, sulpiride, a hydrophilic antagonist ligand and membrane-impermeable, is restricted to bind only to the cell surface receptor of intact cells (12, 28, 32). The internalization of D3R or D2R is determined as the following: total receptor = ([3H]methylspiperone binding (dpm) -([3H]methylspiperone binding with haloperidol (dpm, nonspecific binding); surface receptor = ([3H]methylspiperone binding (dpm) - ([3H]methylspiperone binding with sulpiride (dpm); intracellular receptor = total receptor (dpm)-surface receptor (dpm). Surface receptor change (the percentage of receptor internalization or upregulation) = (surface receptor of drug-treated group - surface receptor of vehicle-treated control)/surface receptor of vehicle-treated control x100%. Pharmacological sequestration of D3R was determined based on published procedures [12,22]. CHO-D3R cells were pretreated with vehicle, DA or test compounds for 60 min or indicated time. Cells were then chilled and washed to remove the bound ligands with cold PBS buffer three times at 4 °C (10 min/each time on ice) and harvested with cold Versene buffer and then cell number was determined by a Z1 cell and particle counter (Beckman Coulter). [3H]sulpiride (about 2 nM) binding was carried out on intact cells with the same numbers of cells (about 0.8–1 × 105 cells/each reaction tube) in Kreb’s buffer solution at 4 °C for 2 h (the binding has reached a plateau for 2 h at 4 °C). Nonspecific binding was defined as the binding in the presence of 10 μM haloperidol. The percentage of pharmacological sequestration is calculated as [(drug-treated specific binding -vehicle-treated specific binding)/vehicle-treated specific binding] X 100%.

receptor by the pCMV6 plasmid vector, HEK-293 cells or SY-SY5Y cells stably expressing human dopamine D3 receptor by the pcDNA 3.1 or pEGFP-N1 plasmid vector respectively, were grown in 100-mm culture dishes in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin in a humidified atmosphere consisting of 5% CO2 and 95% air at 37 °C. HEK-D3R cells, which have the CMV promoter controlled by a Tet-on mechanism, were grown in the presence of 50 μg/ml hygromycin and 5 μg/ml blasticidin to maintain T-rex and receptor selection, respectively. The HEK-D3R cells were induced to express the D3R with 1 μg/ml tetracycline for 12–24 h before binding or functional assays. CHO-D3R cells, SY-SY5Y-D3R cells, or CHO-D2R cells, were grown in the presence of 0.1 mg/ml G418 to maintain the receptor expression. For transient transfection experiments, 0.1 × 106 CHO-D3R cells were seeded in 6-well plates and cultured in 3 ml DMEM complete growth medium for 48 h and then transiently transfected with mammalian expression vectors for target genes or mock vectors using Lipofectamine 3000 transfection kit following the manufacturer’s instructions. Twenty four hours later, the vector expression for β-arrestin1 dominant negative mutant-GFP (V53D) and β-arrestin-2 dominant negative mutant-GFP (V54D) and ECFP-GIT1 were validated by fluorescent microscope and used for the following pharmacological studies. 3.3. Validation of over-expression of the transfected proteins After transient transfection of β-arrestin 1 or 2 dominant negative mutants, GIT1, GRK2 dominant negative mutant or mock vectors for 48 h, CHO-D3R cells in 6-well plates were validated by fluorescent microscope and then solubilized with 100 μl/well lysis buffer (Thermo Scientific) with 1x Protease Inhibitor Cocktail and 1x Phosphatase Inhibitor Cocktail and 1 mM PMSF. Protein content was assayed with DC Protein Assay Kit II (Bio-Rad). The protein samples were added loading buffer and homogenized using a Pellet Pestle Motor Homogenizer (10 strokes), and then 40 μg total protein/lane was loaded to Novex® 4–20% Tris-Glycine Mini Gel for separation (Invitrogen) and transferred to a PVDF membrane (Thermo Scientific). Immunoblotting was performed with rabbit anti β-arrestin 1, β-arrestin 2, GIT 1 or GRK 2 polyclonal antibody (1:1,000, Cell Signaling Technology) and peroxidase-conjugated goat anti-rabbit Ig G (H + L) (1:5000, Jackson Immuno Research Laboratories, INC). Plasma membrane marker GAPDH was also determined with rabbit anti- GAPDH polyclonal antibody (1:5,000, ThermoFisher Scientific) after stripping the blots of the same PVDF membrane.

3.5. Immunoblot analysis of D3R HEK-FLAG-D3R cells grown in 100 mm cell culture dish were washed with PBS and incubated with serum-free DMEM for 30 min and then treated with vehicle, 1 μM DA, 0.1 μM PMA or 3 μM SK608 for 30 min. Cells were collected immediately on ice and membranes were made according to our published procedures (Xu, et al, 2017) and protein content was assayed. The protein samples were added loading buffer and homogenized using a Pellet Pestle Motor Homogenizer (10 strokes), and then 10 μg total protein/lane was loaded to Novex® 4–20% Tris-Glycine Mini Gel for separation (Invitrogen) and transferred to a PVDF membrane (Thermo Scientific). Immunoblotting was performed with M1 anti FLAG monoclonal antibody (1:1,000, Sigma) and peroxidase-conjugated goat anti-mouse Ig G (H + L) (1:5000, Jackson Immuno Research Laboratories, INC). Plasma membrane marker GAPDH was also determined with rabbit anti- GAPDH polyclonal antibody (1:5,000, ThermoFisher Scientific) after stripping the blots of the same PVDF membrane. Chemiluminescence detection was performed using the SuperSignal West Dura Extended Duration Substrate detection kit (Thermo Scientific). Glycosylated and partially glycosylated forms of D3R immunoblots were quantified by densitometry with ImageQuant LAS4000 (GE Healthcare Bio-Sciences, Pittsburgh, PA) and normalized to vehicle treatment and GAPDH.

3.4. Internalization and pharmacological sequestration assays Internalization of D3R or D2R was determined using a well-established procedure with radiolabeled hydrophobic ligands combined with hydrophilic ligands which has been demonstrated for several GPCRs including D2R or D3R [12,22,32,34,35], beta-adrenergic receptor [3,5,50] and opioid receptors [31,85,86]. CHO-D3R cells, CHO-D2R cells, HEK-D3R cells or SH-SY5Y-D3R cells were pretreated with vehicle, DA or test compounds for 60 min or indicated time at 37 °C. Cells were then chilled and washed to remove the bound ligands with cold PBS (phosphate buffered saline) three times (10 min/each time on ice) and harvested with cold Versene buffer (0.54 mM EDTA, 140 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.46 mM KH2PO4, and 1 mM glucose, pH 7.4) and the cell numbers were determined by a Z1 cell and particle counter (Beckman Coulter). [3H]methylspiperone (about 1 nM) binding was carried out on intact cells with the same numbers of cells (about 0.8–1 × 105 cells/each reaction tube) in Kreb’s buffer solution (130 mM NaCl, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 4.2 mM NaHCO3, 1.3 mM CaCl2, 10 mM glucose, and 25 mM HEPES, pH 7.4) at 4 °C for 2 h (the binding curve plateaus at 2 h under 4 °C). For total and cellsurface receptors, nonspecific binding was defined as the binding in the

3.6. Immunofluorescence staining of D3R internalization Internalization of D3R was validated using immunofluorescence staining according to the published procedure (Peng, et al, 2013; Chen, et al,2016). HEK-FLAG-D3R cells grown on culture slides for 48 h were washed and incubated with serum-free DMEM for 30 min. Cells were incubated with M1 mouse anti-FLAG antibody (2.0 μg/ml, Sigma) for 30 min at 37° and then treated with vehicle, 1 μM DA, 0.1 μM PMA or 3 μM SK608 for 30 min. Cells were washed with TBS (50 mM Tris, 150 mM NaCl, 1 mM, CaCl2, pH 7.4), fixed with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 15 min at room temperature (RT), permeabilized with 0.1% Triton x100 (sigma) in a blocking solution (5% normal goat serum in TBS for 10 m, incubated 55

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with goat anti-mouse IgG (H + L) conjugated with Alexa Fluor® 488 (1:500 dilution in TBS, Molecular Probes) for 1 h at RT. Cells were washed with TBS and mounted with Slow Fade mounting medium (Molecular Probes) and coverslips were sealed with nail polish and examined under a fluorescence microscope and images were captured.

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3.7. Statistical analysis Data are presented as the mean ± s.e.m unless specified otherwise. For comparison of multiple groups, data was analyzed by one-way ANOVA followed by a Dunnett’s multiple comparison test. For comparison of two groups, Student's t test was performed. P < 0.05 was defined as statistically significant difference. Conflict of interestfor S. Kortagere SK608 is a novel compound that was discovered in our laboratory and is currently listed as a lead molecule in two patent applications 13/ 764,623 and 15/028,654 filed by Drexel University. Both the patents are licensed to Polycore Therapeutics LLC. Polycore Therapeutics did not fund or influence any aspect of this study. SK is a co-founder and holds equity in Polycore Therapeutics LLC. Authors: W.Xu, Reith MEA and Liu-Chen LY have no conflicts of interest to declare. Author contributions Participated in research design: Xu, Reith, Liu-Chen and Kortagere. Contributed reagents: Reith, Liu-Chen and Kortagere. Conducted experiments: Xu. Performed data analysis: Xu and Kortagere. Writing the manuscript: Xu, Reith, Liu-Chen and Kortagere. Acknowledgments We thank Drs. Chiu and Huang and Mr. CC Chen of Temple University School of Medicine, Philadelphia, PA, for supporting binding assays and helpful discussions, Dr. Marc Caron for cDNA clones of βarrestin-1 dominant negative mutant (V53D) and β-arrestin-2 dominant negative mutant (V54D). SK acknowledges funding support from Drexel University Innovation fund, Drexel-Coulter translational research grants and faculty development funds from Department of Microbiology & Immunology, Drexel University. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.phrs.2019.03.003. References [1] R.R. Gainetdinov, R.T. Premont, L.M. Bohn, R.J. Lefkowitz, M.G. Caron, Desensitization of G protein-coupled receptors and neuronal functions, Annu. Rev. Neurosci. 27 (2004) 107–144. [2] M. Von Zastrow, D.E. Keith Jr., C.J. Evans, Agonist-induced state of the delta-opioid receptor that discriminates between opioid peptides and opiate alkaloids, Mol. Pharmacol. 44 (1993) 166–172. [3] M. von Zastrowand, B.K. Kobilka, Ligand-regulated internalization and recycling of human beta 2-adrenergic receptors between the plasma membrane and endosomes containing transferrin receptors, J. Biol. Chem. 267 (1992) 3530–3538. [4] J. Lameh, M. Philip, Y.K. Sharma, O. Moro, J. Ramachandran, W. Sadee, Hm1 muscarinic cholinergic receptor internalization requires a domain in the third cytoplasmic loop, J. Biol. Chem. 267 (1992) 13406–13412. [5] S.S. Yu, R.J. Lefkowitz, W.P. Hausdorff, Beta-adrenergic receptor sequestration. A potential mechanism of receptor resensitization, J. Biol. Chem. 268 (1993) 337–341. [6] J.L. Seachristand, S.S. Ferguson, Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases, Life Sci. 74 (2003) 225–235. [7] M.T. Duvernay, C.M. Filipeanu, G. Wu, The regulatory mechanisms of export trafficking of G protein-coupled receptors, Cell. Signal. 17 (2005) 1457–1465.

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