Activation, internalization, and recycling of theserotonin 2A receptor by dopamine
, 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 92697Contributed by Ricardo Miledi, August 1, 2006
Serotonergic and dopaminergic systems, and their functional in-teractions,havebeenimplicatedinthepathophysiologyofvariousCNS disorders. Here, we use recombinant serotonin (5-HT) 2A(5-HT
) receptors to further investigate direct interactions be-tween dopamine and 5-HT receptors. Previous studies in
oocytes showed that dopamine, although not the cognate ligandfor the 5-HT
receptor, acts as a partial-efﬁcacy agonist. Atmicromolarconcentrations,dopaminealsoactsasapartial-efﬁcacyagonist on 5-HT
receptors in HEK293 cells. Like 5-HT, dopaminealso induces receptor-internalization in these cells, although atsigniﬁcantly higher concentrations than 5-HT. Interestingly, if thereceptors are ﬁrst sensitized or ‘‘primed’’ by subthreshold concen-trations of 5-HT, then dopamine-induced internalization occurs atconcentrations
10-foldlowerthanwhendopamineisusedalone.Furthermore, unlike 5-HT-mediated internalization, dopamine-me-diated receptor internalization, alone, or after sensitization by5-HT, does not depend on PKC. Dopamine-internalized receptorsrecycle to the surface at rates similar to those of 5-HT-internalizedreceptors.Ourresultssuggestapreviouslyuncharacterizedrolefordopamine in the direct activation and internalization of 5-HT
receptors that may have clinical relevance to the function ofserotonergic systems in anxiety, depression, and schizophreniaand also to the treatment of these disorders.
opamine and serotonin (5-HT) have been implicated in anumber of pathological psychiatric disorders, includingdepression, anxiety, bipolar disorder, schizophrenia, and drugabuse (1–6). Many antipsychotic drugs bind to both dopamineand 5-HT receptors (4, 7), and several studies have indicated that5-HT, acting through its receptors, can modulate dopaminefunction (8–14). In particular, the 5-HT
receptor has beenshown to be present on dopaminergic neurons in various regionsof the brain; e.g., the ventral tegmental area in rats and humans(15, 16). These findings suggest that dopamine and 5-HT,interacting through their respective receptors and signal trans-duction pathways, may modulate each other’s response. Although dopamine is not the cognate ligand for 5-HTreceptors, it has been shown to directly activate 5-HT
receptors (17, 18). Dopamine acts as a partial-efficacyagonist at these receptors, i.e., it activates these receptors to alesserextentthandoes5-HT.Althoughdopamineactivates5-HTreceptors directly, the potency and efficacy of its binding varysignificantly among subtypes. Early work showed that dopamineacts as a partial-efficacy agonist at the rat 5-HT
oocytes (17), although micromolar con-centrations of dopamine are required to activate the 5-HT
receptor. More recent studies on the interactions of dopamine with 5-HT
, and 5-HT
receptors indicate that theaffinity of the ligand to the receptor does not correlate with theefficacy of the ligand for activation (18). In addition, a numberof dopamine-receptor antagonists (atypical antipsychotic drugs) were also found to internalize the 5-HT
receptor, some atnanomolar concentrations.To date, there have been no detailed studies on the effects of dopamine on 5-HT
receptors, studies which are important tomore completely understand the potential for direct cross-talkbetween serotonergic and dopaminergic systems. Because5-HT
receptors localize to some dopaminergic neurons, and anumber of clinically used drugs bind to both 5-HT and dopaminereceptors, modulation of 5-HT
-receptor responses by dopa-mine could play an important role in the CNS. The current studyusesmammaliancelllinestocharacterizereceptoractivationandtrafficking using a full-length rat 5-HT
receptor tagged at theC terminus with EGFP (SR2-GFP receptor), stably expressed inHEK293 cells. An earlier study using this cell line showed thatthe tagged receptor is functional, easily visualized, and can beused to study trafficking and activation of the receptor (19).
Dopamine Activates the Rat 5-HT
Receptor in HEK293 Cells.
oocytes, rat 5-HT
receptors stimulate the phospho-lipase C-IP
pathway upon activation by dopamine (17). Todetermine whether dopamine activates rat 5-HT
receptorsexpressed in mammalian cells, intracellular Ca
levels weremonitored by using the Ca
-sensitive Rhod2-AM dye in SB1cells (i.e., HEK293 cells stably expressing SR2-GFP receptors)after exposure to dopamine. Application of 10
M dopamineelicited an increase in intracellular Ca
levels in SB1 cells (Fig.1). This increase in Ca
levels was not seen in untransfectedHEK293 cells, indicating that the 5-HT
receptor mediates theresponse. Concentrations of dopamine
M did not cause adetectable increase in intracellular Ca
(data not shown). Theincrease in the levels of Ca
-dependent intracellular fluores-cence upon application of 10
M dopamine was significantlysmaller than that produced by 10
M 5-HT (Fig. 1
). This resultis consistent with the reduced efficacy of activation of the5-HT
receptor by dopamine observed earlier in
oocytes (17). These experiments suggest that, at micromolarconcentrations, dopamine is a partial-efficacy agonist also at rat5-HT
receptors in HEK293.
Dopamine-Activated Rat 5-HT
Receptors Internalize in HEK293 Cells.
Because dopamine activated the rat 5-HT
receptor, we exam-ined whether it would also induce receptor internalization, asdoes serotonin (17). Cycloheximide-treated SB1 cells were stim-ulated with different concentrations of dopamine, and the cells were imaged to check for the presence of an internalized pool of receptors. These receptors were observed to internalize atdopamine concentrations
M (Fig. 2), whereas 5-HT pro-
Author contributions: S.B., R.M., and M.M.P. designed research; S.B., I.R., and A.B. per-formed research; S.B., I.R., A.B., and M.M.P. analyzed data; and S.B., I.R., A.B., R.M., andM.M.P. wrote the paper.The authors declare no conﬂict of interest.Abbrreviation: 5-HT, serotonin.
Present address: Department of Psychiatry and Behavioral Sciences, Stanford University,Stanford, CA 94305.
To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or email@example.com.© 2006 by The National Academy of Sciences of the USA
October 10, 2006
no. 41 www.pnas.org