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

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

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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
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

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04/25/2012

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Activation, internalization, and recycling of theserotonin 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 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
2A
) receptors to further investigate direct interactions be-tween dopamine and 5-HT receptors. Previous studies in
Xenopus
oocytes showed that dopamine, although not the cognate ligandfor the 5-HT
2A
receptor, acts as a partial-efficacy agonist. Atmicromolarconcentrations,dopaminealsoactsasapartial-efficacyagonist on 5-HT
2A
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
2A
receptors that may have clinical relevance to the function ofserotonergic systems in anxiety, depression, and schizophreniaand also to the treatment of these disorders.
receptor priming
receptor recycling
D
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
2A
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
1A
, 5-HT
2C
,and 5-HT
3
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
2A
receptorexpressed in
Xenopus
oocytes (17), although micromolar con-centrations of dopamine are required to activate the 5-HT
2A
receptor. More recent studies on the interactions of dopamine with 5-HT
1A
, 5-HT
2C
, and 5-HT
3
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
2A
receptor, some atnanomolar concentrations.To date, there have been no detailed studies on the effects of dopamine on 5-HT
2A
receptors, studies which are important tomore completely understand the potential for direct cross-talkbetween serotonergic and dopaminergic systems. Because5-HT
2A
receptors localize to some dopaminergic neurons, and anumber of clinically used drugs bind to both 5-HT and dopaminereceptors, modulation of 5-HT
2A
-receptor responses by dopa-mine could play an important role in the CNS. The current studyusesmammaliancelllinestocharacterizereceptoractivationandtrafficking using a full-length rat 5-HT
2A
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).
Results
Dopamine Activates the Rat 5-HT
2A
Receptor in HEK293 Cells.
In
 Xenopus
oocytes, rat 5-HT
2A
receptors stimulate the phospho-lipase C-IP
3
pathway upon activation by dopamine (17). Todetermine whether dopamine activates rat 5-HT
2A
receptorsexpressed in mammalian cells, intracellular Ca
2
levels weremonitored by using the Ca
2
-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
2
levels in SB1 cells (Fig.1). This increase in Ca
2
levels was not seen in untransfectedHEK293 cells, indicating that the 5-HT
2A
receptor mediates theresponse. Concentrations of dopamine
5
M did not cause adetectable increase in intracellular Ca
2
(data not shown). Theincrease in the levels of Ca
2
-dependent intracellular fluores-cence upon application of 10
M dopamine was significantlysmaller than that produced by 10
M 5-HT (Fig. 1
C
). This resultis consistent with the reduced efficacy of activation of the5-HT
2A
receptor by dopamine observed earlier in
Xenopus
oocytes (17). These experiments suggest that, at micromolarconcentrations, dopamine is a partial-efficacy agonist also at rat5-HT
2A
receptors in HEK293.
Dopamine-Activated Rat 5-HT
2A
Receptors Internalize in HEK293 Cells.
Because dopamine activated the rat 5-HT
2A
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
5
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: panic@ncbs.res.in or rmiledi@uci.edu.© 2006 by The National Academy of Sciences of the USA
15248–15253
PNAS
October 10, 2006
vol. 103
no. 41 www.pnas.org
cgi
doi
10.1073
pnas.0606578103
 
duced internalization at 100 nM (Fig. 7, which is published assupporting information on the PNAS web site). Interestingly,only 70–75% of the cells showed receptor internalization uponapplication of dopamine, as compared with
100% for 5-HT.The reasons why 25–30% of the cells seem to be refractory todopamine-mediated internalization remain to be determined.
Dopamine-Internalized 5-HT
2A
Receptors Recycle to the Cell Surfacewith the Same Kinetics as 5-HT-Internalized 5-HT
2A
Receptors.
Todetermine whether dopamine-internalized 5-HT
2A
receptorsrecycle to the cell surface, 10
M dopamine was applied for 10min to SB1 cells after the initial cycloheximide treatment. Cells were then washed of dopamine and followed for various times inthe continued presence of cycloheximide (Fig. 3). Receptors thatinternalized upon application of dopamine were seen to localizeto the perinuclear region in 10 min, i.e., the recycling endosome(Fig. 8, which is published as supporting information on thePNAS web site). After a 1.5-h wash, receptors were seen toredistribute within the cytoplasm, and, at 2.5 h, receptorsreturned to the cell surface, as evidenced by the disappearanceof 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 recep-tor localization in the above experiment suggest that, afterdopamine-mediated internalization, receptors recycle to the cellsurface in 2.5 h in SB1 cells, similar to 5-HT-internalizedreceptors (19).
5-HT
2A
Receptors Primed by 5-HT Require Lower Concentrations ofDopaminetoInternalize.
Totestwhetherpriorexposureof5-HT
2A
receptors to serotonin would induce internalization by lowerconcentrations of dopamine (
5
M), SB1 cells were incubated with 50 nM 5-HT, a subthreshold concentration for internaliza-tion,for10minbeforetheapplicationofdopamine.Asexpected,50 nM 5-HT, by itself, did not induce internalization of theSR2-GFP receptors (Fig. 4
 B
). Subsequently, various concentra-tions of dopamine were applied in the continued presence of 5-HT for an additional 10 min, and receptors were observed tointernalizefromconcentrationsaslowas500nMdopamine(Fig.4
C
).Thus,sensitizationor‘‘priming’’ofSR2-GFPreceptorswithconcentrations of 5-HT subthreshold for internalization de-creased the threshold concentration of dopamine required toinduce internalization by
10-fold. As seen in internalizationexperiments with dopamine alone, SR2-GFP receptors internal-
Fig.1.
Dopamine(Dop)activatestheSR2-GFPreceptorinHEK293cells.(
 A
and
B
) Untransfected HEK293 cells or SB1 cells were loaded with the Rhod2-AM, aCa
2
-sensitive fluorescent dye. Ten micromolar dopamine was then applied tothe cells. Untransfected cells showed no change in intracellular fluorescencebefore (
 Ai 
) and after (
 Aii 
) the addition of dopamine. (
Bi 
) The basal level offluorescencebeforetheadditionofdopamineinSB-1cells.(
Bii 
)SB1cellsshowedanincreaseintheintracellularCa
2
levelsaftertheadditionof10
Mdopamine.(
)Therelativeincreasesinintracellularfluorescenceaftertheapplicationof5-HTor dopamine normalized to basal levels of fluorescence seen in SB1 cells beforeapplication of the ligand. (Scale bar, 50
m.)
Fig. 2.
Dopamine induces SR2-GFP-receptor internalization in SB1 cells.(
 A
D
) Most 5-HT
2A
receptors were localized to the plasma membrane of SB1cells after cycloheximide treatment. Cells were then incubated in variousconcentrations of dopamine for 10 min. (
and
) Internalization of thereceptor is seen at 5
M dopamine (
) and increased on application of 10
Mdopamine(
).Lessthan5
Mdopamine,i.e.,1
M(
B
),3
M(
),and4
M(
D
)dopamine, did not induce any observable internalization of the receptor.(Scale bar, 50
m.)
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 thereceptors internalized upon application of 10
M dopamine for 10 min (
B
),afterwhich,cellswerewashedfreeofdopamineandincubatedat37°Cintheabsenceofdopamineandinthepresenceofcycloheximidefor1h(
),1.5h(
D
),2 h (
), and 2.5 h (
). Almost all receptors recycled to the cell surface in 2.5 h(
). (Scale bar, 50
m.)
Bhattacharyya
et al.
PNAS
October 10, 2006
vol. 103
no. 41
15249
      N      E      U      R       O       S       C      I      E      N       C      E
 
ized in only 70–75% of the cells upon application of dopamine,even after prior sensitization by 5-HT. This priming was furtherinvestigated in terms of both the sequence and duration of application of the ligands. No internalization was seen when SB1cells were treated with subthreshold levels of dopamine (i.e., 1
M dopamine), followed by 50 nM 5-HT (data not shown).Internalization was also not observed when two separate pulsesof 50 nM 5-HT were applied to SB1 cells (data not shown),suggesting that the priming phenomenon is not the same as meresensitization, i.e., increased sensitivity, of the receptor to anagonist. Increased sensitivity seems to be specifically confined todopamine, the interaction of both 5-HT and dopamine with thereceptor seems essential, and the interaction has to occur in adefined temporal sequence. We then went on to test the durationfor which the receptor remains primed. SB1 cells were incubated with 50 nM 5-HT for 10 min, after which the 5-HT was washedoff, and cells were incubated for varying periods of time beforethe application of 500 nM dopamine. If the interval between thetwo applications exceeded 15 min, no internalization took place.This result indicated that the primed receptors retained in-creased sensitivity to dopamine for 15 min after the wash-out of 5-HT(Fig.9,whichispublishedassupportinginformationonthePNAS web site).
5-HT
2A
Receptors Sensitized by 5-HT Are also Activated by LowerConcentrations of Dopamine.
Because priming of the receptor by5-HT facilitated dopamine-mediated internalization, we nextasked whether priming also activated the receptor or causedinternalization without activation. To determine this, SB1 cells were loaded with the Ca
2
-sensitive Rhod2-AM dye, and 50 nM5-HT was applied. No increase in the Ca
2
-dependent intracel-lular fluorescence could be measured, suggesting that 50 nM5-HT did not result in an observable activation of SR2-GFPreceptors under these conditions. Subsequently, various concen-trations of dopamine were applied in the continued presence of 5-HT. Increases in the intracellular Rhod2-AM fluorescence,i.e., increases in Ca
2
levels were observed at dopamine con-centrations starting at 500 nM, indicating that SR2-GFP recep-tors were activated by concentrations of 500 nM dopamine if subthreshold concentrations of 5-HT were applied before do-pamine (Fig. 5). Dopamine at lower levels than 500 nM did notresult in an observable increase in intracellular Ca
2
levels.To study further similarities
differences between the twoforms (dopamine alone and 5-HT-primed) of dopamine-mediated internalization of the rat 5-HT
2A
receptor, the timecourse of recycling of 50 nM 5-HT-primed 500 nM dopamine-exposed receptors was examined (Fig. 10, which is published assupporting information on the PNAS web site). It was found that5-HT-primed dopamine-internalized receptors take 2.5 h torecycle to the surface, similar to the time taken by receptorsactivated by 10
M 5-HT or dopamine alone.
Dopamine-MediatedInternalizationoftheRat5-HT
2A
ReceptorIsBothCa
2
-andPKC-Independent.
The5-HT-mediatedinternalizationof 5-HT
2A
receptors depends on PKC (19). To determine the roleof PKC in dopamine-mediated internalization of 5-HT
2A
recep-tors, experiments were repeated after prior incubation in 50
Msphingosine for 10 min. This concentration of sphingosinecompletely inhibits 10
M 5-HT-mediated internalization of thereceptor, thus serving as a positive control (19). Subsequently,cells were either treated with varying concentrations of dopa-mine alone or primed with 50 nM 5-HT, followed by varyingconcentrations of dopamine. Inhibition of PKC did not block
Fig. 4.
Prior application of subthreshold concentration of 5-HT leads to theSR2-GFPreceptorinternalizationatlesserconcentrationofdopamine(Dop)inSB1cells.(
 A
and
B
)ControlcellshaveSR2-GFPreceptorsatthecellsurface(
 A
),and application of 50 nM 5-HT for 10 min did not induce any observableinternalization of the receptor (
B
). (
and
D
) Receptors were observed tointernalizeatlowerconcentration,i.e.,500nM(
)and1
M(
D
)dopamine,ifcells were incubated in 50 nM 5-HT for 10 min before the application ofdopamine. (Scale bar, 50
m.)
Fig. 5.
Lesser concentrations of dopamine (Dop) can activate SR2-GFPreceptorsifasubthresholdconcentrationof5-HTisappliedbeforedopamine.(
 A
)SB1cellswereloadedwiththeRhod2-AMCa
2
-sensitivedye.Cellsdidnotshow a change in the intracellular fluorescence before (
 Ai 
) and after (
 Aii 
) theadditionof50nM5-HT.(
B
)Fivehundrednanomolardopaminecouldactivatethe SR2-GFP receptor, as seen by the increase in the intracellular Ca
2
levels,ifreceptorswereexposedto50nM5-HTbeforetheapplicationofdopamine.(Scale bar, 50
m.)
15250
www.pnas.org
cgi
doi
10.1073
pnas.0606578103 Bhattacharyya
et al.

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