Activation, Internalization, and Recycling of the Serotonin 2A Receptor by Dopamine

Activation, internalization, and recycling of the serotonin 2A receptor by dopamine

Samarjit Bhattacharyya*†, Ishier Raote*, Aditi Bhattacharya*, Ricardo Miledi‡§, and Mitradas M. Panicker*§

*National Centre for Biological Sciences, Tata Institute of Fundamental Research, University of Agricultural Sciences–Gandhi Krishi Vignana Kendra Campus, Bellary Road, Bangalore-560065, Karnataka, India; and ‡Department of Neurobiology and Behavior, University of California, Irvine, CA 92697

Contributed by Ricardo Miledi, August 1, 2006 Serotonergic and dopaminergic systems, and their functional in- To date, there have been no detailed studies on the effects of teractions, have been implicated in the pathophysiology of various dopamine on 5-HT2A receptors, studies which are important to CNS disorders. Here, we use recombinant serotonin (5-HT) 2A more completely understand the potential for direct cross-talk (5-HT2A) receptors to further investigate direct interactions be- between serotonergic and dopaminergic systems. Because tween dopamine and 5-HT receptors. Previous studies in Xenopus 5-HT2A receptors localize to some dopaminergic neurons, and a oocytes showed that dopamine, although not the cognate ligand number of clinically used drugs bind to both 5-HT and dopamine for the 5-HT2A receptor, acts as a partial-efficacy agonist. At receptors, modulation of 5-HT2A-receptor responses by dopa- micromolar concentrations, dopamine also acts as a partial-efficacy mine could play an important role in the CNS. The current study agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine uses mammalian cell lines to characterize receptor activation and also induces receptor-internalization in these cells, although at trafficking using a full-length rat 5-HT2A receptor tagged at the significantly higher concentrations than 5-HT. Interestingly, if the C terminus with EGFP (SR2-GFP receptor), stably expressed in receptors are first sensitized or ‘‘primed’’ by subthreshold concen- HEK293 cells. An earlier study using this cell line showed that trations of 5-HT, then dopamine-induced internalization occurs at the tagged receptor is functional, easily visualized, and can be concentrations Ϸ10-fold lower than when dopamine is used alone. used to study trafficking and activation of the receptor (19). Furthermore, unlike 5-HT-mediated internalization, dopamine-me- Results diated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors Dopamine Activates the Rat 5-HT2A Receptor in HEK293 Cells. In recycle to the surface at rates similar to those of 5-HT-internalized Xenopus oocytes, rat 5-HT2A receptors stimulate the phospho- receptors. Our results suggest a previously uncharacterized role for lipase C-IP3 pathway upon activation by dopamine (17). To determine whether dopamine activates rat 5-HT receptors dopamine in the direct activation and internalization of 5-HT 2A 2A expressed in mammalian cells, intracellular Ca2ϩ levels were receptors that may have clinical relevance to the function of monitored by using the Ca2ϩ-sensitive Rhod2-AM dye in SB1 serotonergic systems in anxiety, depression, and schizophrenia cells (i.e., HEK293 cells stably expressing SR2-GFP receptors) and also to the treatment of these disorders. after exposure to dopamine. Application of 10 ␮M dopamine elicited an increase in intracellular Ca2ϩ levels in SB1 cells (Fig. ͉ receptor priming receptor recycling 1). This increase in Ca2ϩ levels was not seen in untransfected HEK293 cells, indicating that the 5-HT2A receptor mediates the opamine and serotonin (5-HT) have been implicated in a response. Concentrations of dopamine Ͻ5 ␮M did not cause a Dnumber of pathological psychiatric disorders, including detectable increase in intracellular Ca2ϩ (data not shown). The depression, anxiety, bipolar disorder, schizophrenia, and drug increase in the levels of Ca2ϩ-dependent intracellular fluores- abuse (1–6). Many antipsychotic drugs bind to both dopamine cence upon application of 10 ␮M dopamine was significantly and 5-HT receptors (4, 7), and several studies have indicated that smaller than that produced by 10 ␮M 5-HT (Fig. 1C). This result 5-HT, acting through its receptors, can modulate dopamine is consistent with the reduced efficacy of activation of the function (8–14). In particular, the 5-HT2A receptor has been 5-HT2A receptor by dopamine observed earlier in Xenopus shown to be present on dopaminergic neurons in various regions oocytes (17). These experiments suggest that, at micromolar of the brain; e.g., the ventral tegmental area in rats and humans concentrations, dopamine is a partial-efficacy agonist also at rat (15, 16). These findings suggest that dopamine and 5-HT, 5-HT2A receptors in HEK293. interacting through their respective receptors and signal transduction pathways, may modulate each other’s response. Dopamine-Activated Rat 5-HT2A Receptors Internalize in HEK293 Cells. Although dopamine is not the cognate ligand for 5-HT Because dopamine activated the rat 5-HT2A receptor, we examined whether it would also induce receptor internalization, as receptors, it has been shown to directly activate 5-HT1A, 5-HT2C, does serotonin (17). Cycloheximide-treated SB1 cells were stim- and 5-HT3 receptors (17, 18). Dopamine acts as a partial-efficacy agonist at these receptors, i.e., it activates these receptors to a ulated with different concentrations of dopamine, and the cells lesser extent than does 5-HT. Although dopamine activates 5-HT were imaged to check for the presence of an internalized pool of receptors. These receptors were observed to internalize at receptors directly, the potency and efficacy of its binding vary Ն ␮ significantly among subtypes. Early work showed that dopamine dopamine concentrations 5 M (Fig. 2), whereas 5-HT pro- acts as a partial-efficacy agonist at the rat 5-HT2A receptor

expressed in Xenopus oocytes (17), although micromolar con- Author contributions: S.B., R.M., and M.M.P. designed research; S.B., I.R., and A.B. per- centrations of dopamine are required to activate the 5-HT2A formed research; S.B., I.R., A.B., and M.M.P. analyzed data; and S.B., I.R., A.B., R.M., and receptor. More recent studies on the interactions of dopamine M.M.P. wrote the paper. with 5-HT1A, 5-HT2C, and 5-HT3 receptors indicate that the The authors declare no conﬂict of interest. affinity of the ligand to the receptor does not correlate with the Abbrreviation: 5-HT, serotonin. efficacy of the ligand for activation (18). In addition, a number †Present address: Department of Psychiatry and Behavioral Sciences, Stanford University, of dopamine-receptor antagonists (atypical antipsychotic drugs) Stanford, CA 94305. § were also found to internalize the 5-HT2A receptor, some at To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. nanomolar concentrations. © 2006 by The National Academy of Sciences of the USA

15248–15253 ͉ PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606578103 Downloaded by guest on September 26, 2021 Fig. 3. SR2-GFP receptors recycle to the cell surface after dopamine- mediated internalization in SB1 cells. Before dopamine (Dop) application, control cells have the SR2-GFP receptors at the cell surface (A), and the receptors internalized upon application of 10 ␮M dopamine for 10 min (B), after which, cells were washed free of dopamine and incubated at 37°C in the absence of dopamine and in the presence of cycloheximide for1h(C), 1.5 h (D), 2h(E), and 2.5 h (F). Almost all receptors recycled to the cell surface in 2.5 h (F). (Scale bar, 50 ␮m.)

application of dopamine, as compared with Ϸ100% for 5-HT. Fig. 1. Dopamine (Dop) activates the SR2-GFP receptor in HEK293 cells. (A and The reasons why 25–30% of the cells seem to be refractory to B) Untransfected HEK293 cells or SB1 cells were loaded with the Rhod2-AM, a dopamine-mediated internalization remain to be determined. Ca2ϩ-sensitive fluorescent dye. Ten micromolar dopamine was then applied to the cells. Untransfected cells showed no change in intracellular fluorescence before (Ai) and after (Aii) the addition of dopamine. (Bi) The basal level of Dopamine-Internalized 5-HT2A Receptors Recycle to the Cell Surface fluorescence before the addition of dopamine in SB-1 cells. (Bii) SB1 cells showed with the Same Kinetics as 5-HT-Internalized 5-HT2A Receptors. To ϩ an increase in the intracellular Ca2 levels after the addition of 10 ␮M dopamine. determine whether dopamine-internalized 5-HT2A receptors (C) The relative increases in intracellular fluorescence after the application of 5-HT recycle to the cell surface, 10 ␮M dopamine was applied for 10 or dopamine normalized to basal levels of fluorescence seen in SB1 cells before min to SB1 cells after the initial cycloheximide treatment. Cells application of the ligand. (Scale bar, 50 ␮m.) were then washed of dopamine and followed for various times in the continued presence of cycloheximide (Fig. 3). Receptors that internalized upon application of dopamine were seen to localize duced internalization at 100 nM (Fig. 7, which is published as to the perinuclear region in 10 min, i.e., the recycling endosome supporting information on the PNAS web site). Interestingly, (Fig. 8, which is published as supporting information on the only 70–75% of the cells showed receptor internalization upon PNAS web site). After a 1.5-h wash, receptors were seen to redistribute within the cytoplasm, and, at 2.5 h, receptors returned to the cell surface, as evidenced by the disappearance of the internal fluorescence and a coincident reappearance of surface fluorescence. Because there was no new protein synthe- sis during the experimental time period, the dynamics of receptor localization in the above experiment suggest that, after dopamine-mediated internalization, receptors recycle to the cell surface in 2.5 h in SB1 cells, similar to 5-HT-internalized receptors (19).

5-HT2A Receptors Primed by 5-HT Require Lower Concentrations of Dopamine to Internalize. To test whether prior exposure of 5-HT2A receptors to serotonin would induce internalization by lower concentrations of dopamine (Ͻ5 ␮M), SB1 cells were incubated with 50 nM 5-HT, a subthreshold concentration for internalization, for 10 min before the application of dopamine. As expected, 50 nM 5-HT, by itself, did not induce internalization of the SR2-GFP receptors (Fig. 4B). Subsequently, various concentrations of dopamine were applied in the continued presence of Fig. 2. Dopamine induces SR2-GFP-receptor internalization in SB1 cells. 5-HT for an additional 10 min, and receptors were observed to (A–D) Most 5-HT2A receptors were localized to the plasma membrane of SB1 internalize from concentrations as low as 500 nM dopamine (Fig. cells after cycloheximide treatment. Cells were then incubated in various 4C). Thus, sensitization or ‘‘priming’’ of SR2-GFP receptors with concentrations of dopamine for 10 min. (E and F) Internalization of the receptor is seen at 5 ␮M dopamine (E) and increased on application of 10 ␮M concentrations of 5-HT subthreshold for internalization de- dopamine (F). Less than 5 ␮M dopamine, i.e., 1 ␮M(B), 3 ␮M(C), and 4 ␮M(D) creased the threshold concentration of dopamine required to

dopamine, did not induce any observable internalization of the receptor. induce internalization by Ϸ10-fold. As seen in internalization NEUROSCIENCE (Scale bar, 50 ␮m.) experiments with dopamine alone, SR2-GFP receptors internal-

Bhattacharyya et al. PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 ͉ 15249 Downloaded by guest on September 26, 2021 Fig. 4. Prior application of subthreshold concentration of 5-HT leads to the SR2-GFP receptor internalization at lesser concentration of dopamine (Dop) in SB1 cells. (A and B) Control cells have SR2-GFP receptors at the cell surface (A), and application of 50 nM 5-HT for 10 min did not induce any observable Fig. 5. Lesser concentrations of dopamine (Dop) can activate SR2-GFP internalization of the receptor (B). (C and D) Receptors were observed to receptors if a subthreshold concentration of 5-HT is applied before dopamine. ␮ ϩ internalize at lower concentration, i.e., 500 nM (C) and 1 M(D) dopamine, if (A) SB1 cells were loaded with the Rhod2-AM Ca2 -sensitive dye. Cells did not cells were incubated in 50 nM 5-HT for 10 min before the application of show a change in the intracellular ﬂuorescence before (Ai) and after (Aii) the ␮ dopamine. (Scale bar, 50 m.) addition of 50 nM 5-HT. (B) Five hundred nanomolar dopamine could activate the SR2-GFP receptor, as seen by the increase in the intracellular Ca2ϩ levels, if receptors were exposed to 50 nM 5-HT before the application of dopamine. ized in only 70–75% of the cells upon application of dopamine, (Scale bar, 50 ␮m.) even after prior sensitization by 5-HT. This priming was further investigated in terms of both the sequence and duration of application of the ligands. No internalization was seen when SB1 receptors under these conditions. Subsequently, various concen- cells were treated with subthreshold levels of dopamine (i.e., 1 trations of dopamine were applied in the continued presence of ␮M dopamine), followed by 50 nM 5-HT (data not shown). 5-HT. Increases in the intracellular Rhod2-AM fluorescence, ϩ Internalization was also not observed when two separate pulses i.e., increases in Ca2 levels were observed at dopamine con- of 50 nM 5-HT were applied to SB1 cells (data not shown), centrations starting at 500 nM, indicating that SR2-GFP recep- suggesting that the priming phenomenon is not the same as mere tors were activated by concentrations of 500 nM dopamine if sensitization, i.e., increased sensitivity, of the receptor to an subthreshold concentrations of 5-HT were applied before do- agonist. Increased sensitivity seems to be specifically confined to pamine (Fig. 5). Dopamine at lower levels than 500 nM did not ϩ dopamine, the interaction of both 5-HT and dopamine with the result in an observable increase in intracellular Ca2 levels. receptor seems essential, and the interaction has to occur in a To study further similarities͞differences between the two defined temporal sequence. We then went on to test the duration forms (dopamine alone and 5-HT-primed) of dopamine- for which the receptor remains primed. SB1 cells were incubated mediated internalization of the rat 5-HT2A receptor, the time with 50 nM 5-HT for 10 min, after which the 5-HT was washed course of recycling of 50 nM 5-HT-primed 500 nM dopamine- off, and cells were incubated for varying periods of time before exposed receptors was examined (Fig. 10, which is published as the application of 500 nM dopamine. If the interval between the supporting information on the PNAS web site). It was found that two applications exceeded 15 min, no internalization took place. 5-HT-primed dopamine-internalized receptors take 2.5 h to This result indicated that the primed receptors retained in- recycle to the surface, similar to the time taken by receptors creased sensitivity to dopamine for 15 min after the wash-out of activated by 10 ␮M 5-HT or dopamine alone. 5-HT (Fig. 9, which is published as supporting information on the PNAS web site). Dopamine-Mediated Internalization of the Rat 5-HT2A Receptor Is Both Ca2؉- and PKC-Independent. The 5-HT-mediated internalization of 5-HT2A Receptors Sensitized by 5-HT Are also Activated by Lower 5-HT2A receptors depends on PKC (19). To determine the role Concentrations of Dopamine. Because priming of the receptor by of PKC in dopamine-mediated internalization of 5-HT2A recep- 5-HT facilitated dopamine-mediated internalization, we next tors, experiments were repeated after prior incubation in 50 ␮M asked whether priming also activated the receptor or caused sphingosine for 10 min. This concentration of sphingosine internalization without activation. To determine this, SB1 cells completely inhibits 10 ␮M 5-HT-mediated internalization of the were loaded with the Ca2ϩ-sensitive Rhod2-AM dye, and 50 nM receptor, thus serving as a positive control (19). Subsequently, 5-HT was applied. No increase in the Ca2ϩ-dependent intracel- cells were either treated with varying concentrations of dopa- lular fluorescence could be measured, suggesting that 50 nM mine alone or primed with 50 nM 5-HT, followed by varying 5-HT did not result in an observable activation of SR2-GFP concentrations of dopamine. Inhibition of PKC did not block

15250 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606578103 Bhattacharyya et al. Downloaded by guest on September 26, 2021 receptor internalization driven by extrasynaptic concentrations of dopamine. We found that, although dopamine alone can activate and internalize rat 5-HT2A receptors expressed in HEK293 cells, it does so only at micromolar concentrations high enough to argue against having major physiological relevance. Both 5-HT and dopamine-mediated internalization cause receptors to enter a recycling compartment and, apparently, recycle to the surface in 2.5 h (19). Thus, 5-HT- and dopamine-mediated recycling appear to share common features. Moreover, because both ligands activate the phospholipase C-IP3 pathway, it is likely that com- ponents of the internalization pathways are also common to both processes. PKC activation is known to be sufficient and necessary for 5-HT-mediated internalization of the majority of the 5-HT2A receptors (19). Interestingly, however, the present study shows that dopamine-mediated internalization of the receptor does not depend on PKC. This result indicates that, although some aspects of the internalization and recycling may be similar, there are also fundamental differences in the mechanisms of 5-HT- and dopamine-mediated internalization of 5-HT2A receptors. The 5-HT2A receptor has been reported to also activate other signaling pathways (20–24), e.g., ERK MAPK in many cell types. In aortic myocytes, this activation is brought about by the activation of Src kinase, and, in PC12 cells, the 5-HT2A receptor has been shown to activate tyrosine kinases in a Ca2ϩ͞calmod- ulin-dependent manner. Differential activation of one signaling Fig. 6. Inhibition of PKC does not inhibit the dopamine-mediated internalization or the sensitization of the SR2-GFP receptor by 5-HT. (A) Control cells pathway versus another, independent of ligand affinity (func- showed receptors localized to the cell surface after the initial cycloheximide tional selectivity), has been observed with 5-HT2 receptors, and treatment. (B) The receptor internalized upon application of 10 ␮M dopamine it is possible that dopamine differentially activates alternate (Dop). (C) Internalization of SR2-GFP receptors by 10 ␮M dopamine was not effector pathways (25, 26) whose role in dopamine-mediated inhibited by 50 ␮M sphingosine. (D) SR2-GFP receptors sensitized by prior internalization remains to be explored. application of 50 nM of 5-HT internalized upon application of 500 nM dopa- The subthreshold (50 nM) concentrations of 5-HT used in our mine in the sphingosine-treated SB1 cells. (Scale bar, 50 ␮m.) experiments did not initiate detectable internalization or show an increase in intracellular Ca2ϩ. Still, it is possible that Ca2ϩ ␮ levels may have risen, but the sensitivity of the assay was internalization of the receptor by 10 M dopamine alone or insufficient to detect such an increase (27). Whether signal internalization of 5-HT-primed receptors stimulated with 500 transduction pathways were activated or not, subthreshold con- nM dopamine (Fig. 6). Lower concentrations (10 ␮M) of sphin- centrations of 5-HT clearly had a priming action at the 5-HT2A gosine also did not inhibit the sensitization of the receptor by receptors, such that they require 10-fold-lesser amounts of 5-HT. These results suggest that PKC is not involved in the dopamine to cause receptor internalization and activation. This sensitization and dopamine-mediated internalization of 5-HT2A priming is not a mere sensitization of the receptor to an agonist; receptors. an interaction of both 5-HT and dopamine with the receptor is Because 5-HT-primed receptors are activated and internalized essential, and it has to occur in a defined temporal sequence. by 500 nM dopamine, it was possible that Ca2ϩ surges resulting Such concentrations of dopamine as are required to internalize from receptor activation by dopamine could be responsible for the 5-HT-primed receptor are within the range of concentrations internalization. To test this hypothesis, SB1 cells were loaded of dopamine present at synapses in the brain. For example, with 25 ␮M BAPTA-AM to chelate cytosolic Ca2ϩ. When these extracellular concentrations of dopamine in conscious rats has cells were stimulated with dopamine after being primed by 5-HT, been reported to be Ϸ1 ␮M in rat striatum (28). Potential direct internalization was not inhibited (Fig. 11, which is published as interactions of dopamine on 5-HT2A receptors would be most supporting information on the PNAS web site). This result was likely in areas of the brain where serotonergic and dopaminergic consistent with the observation that no internalization of recep- neurons coexist, e.g., the ventral tegmental area (VTA) (29). tors took place when SB1 cells were treated with the Ca2ϩ Because 5-HT receptors are most likely presynaptic to seroto- ionophore A23187, alone or followed by 500 nM dopamine (data nergic neurons, it is likely that prevailing concentrations of 5-HT not shown). These results indicate that, although there is a Ca2ϩ are sufficient to sensitize the 5-HT2A receptors before activation by dopamine, and this could result in an altered and increased surge upon activation of the receptor by 50 nM 5-HT followed sensitivity to dopamine. Interestingly, 5-HT receptors are by 500 nM dopamine, 5-HT, or dopamine alone, the Ca2ϩ rise 2A present presynaptically on dopaminergic neurons of the VTA, makes little or no contribution to the internalization of the and the interaction of these receptors with some antipsychotic receptor under these conditions. drugs regulates the release of dopamine (30). This dopamine- in Discussion mediated partial activation and internalization could function vivo, as a partial desensitization of synaptic 5-HT2A receptors. In The major findings of this study are twofold: (i) Dopamine, at addition, this process could act as a molecular AND gate, micromolar concentrations, is a partial-efficacy agonist at re- exhibiting activity only in the presence of both 5-HT and combinant rat 5-HT2A receptors expressed in HEK293 cells and dopamine in a defined temporal sequence. can induce receptor internalization and subsequent recycling; The priming of 5-HT2A receptors by 5-HT shows some im- and (ii) interactions between dopamine and 5-HT2A receptors portant features. After internalization, the primed receptor

could be physiologically relevant when the receptor is previously recycles to the cell surface in the same time as taken for 5-HT- NEUROSCIENCE primed by subthreshold concentrations of 5-HT, resulting in or dopamine-mediated recycling. Furthermore, our experiments

Bhattacharyya et al. PNAS ͉ October 10, 2006 ͉ vol. 103 ͉ no. 41 ͉ 15251 Downloaded by guest on September 26, 2021 indicate that dopamine-mediated internalization of primed re- Tissue-culture plastic wares were obtained from Nalge Nunc In- ceptors is independent of PKC activation and independent also ternational (Roskilde, Denmark). Coverslips (Gold seal cover glass, of the Ca2ϩ transients produced in the cells. Nonetheless, primed No. 1) were purchased from Clay Adams (Franklin Lakes, NJ). receptors recycle to the cell surface in the same time as that taken for receptors internalized by 5-HT or dopamine alone. Functional and Internalization Studies. The functional SR2-GFP These results suggest that 5-HT2A receptors internalized by receptor was constructed by tagging EGFP at the C terminus of the dopamine, with or without priming by 5-HT, are internalized by full-length rat 5-HT2A receptor, and a stable line, SB1, expressing a different mechanism from that used by 5-HT. The results also fluorescent SR2-GFP receptors, was established in the HEK293 suggest that, when 5-HT occupies the receptor at subthreshold cell line, as described earlier (19). The effects of dopamine and concentrations, pathways other than a PKC-dependent pathway 5-HT on the activation of SR2-GFP receptors were studied in SB1 may be activated. cells by monitoring increases in intracellular Ca2ϩ levels as de- The activation and internalization of 5-HT2A receptors may have scribed (19). Briefly, SB1 cells were grown to 65–70% confluence an important physiological function. For example, this process on 35-mm coverslip dishes coated with 20 ␮g͞ml poly (DL- would be different from interactions of a receptor with an antag- ornithine), at 37°C and loaded with Rhod2-AM, a Ca2ϩ-sensitive onist or inverse agonist, where the second-messenger system is not fluorescent dye. Fluorescence was monitored before and after the activated or the basal activity is reduced. The order of application addition of the ligand to determine functional activation of of the agonist (5-HT) and partial-efficacy agonist (dopamine), the SR2-GFP receptors. Untransfected HEK293 cells served as the having very different effects, would also result in a system with the control. To study the effect of dopamine on receptor activation capability of responding to a specific sequence of events, i.e., when receptors were preexposed to subthreshold concentration (for temporal coding, which is evident from the sequence of neuro- activation) of 5-HT, SB1 cells were loaded with Rhod2-AM dye and transmitter application required for priming to occur. incubated with 50 nM 5-HT. Subsequently, various concentrations One mechanism by which priming could occur is through of dopamine were applied to the cells, and fluorescence changes 5-HT2A-receptor multimers. Like other 5-HT receptors and were monitored before and after the addition of dopamine. In all many other seven-transmembrane receptors, 5-HT2A receptors experiments, total fluorescence in cells under various experimental may form multimers (31–33). It has been shown that, in some conditions was determined by using a confocal microscope and receptor homodimers and heterodimers, binding of ligand to one quantified by using ImageJ software (National Institutes of Health, receptor site changes the conformation of the binding pocket of Bethesda, MD). the other receptor partner (34, 35). One could, therefore, Internalization studies were done as described (19). Briefly, SB1 propose a model where, at subthreshold concentrations of 5-HT, cells were treated with 100 ␮g͞ml cycloheximide for 5–6 h to clear one receptor subunit is occupied by 5-HT, and the other subunit internal fluorescence. At the end of this treatment, receptor then acquires a conformation having higher affinity for dopa- fluorescence was entirely confined to the cell surface, with no mine. This situation could result in dopamine binding at lower intracellular fluorescence. Various concentrations of dopamine (1, concentrations. Alternatively, partially activated or modified 3, 4, 5, and 10 ␮M) were applied to the cells for 10 min at 37°C. Cells receptor states may exist at concentrations of 5-HT that we were fixed in 4% paraformaldehyde in phosphate buffer for 30 min, empirically define as subthreshold under our assay conditions. washed with PBS, and imaged by using a confocal microscope, as Because all of the experiments used a GFP-tagged receptor, detailed below. The method of fixation used was identical for all it is important to stress that the C-terminal addition of EGFP did not appear to be responsible for introducing artifacts in our experimental procedures. To study dopamine-mediated internal- observations, because the internalization of an N-terminal- ization of the receptor when receptors were exposed to subthresh- tagged myc-5-HT receptor was similar to what was observed old concentration (of internalization) of 5-HT before the 2A application of dopamine, SB1 cells were incubated with 50 nM with GFP-tagged receptors (data not shown). We therefore ␮ conclude that the EGFP tag does not significantly affect any of 5-HT for 10 min. Subsequently, dopamine (500 nM and 1 M) were our results with dopamine, although it has been reported that the applied for 10 min. Cells were fixed and imaged. dendritic localization of this receptor may depend partially on a To determine whether two pulses of 50 nM 5-HT, one after the C-terminal PDZ-binding domain (36). other, were sufficient to cause internalization of the 5-HT2A In summary, there is extensive literature describing interac- receptor, SB1 cells were treated with cycloheximide for 6 h. This tions between dopaminergic and serotonergic systems in mam- procedure was followed by a 10-min pulse of 50 nM 5-HT. malian brain (37, 38). The present study suggests that some of Subsequently, the cells were washed twice to remove any trace these results could be explained by direct interactions of dopa- of 5-HT. Then, another pulse of 50 nM 5-HT was provided for mine with 5-HT receptors. The differential regulation of the 10 min. Cells were fixed and imaged. To determine the time for which the receptor stayed primed 5-HT2A receptors by an agonist (5-HT) or a partial-efficacy agonist (dopamine) adds another dimension to the control of after being exposed to 50 nM 5-HT, SB1 cells were treated with dopamine release and function. Such direct interactions could cycloheximide for 5 h, after which they were exposed to 50 nM have implications for our understanding of the effects of a broad 5-HT in DMEM for 10 min. The cells were then washed twice range of neuropsychiatric therapeutics on 5-HT and dopamine with DMEM and kept in plain DMEM (with cycloheximide) for receptors as well as on normal brain function. varying amounts of time (0–20 min). Dopamine (500 nM) was applied for 10 min. Cells were fixed and imaged. Materials and Methods To determine the role of PKC in dopamine-mediated receptor ␮ Materials. The rat 5-HT2A-receptor full-length cDNA construct was internalization, SB1 cells were preincubated for 10 min in 10 M a gift from David Julius (University of California, San Francisco, and 50 ␮M sphingosine (an inhibitor of PKC), after which 10 ␮M CA). pEGFP-N1 and pCruz-Myc vectors were purchased from 5-HT or 10 ␮M dopamine was applied for an additional 10 min Clontech (Mountain View, CA) and Santa Cruz Biotechnology in the continued presence of sphingosine. Cells were then fixed (Santa Cruz, CA), respectively. Tissue-culture reagents were pur- and imaged. Similar experiments were done by using calphostin chased from Life Technologies (Carlsbad, CA) and Sigma (St. C, another specific inhibitor of PKC. To determine whether Louis, MO). Rhod2-AM dye, BAPTA-AM, and pluronic were inhibition of PKC inhibits the sensitization of the receptor by from Molecular Probes (Eugene, OR). Poly (DL-ornithine), 5-HT, 5-HT, SB1 cells were treated with 10 ␮M and 50 ␮M sphingosine dopamine, cycloheximide, sphingosine, calphostin C, and 1,4- for 10 min after the initial cycloheximide treatment. Subse- diazabicyclo[2.2.2]octane (DABCO) were purchased from Sigma. quently, 50 nM 5-HT was applied for 10 min. After that, 500 nM

15252 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606578103 Bhattacharyya et al. Downloaded by guest on September 26, 2021 and 1 ␮M dopamine was applied for an additional 10 min. Cells To study recycling after priming, followed by dopamine- were fixed and imaged in the confocal microscope. mediated internalization, SB1 cells were treated with cycloheximide To determine the role played by Ca2ϩ, SB1 cells were first for6htoclear internal fluorescence. A 10-min pulse of 50 nM 5-HT treated with cycloheximide for 5 h. They were then loaded with was applied to the cells, after which the cells were incubated with 25 ␮M BAPTA-AM for 45 min. Cells were washed free of 500 nM dopamine for 10 min. Cells were washed free of dopamine BAPTA-AM and incubated at room temperature for 30 min, and incubated at 37°C for various times (0.5–2.5 h) in the continued followed by 50 nM 5-HT for 10 min and, subsequently, 500 nM presence of cycloheximide, fixed, and imaged. dopamine for 10 min. Cells were then fixed and imaged. Also, cells were preincubated with the Ca2ϩ ionophore Confocal Microscopy. A laser-scanning confocal microscope A-23187 (20 ␮M) for 15 min, and then the SB1 cells were (Model MRC1024; Bio-Rad) attached to an inverted microscope incubated with 50 nM 5-HT for 10 min or with 500 nM dopamine. (Eclipse TE300; Nikon, East Rutherford, NJ), and Lasersharp ϫ Cells were then fixed and imaged. acquisition software was used for imaging. A 60 oil-immersion ϭ In all experiments, cycloheximide was present until the cells objective (N.A. 1. 4) was used with laser power at 30% in all ͞ were fixed. Typically 100–150 cells were chosen randomly and studies. GFP excitation emission was achieved with a filter set ͞ imaged in any one experiment. All experiments were repeated at (488 nm 510 nm) designed for fluorescein detection. Images least three times. were processed with Photoshop (Adobe Systems, Mountain View, CA) by using identical values for contrast and brightness. For Figs. 9–11, confocal images were acquired on the Olympus Colocalization Studies. To determine whether 5-HT2A receptors are targeted to the recycling compartment upon dopamine- (Melville, NY) FV 1000 confocal setup attached to an Olympus inverted microscope (IX81). FV10-ASW 1.3 acquisition mediated internalization, transferrin coupled to Alexa Fluor 568 software was used for imaging. A ϫ60 oil-immersion objective was used as a marker of recycling endosomes (39–41). SB1 cells (N.A. ϭ 1. 4) was used with laser power at 7% of the 488-nm line were treated with cycloheximide as described before. Dopamine ␮ ␮ ͞ of the argon ion laser. GFP excitation was achieved with a (10 M) and 10 g ml transferrin (conjugated to Alexa Fluor primary dichroic mirror 405͞488. Fluorescent signals were ac- 568) were applied to the cells for 15 min. Subsequently, the cells quired by using a spectral detector whose bandwidth was auto- were kept on ice, washed twice with cold DMEM, and treated matically set by using the on-line dye database option for FITC. with 200 ␮l of ascorbate buffer (pH 4.5) (140 mM sodium ͞ ͞ ͞ Images were processed by using Photoshop with identical values ascorbate 65 mM ascorbic acid 1 mM CaCl2 1 mM MgCl2) for for contrast and brightness. 10 min to remove membrane-bound noninternalized transferrin. After 10 min, the cells were fixed with 4% ice-cold paraformal- We thank members of our laboratory, particularly Saptarshi Mandal, for dehyde for 30 min, washed with PBS, and stored in DABCO at valuable discussions and support; Gaiti Hasan and Satyajit Mayor for 4°C until they were imaged. related discussions; the National Centre for Biological Sciences Confocal Facility and Dr. H. Krishnamurthy for imaging-related discussions; and Recycling Studies. To study recycling after dopamine-mediated Rahul Chadda and Satyajit Mayor (National Centre for Biological internalization, a 10-min pulse of 10 ␮M dopamine was applied Sciences, Tata Institute of Fundamental Research) for the generous gift ␮ ͞ of Alexa Fluor 568-labeled transferrin. This work was supported by the to the SB1 cells after 100 g ml cycloheximide treatment for 5–6 National Centre for Biological Sciences, Tata Institute of Fundamental h. Cells were then washed free of dopamine and incubated at Research. S.B. and A.B. were recipients of the Kanwal Rekhi Career 37°C for various times (1, 1.5, 2, and 2.5 h) in the presence of Development Fellowship from the Tata Institute for Fundamental cycloheximide, fixed, and imaged. Research endowment fund.

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