Dopamine D2 Receptors Internalize in Their Low-A⁄Nity State

NEUROPHARMACOLOGYAND NEUROTOXICOLOGY NEUROREPORT

Dopamine D2 receptors internalize in their low-a⁄nity state

F. Ko,CA P. Seeman, W. S. Sun1 and S. Kapur1

Department of Pharmacology, Medical Science Building, Room 4344,University of Toronto,Toronto,Ont. M5S1A8; 1Department of Psychiatry, Centre for Addiction and Mental Health,Clarke site, 250 College Street,Toronto,Ontario,Canada M5T1R8

CACorresponding Author

Received 28 January 2002; accepted 7 April 2002

The present study was done in order to determine whether dopa- converted to low-a⁄nity states by guanine nucleotide, the latter mine internalizes D2 receptors in CHO cells and whether the high- had no e¡ect in blocking the dopamine-induced reduction a⁄nity or the low-a⁄nity state of the dopamine D2 receptor is in [3H]sulpiride binding, indicating that the dopamine-induced associated with dopamine-induced internalization of dopamine internalization of D2 receptors occurred with D2 in the D2 receptors. Using [3H]sulpiride to label D2Long receptors in low-a⁄nity state. NeuroReport 13:1017^1020 c 2002 Lippincott CHO cells, it was found that dopamine lowered the binding of Williams & Wilkins. [3H]sulpiride by 20%. Although the high-a⁄nity states of D2 were

Key words: D2 receptor; Dopamine; High-a⁄nity state of D2; Internalization; Schizophrenia

INTRODUCTION pre-synaptically, this would lead to a heightened displace- Many types of G protein-linked receptors become desensi- ment or decrease in radioligand binding. If such a fall in tized upon continuous exposure to agonists. A variety of receptor density is attributable to internalization, then it is molecular events occurs during the course of desensitization important to know the factors that contribute to the process [1,2], including receptor phosphorylation [3], receptor of internalization. For example, although it is known that conversion from the high-affinity to the low-affinity state b2-adrenoceptors are internalized in their low-affinity state, [4], receptor internalization or endocytosis [5], and down- no such information is available for dopamine D2 receptors regulation of receptor site density [6]. The internalization of [5]. Because it is known that the high-affinity state of the receptors appears necessary for the re-sensitization of dopamine D2 receptor is the functional state of the D2 receptors [5,7]. Although dopamine receptors become receptor in controlling the release of prolactin [18], one desensitized upon exposure to dopamine agonists [8,9], might expect that the functional state of the dopamine D2 there is little information on which of these various receptors in the brain and tissue culture cells would also molecular events are associated with desensitization to function in their high-affintiy state. Therefore, one would dopamine. In particular, although there is information on expect that dopamine D2 receptors would be internalized in desensitization of dopamine D1 receptors [9–11], there are their high-affinity state upon exposure to dopamine. The only a few studies on dopamine D2 receptors [12–14]. These present findings, however, reveal that the dopamine D2 latter studies looked at the sequestration of the D2 receptors receptors are internalized in their low-affinity state. through the G-protein-coupled receptor kinases (GRK)- mediated pathways by co-expression of G-protein-coupled receptor kinases and dopamine D2 receptors, then examin- ing the subsequent [3H]sulpiride binding. METHODS AND MATERIALS It is of considerable clinical interest to determine the basic Cell culture: CHO cells stably transfected with the D2Long mechanism of dopamine D2 receptor down-regulation, receptor were grown on large Falcon tissue culture dishes because Laruelle et al. and Breier et al. [15,16] have found (100 Â 20 mm) to B90% confluence in Alpha- that the dopamine-releasing action of amphetamine causes a MEM þ penicillin/streptomycin (Tissue Culture Media Pre- greater apparent fall in D2 receptors in schizophrenia paration Service, University of Toronto, Toronto, ON) and patients than in control individuals. The imaging data from 10% fetal bovine serum; Gibco-BRL products, Life Technol- these studies suggest that there is a higher release of ogies, Burlington, ON, containing 1 mg/ml Geneticin endogenous dopamine in schizophrenia patients, a finding (GIBCO-BRL). Cells were maintained at 371C in a CO2 compatible with the dopamine over-activity hypothesis of incubator. For each experiment, unless otherwise men- schizophrenia [17]. With an increase in dopamine release tioned, 24-well plates were seeded with the CHO cells

0959-4965 c Lippincott Williams & Wilkins Vol 13 No 8 12 June 2002 1017 NEUROREPORT F. KO ETAL. stably transfected with the D2Long receptor and allowed to incubated for 2 h at room temperature (201C), after which grow for an additional 3 days at 371C. the incubates were filtered, using a 12-well cell harvester (Titertek, Skatron, Lier, Norway) and buffer-presoaked glass Radioligand binding to D2 receptors in intact tissue culture fiber filter mats (No. 7034, Skatron, Sterling, VA). After cells: On the day of the experiment, cells adhering to the filtering the incubate, the filter mat was rinsed with buffer bottom of the 24-well plates were washed three times with for 15 s (7.5 ml buffer). The filters were pushed out and 0.5 ml cold phosphate-buffered saline (PBS), after which placed in scintillation minivials. The minivials received 4 ml 0.5 ml buffer (50 mM Tris–HCl, pH 7.4 at 201C, 5 mM KCl, each of scintillant and were monitored 6 h later for tritium in a Packard 4660 scintillation spectrometer at 55% efficiency. 1.5 mM CaCl2, 4 mM MgCl2.6H2O, 1 mM EDTA and 120 mM NaCl) or dopamine (supplemented with 5.7 mM L-ascorbic Non-specific binding for dopamine D2 receptors was acid) was added. The dopamine concentrations tested were defined as that which occurred in the presence of 10 mM 100 nM, 200 nM, 500 nM and 1 mM. Cells were exposed to S-sulpiride. dopamine at both room temperature and at 371C and for varying time periods ranging from 5 min to 60 min. After D2High state in intact CHO cells: CHO cells with the exposure to dopamine, the buffer containing dopamine was D2Long receptor were seeded on 24-well plates. Competi- removed and the wells were washed three times with 0.5 ml tion experiments were carried out between dopamine and cold PBS to remove the dopamine. Preliminary tests were [3H]raclopride on the intact cells in the 24-well plates. Cells carried out using [3H]dopamine to ensure that all the adhering to the bottom of the wells were washed three times dopamine was removed from the wells. Non-specific with 0.5 ml cold PBS, and the following were added to each binding for dopamine D2 receptors was defined by the well: 0.5 ml buffer containing increasing concentrations of presence of 100 nM haloperidol or 100 nM domperidone. dopamine (or a final concentration of 10 mM S-sulpiride), In order to determine the optimal concentration of the 0.5 ml [3H]raclopride (76.8 Ci/mmol, final concentration radioligand [3H]sulpiride (70.3 Ci/mmol; NEN Life Science 2 nM) and 1 ml buffer (or Gpp[NH]p, final concentration Products, Boston, MA) to label dopamine D2 receptors in 200 mM). The wells, containing a total volume of 2 ml, were the CHO cells, preliminary experiments were done using incubated for 2 h at room temperature (201C), after which 3 [ H]sulpiride concentrations ranging from 1.4 nM to 10 nM they were washed three times with 0.5 ml cold PBS, (final concentrations). A final concentration of 5 nM trypsinized with trypsin–EDTA (Gibco-BRL) and scraped 3 [ H]sulpiride (KD 5 nM) was used to measure the binding off the wells and placed into the minivials. Four ml of of [3H]sulpiride to the D2-transfected CHO cells. scintillant was added per minivial and these were then The cells were incubated with the radioligand for 4 h at monitored for tritium in a Beckman scintillation spectro- 41C, after which trypsin–EDTA (Gibco-BRL) was added to meter at 55% efficiency. the cells. The cells were scraped off the wells and placed into minivials (Packard Instruments, Chicago, IL). Scintillant (4 ml; CytoScint, ICN, CA) was added per minivial and the RESULTS vials were then monitored for tritium in a Beckman Optimizing conditions to study the in vitro action of scintillation spectrometer at 55% efficiency. dopamine on radioligand binding to D2 receptors in intact tissue culture cells: In order to study the dopamine- Guanine nucleotide effect in intact tissue culture cells: The induced internalization, conditions were optimized. To same experiments as described above were carried out in establish the conditions under which dopamine exposure the presence of 200 mM final concentration of guanilylimi- would have a consistent effect in the range 20–30%, we dodiphosphate (Gpp[NH]p) included with dopamine dur- tested the variables of dopamine concentration, tempera- ing its exposure with the intact tissue culture cells. ture, and length of exposure time to dopamine. After testing each variable, we found that exposure to 1 mM dopamine for 1 h at room temperature produced the maximal decrease in Effect of sucrose and concanavalin A on intact tissue culture [3H]sulpiride binding. The optimum concentration of cells: The same experiments as described above were [3H]sulpiride was 5 nM, which in fact was the K of this carried out in the presence of 0.5 mg/ml concanavalin A, D ligand. included with dopamine during its exposure with the intact tissue culture cells. The same experiments were also repeated with 0.6 M final concentration of sucrose. Effects of concanavalin A in the presence of dopamine on [3H]sulpiride binding to D2 receptors in intact tissue culture cells: Table 1 shows that 1 mM dopamine decreased D2High state in disrupted CHO cells: Competition experi- 3 ments were carried out between dopamine and [3H]raclo- [ H]sulpiride binding significantly by 21 7 2.7% and that pride in the following manner. Incubation tubes this decrease in binding was blocked by 0.5 mg/ml (12 Â 75 mm glass) received 0.5 ml buffer (with increasing concanavalin A. concentrations of dopamine or Gpp[NH]p, final concentration 200 mM; or with a final concentration 10 mM S-sulpiride), Establishing the presence of the D2High state in disrupted 0.5 ml [3H]raclopride (76.8 Ci/mmol, final concentration CHO cells: To establish that the high-affinity state of the 2 nM) and 0.5 ml of a suspension of disrupted CHO cells. D2 receptor was present in disrupted CHO cells, competi- The cells were not washed, centrifuged or homogenized, tion experiments were carried out between dopamine and though they were disrupted since they were scraped off the [3H]raclopride. As shown in Fig. 1a, the high-affinity state of plate. The tubes containing a total volume of 1.5 ml were the D2 receptor was present in disrupted cells and this state

1018 Vol 13 No 8 12 June 2002 D2 RECEPTORS IN THEIR LOW-AFFINITY STATE NEUROREPORT

Table1. Dopamine decreased [3H]sulpiride binding (5nM) to D2-transfected cells, an e¡ect blocked by concanavalin A.Values are mean 7 s.e.m. Number of D2-speci¢c binding Decrease in speci¢c % of control % decrease experiments (d.p.m./mg protein)a binding caused by1 mM dopamine (d.p.m./mg protein) Control 63 153500 7 2150 0 100 7 1.4 0 7 1.4 1 mM dopamine 32 121270 7 3300** 32 230 7 3300** 79 7 2.2 21 7 2.7 1 mM dopamine þ 200 mM Gpp[NH]p 44 119 730 7 2000** 33770 7 2000** 78 7 1.3 22 7 1.8 1 mM dopamine þ 0.5 mg/ml Con A 28 153 000 7 4300 B0 100 7 2.8 0 7 2.8 1 mM dopamine þ 0.6 M sucrose 4 155 000 7 6900 B0 100 7 4.5 0 7 4.5 aNon-speci¢c binding in the presence of100 nM haloperidol ¼1200 7 50 d.p.m./mg protein (n ¼ 63). **Statistically signi¢cantly di¡erent from control, p o 0.005 (t-test).

(a) Inhibition of D2 High state in disrupted CHO cells Fig. 1b). The concentration of dopamine for 50% inhibition of [3H]raclopride at the high-affinity site was 10 nM. 100 Guanine nucleotide only partially inhibited the high-affinity 90 with guanine n state receptors (40–15%) [18], as is clearly seen by the 80 competition curve spanning 4 2 log units.

H]raclopride 70 3 60 High-affinity ucleotide 50 Contr Effects of guanine nucleotide and sucrose, respectively, in component the presence of dopamine on [3H]sulpiride binding to D2 40 ol receptors in intact tissue culture cells: Dopamine de- 30 3 20 creased the dopamine D2 receptor binding of [ H]sulpiride 10 by 21 7 2.7% (n ¼ 32) when compared with controls (Table 0 1). Surprisingly, in the presence of dopamine and guanine % Specific binding of [ 1 10 100 1000 10,000 100,000 10 µM nucleotide, the decrease in [3H]sulpiride binding was not nM Dopamine S-Sulpiride reversed, but remained decreased by 22 7 1.8% compared with controls. Sucrose completely blocked the dopamine- 3 (b) Partial inhibition of D2 High state in intact CHO cells induced decrease in [ H]sulpiride binding (Table 1).

100 90 DISCUSSION with guanine n 80 The main observation of this study is that guanine

H]raclopride 70 nucleotide was unable to inhibit the dopamine-induced 3 60 internalization of dopamine D2 receptors labeled by ucleo 3 50 K High = 10 nM [ H]sulpiride, suggesting that the D2 receptors are inter- i Contr 40 tide nalized in their low-affinity state. 30 ol One disadvantage of the study was that dopamine was 20 only able to internalize 21% of the D2Long receptors, as 10 revealed by the reduction of [3H]sulpiride binding. Never-

% Specific binding of [ 0 theless, this reduction was completely inhibited by either 1 10 100 1000 10,000 100,000 10 µM 0.6 M sucrose or 0.5 mg/ml concanavalin A, indicating that nM Dopamine S-Sulpiride dopamine genuinely caused internalization of D2 receptors. Although sucrose inhibited the dopamine-induced reduc- Fig. 1. Demonstrating the existence of high-a⁄nity states of dopamine 3 D2 receptors in CHO cells, and their conversion to low-a⁄nity states. (a) tion in [ H]sulpiride binding, this effect is not especially Disrupted cells: although dopamine recognized high- and low-a⁄nity selective because 0.6 M sucrose is hypertonic and would be states, the high-a⁄nity sites all converted to low-a⁄nity sites in the pre- expected to inhibit a wide variety of cellular processes. The sence of 200 mM guanilylimidodiphosphate (Gpp[NH]p). Representative experiment with sucrose, however, served as an excellent experiment. (b) Intact cells: dopamine recognized high- and low-a⁄nity 3 non-specific control to determine whether dopamine in- sites for D2 receptors, with10 nM dopamine inhibiting 50% of the [ H]ra- duced a non-specific reduction in [3H]sulpiride binding. clopride binding to the high-a⁄nity sites. In the presence of 200 mM Thus, the fact that dopamine did not reduce the binding of Gpp[NH]p, the proportion of high-a⁄nity sites was reduced by 4 50%. 3 Representative experiment. [ H]sulpiride in the presence of sucrose indicated the absence of nonspecific internalization in the presence of dopamine. was converted into the low-affinity state in the presence of Concanavalin A, however, is selective in disaggregating guanine nucleotide. microfibrils responsible for receptor endocytosis and pre- venting receptor dephosphorylation, supporting the inter- pretation that dopamine induces internalization of Establishing the presence of the D2High state in intact CHO dopamine D2 receptors. cells: In intact cells, dopamine recognized both the high- Although the magnitude of the dopamine-induced inter- affinity sites (B40%), and the low-affinity sites (B60%; nalization was only 21–22%, it is possible that this may be

Vol 13 No 8 12 June 2002 1019 NEUROREPORT F. KO ETAL. attributed to the low level (B40%) of the proportion of D2 nalization occurred with the D2 receptors in their low- receptors in the high-affinity state, as indicated by the affinity state for dopamine. [3H]raclopride/dopamine competition experiments. At the same time, it may be argued that guanine nucleotide did not fully eliminate the high-affinity states in the intact cells, and that this incomplete action of guanine nucleotide may REFERENCES explain why the guanine nucleotide was not effective in 1. Ferguson SS. Pharmacol Rev 53, 1–24 (2001). 2. Saunders C and Limbird LE. Pharmacol Ther 84, 193–205 (1999). inhibiting dopamine-induced internalization. 3. Pierce KL and Lefkowitz RJ. Nature Rev Neurosci 2, 727–733 (2001). As noted above, the main finding of this study was that 4. Boundy VA, Pacheco MA, Guan W and Molinoff PB. Mol Pharmacol 48, Gpp[NH]p was unable to inhibit the dopamine-induced 956–964 (1995). internalization of [3H]sulpiride. This negative finding did 5. Yu SS, Lefkowitz RJ and Hausdorff WP. J Biol Chem 268, 337–341 (1993). not occur because the Gpp[NH]p was not effective or not 6. Williams BR, Barber R and Clark RB. Mol Pharmacol 58, 421–430 (2000). capable of entering the cell. In fact, the [3H]raclopride/ 7. Schlador ML, Grubbs RD and Nathanson NM. J Biol Chem 275, 23295– 23302 (2000). dopamine competition experiments clearly showed that 30– 8. Bates MD, Senogles SE, Bunzow JR et al. Mol Pharmacol 39, 55–63 (1991). 40% of the D2 receptors were in the high-affinity state (Fig. 9. Lewis MM, Watts VJ, Lawler CP et al. J Pharmacol Exp Ther 286, 345–353 1a,b) and, in addition, that 200 mM Gpp[NH]p significantly (1998). reduced the high-affinity states in both the disrupted CHO 10. Jiang D and Sibley DR. Mol Pharmacol 56, 675–683 (1999). cells and the intact CHO cells. Nevertheless, although the 11. Dumartin B, Caille I, Gonon F and Bloch B. J Neurosci 18, 1650–1661 presence of 200 mM Gpp[NH]p converted the high-affinity (1998). 12. Iwata K, Ito K, Fukuzaki A . 263, 596–602 (1999). states to low-affinity states, the presence of the guanine et al Eur J Biochem 13. Itokawa M, Toru M, Ito K et al. Mol Pharmacol 49, 560–566 (1996). nucleotide had no effect in blocking the dopamine-induced 14. Ito K, Haga T, Lameh J and Sadee W. Eur J Biochem 260, 112–119 3 reduction (internalization) in [ H]sulpiride binding, indicat- (1999). ing that the dopamine-induced internalization of D2 15. Laruelle M, Abi-Dargham A, Gil R et al. Biol Psychiatry 46, 56–72 receptors was mediated by the low-affinity state of the D2 (1999). receptor. Collectively, these data indicate, therefore, that the 16. Breier A, Su TP, Saunders R et al. Proc Natl Acad Sci USA 94, 2569–2574 (1997). dopamine-induced internalization of D2 receptors was 17. van Rossum J. The significance of dopamine-receptor blockade for the mediated in the low-affinity state of the D2 receptor. action of neuroleptic drugs. In: Brill H, Cole O, Deniker P, Hippius H and Bradley PB, eds. Neuropsychopharmacology, Proceedings 5th Collegium Internationale Neuropsychopharmacologicum. Amsterdam: Excerpta Medica; CONCLUSION 1967, pp. 321–329. Dopamine-induced internalization of dopamine D2 recep- 18. George SR, Watanabe M, Di Paolo T et al. Endocrinology 117, 690–697 tors into CHO cells was not affected by the absence of D2 (1985). receptors in their high-affinity state, and that the inter-

Acknowledgements:This research was supported by NARSAD (the National Alliance for Research on Schizophrenia and Depression), the Ontario Mental Health Foundation, the Canadian Institutes of Health Research, the National Institute on Drug Abuse, the Stanley Foundation of NAMI (National Alliance for the Mentally Ill), the Medland family, and Dr Karolina Jus.

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