Supplemental material to this article can be found at:
http://molpharm.aspetjournals.org/content/suppl/2012/10/03/mol.112.078626.DC1
1521-0111/83/1/42–50$25.00
MOLECULAR PHARMACOLOGY
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/mol.112.078626
Mol Pharmacol 83:42–50, January 2013
Functional Selectivity in Serotonin Receptor 2A (5-HT2A)
Endocytosis, Recycling, and Phosphorylation s
Ishier Raote, Samarjit Bhattacharyya, and Mitradas M. Panicker
National Centre for Biological Sciences, Bangalore, India (I.R., M.M.P.), and Indian Institute of Science Education and Research,
Mohali, India (S.B.)
Received March 6, 2012; accepted September 28, 2012
Introduction
Serotonin (5-hydroxytryptamine; 5-HT) receptors are a family of G protein-coupled receptors (GPCRs) and ligand-gated
ion channels found in the central and peripheral nervous
systems where they mediate neurotransmission (Nichols and
Nichols, 2008). 5-HT receptors influence various processes
and are hence a primary target of several drugs, including
many antidepressants, antipsychotics, anorectics, antiemetics,
and hallucinogens (Nichols, 2004).
Several ligands bind to the serotonin 2A receptor (5-HT2A),
including biogenic amines such as dopamine (DA) and
tryptamine as well as recreational drugs such as psilocybin
and lysergic acid diethylamide (LSD) and therapeutic (antipsychotic) drugs (Meltzer et al., 2003, 2012; Meltzer and
Massey, 2011; Nichols, 2004). As seen with some GPCRs,
ligands acting at the 5-HT2A receptor differentially modulate
This work was supported by grants from the Department of Biotechnology,
Government of India, and intramural funding from the National Centre for
Biological Sciences.
dx.doi.org/10.1124/mol.112.078626.
s This article has supplemental material available at molpharm.
aspetjournals.org.
5-HT and DA, there is ligand-specific requirement for protein
kinase C (PKC) in endocytosis. We now show 5-HT2A phosphorylation by PKC is necessary for 5-HT-mediated and DOI-mediated
receptor endocytosis, but DA-mediated and clozapine-mediated
internalization is not affected if PKC is inhibited. Internalized
receptors are recycled to the cell surface, but there is variability in
the time course of recycling. 5-HT- and DA-internalized receptors
are recycled in 2.5 hours while agonist DOI and antagonist
clozapine bring about recycling in 7.5 hours. Recycling in
response to those ligands that require PKC activation to effect
receptor endocytosis is dependent on receptor dephosphorylation
by protein phosphatase 2A (PP2A). Thus, internalization and
phosphorylation/dephosphorylation cycles may play a significant
role in the regulation of 5-HT2A by functionally and therapeutically
important ligands.
intracellular transduction cascades, a phenomenon commonly
called “functional selectivity” (Raote et al., 2007; Urban et al.,
2007). Functional selectivity arises out of GPCR pleiotropy
with respect to the signaling transduction cascades they
couple to via interactions with different signaling proteins.
Different ligands uniquely modulate sets of signaling pathways by stabilizing specific receptor conformations. Thus,
ligands at a single receptor produce heterogeneous effects on
the subsequent activation of signaling cascades with varying
efficacies, which poses a challenge to classifying a ligand as an
agonist or antagonist (Kenakin, 2011).
The effects of 5-HT2A ligands, other than 5-HT, on subcellular transduction cascades downstream of receptor activation are minimally characterized; how they feed back to the
receptor and affect its signaling and trafficking remains
unknown. After ligand binding at the cell surface, the 5-HT2A
may be endocytosed and transiently sequestered in endosomes,
the functional significance of which remains to be elucidated.
5-HT2A internalization is associated with receptor desensitization in C6 glioma cells (Hanley and Hensler, 2002). However, internalization and desensitization are independent
processes in human embryonic kidney 293 (HEK293) cells
ABBREVIATIONS: BSA, bovine serum albumin; DA, dopamine; DMEM, Dulbecco’s modified Eagle’s medium; DOI, 1-(2,5-dimethoxy-4iodophenyl)-aminopropane; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum;
GPCR, G protein-coupled receptor; GRK, G protein-coupled receptor kinase; HBSS, Hanks’ balanced salt solution; HEK293, human embryonic
kidney 293 cells; 5-HT, 5-hydroxytryptamine serotonin; 5-HT2A, 5-hydroxytryptamine receptor subtype 2A; LSD, lysergic acid diethylamide; MDMA,
3,4-methylenedioxymethamphetamine; OA, okadaic acid; PBS, phosphate-buffered saline; PKC, protein kinase C; PMA, phorbol 12-myristate 13acetate; PP2A, protein phosphatase 2A.
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ABSTRACT
G protein-coupled receptor (GPCR) signaling is modulated by
endocytosis and endosomal sorting of receptors between degradation and recycling. Differential regulation of these processes by endogenous ligands and synthetic drugs is a poorly
understood area of GPCR signaling. Here, we describe remarkable diversity in the regulation of trafficking of GPCR induced by
multiple ligands. We show that the serotonin receptor 2A (5HT2A), a prototypical GPCR in the study of functional selectivity
at a signaling receptor, is functionally selective in endocytosis
and recycling in response to five ligands tested: endogenous
agonists serotonin (5-HT) and dopamine (DA), synthetic agonist
1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (DOI), antagonist ketanserin, and inverse agonist and antipsychotic drug
clozapine. Only four ligands (5-HT, DA, DOI, and clozapine) bring
about receptor endocytosis. As we have earlier described with
Functional Selectivity in 5-HT2A Recycling
Materials and Methods
Reagents, Expression Plasmids. Cloning of the rat-5-HT2A into
pEGFP-N1 was described earlier elsewhere (Bhattacharyya et al.,
2002). All reagents unless otherwise mentioned were obtained from
Sigma-Aldrich (St. Louis, MO).
Cell Lines, Transfection, and Stable Line Generation. HEK293
cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM;
Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine
serum (FBS; Invitrogen) in 5% CO2 at 37°C, as described elsewhere
(Bhattacharyya et al., 2002). All culture dishes, flasks, and other apparatuses were from Nalge Nunc (Roskilde, Denmark) and Sarstedt AG
and Co. (Nümbrecht, Germany). Generation of the SB1 cell population,
expressing wild-type rat-5-HT2A-EGFP (enhanced green fluorescent
protein), was described earlier elsewhere (Bhattacharyya et al., 2002).
Internalization Assay. The internalization assay was performed
as previously described elsewhere (Bhattacharya et al., 2010). In
brief, the cells were plated on poly-DL-ornithine-coated glass coverslip
dishes at a density of 40,000 to 60,000 cells per dish. At 16 hours after
plating, the cells were incubated in serum-free DMEM for 24 hours.
The cells were washed and incubated with 100 mg/ml cycloheximide
for 6 hours. This treatment inhibits protein synthesis and allows
sufficient time for already synthesized receptors to traffic to the cell
surface. Most cells imaged at this point show EGFP-associated
fluorescence at the cell membrane with minimal intracellular
fluorescence. Cells were then stimulated with a ligand at the
described concentration at 37°C in 5% CO2 for 15 minutes. During
the last 5 minutes of the incubation, the cells were exposed to 10 mg/ml
human transferrin tagged to Alexa 568 (Invitrogen) in DMEM as an
endocytic tracer. Surface-associated transferrin was then stripped
with ice-cold ascorbate buffer at pH 4.5 and fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature. Receptor internalization in the cells was confirmed by the
appearance of a subcellular pool of fluorescence from the internalized
receptors, whereas the control (unstimulated) cells had fluorescence
associated primarily with the plasma membrane.
If they were used, the specific inhibitors of intracellular molecules
(sphingosine, okadaic acid) were added 15 minutes before stimulation
of the receptor-expressing cells with a ligand, unless otherwise
mentioned. The internalization assay was then performed as already
described but in the continued presence of the inhibitor.
Recycling Assay. The SB1 cells, grown in 35-mm coverslip
dishes, were serum starved for 24 hours and treated with cycloheximide (100 mg/ml) to clear the internal fluorescence, as already
mentioned in the description of the internalization assay. Subsequently, 10 mM 5-HT, 10 mM DA, 100 nM DOI, or 100 nM clozapine
was applied for 15 minutes. The cells were then washed free of the
ligand and were incubated for various time periods in the absence of
ligand or serum and in the continued presence of cycloheximide. Five
minutes before fixation, the cells were treated with Alexa 568–tagged
transferrin. The surface transferrin was stripped using ice-cold
ascorbate buffer (pH 4.5), and then the cells were fixed in ice-cold
4% paraformaldehyde for 20 minutes and imaged.
As in the internalization assay, any inhibitors used (sphingosine,
okadaic acid) were added 15 minutes before the stimulation of the
receptor-expressing cells with a ligand. Recycling assays were then
performed (as already described) in the continued presence of the
inhibitor unless otherwise noted.
ELISA-Based Quantification of Receptor Recycling. The
SB1 cells were grown in poly-DL-ornithine-coated wells on a 96-well
plate initially in DMEM supplemented with 10% FBS. At 24 hours
after plating, the medium was changed to DMEM supplemented with
10% serum, dialyzed against PBS. The cells were maintained in this
medium for 48 hours. Ligands were then added for 15 minutes at
37°C. The cells were subsequently washed three times and incubated
in DMEM supplemented with 10% dialyzed FBS for different periods,
ranging from 0 minutes to 7.5 hours. At each time point, the cells were
fixed for 8 minutes on ice in ice-cold 2% paraformaldehyde (PFA,
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(Gray et al., 2001). On the basis of studies with other GPCRs
(Gagnon et al., 1998; Kallal et al., 1998; Ko et al., 1999; Law
et al., 1984; Tsao et al., 2001), receptor internalization and
subsequent lysosomal targeting is believed to be the mechanism behind 5-HT2A downregulation by the antagonist
clozapine.
In addition to downregulation and desensitization, another
consequence of receptor internalization could be the formation of a signaling endosome that modulates transduction
pathways distinct from those regulated at the cell surface
(Calebiro et al., 2009; Rosen et al., 2009). These processes
could involve a reversion of covalent changes, such as
receptor phosphorylation (after ligand binding), enabled
by a change in the receptor’s local environment from the
cell surface to an acidified endosome. Cycles of phosphorylation and dephosphorylation are important in GPCR
trafficking, signaling, and subcellular targeting (Moro
et al., 1993; Sibley et al., 1986). Phosphorylation enables
receptors to interact with cellular machinery allowing for
trafficking, resensitization, and the formation of a scaffold
involved in signaling. While it is conventionally believed
that kinases involved in GPCR phosphorylation are GRKs
(Benovic et al., 1989; Lorenz et al., 1991), other kinases,
including protein kinase C (PKC), have been shown to play
a significant role (Blaukat et al., 2001; Pollok-Kopp et al.,
2003).
Universally used readouts of signaling and regulation of
canonical GPCRs such as the b2-adrenoceptor, GRK-mediated
receptor phosphorylation, and direct association with b-arrestin2 are not involved in rat 5-HT2A regulation in HEK293 cells
(Bhatnagar et al., 2001).
Although it was initially believed that the 5-HT2A receptor
does not undergo phosphorylation, subsequent reports have
demonstrated that phosphorylation by ribosomal S6 kinase 2
(Strachan et al., 2009) dramatically modulates 5-HT2A
signaling (Sheffler et al., 2006; Strachan et al., 2010a,b). 5HT2A phosphorylation at residues Ser291 and Thr386 by PKC
has been shown in an in vitro assay (Turner and Raymond,
2005) and is hypothesized to play a role in modulating
receptor signaling.
We have previously shown that PKC activation is necessary
for 5-HT-mediated 5-HT2A internalization and is sufficient to
bring about receptor endocytosis (Bhattacharyya et al., 2002,
2006). Subsequently, by showing that DA-mediated 5-HT2A
endocytosis is independent of PKC activation, we described
functional selectivity in 5-HT2A signaling (Bhattacharyya
et al., 2006). These observations led us to the hypothesis that
5-HT2A functional selectivity extends to both proximal events
in receptor signaling as well as distal events during receptor
recycling.
Using five compounds that act via the 5-HT2A receptor—
cognate ligand (5-HT), synthetic agonist [1-(2,5-dimethoxy-4iodophenyl)-aminopropane; DOI], partial-efficacy agonist (DA),
antagonist (ketanserin), and inverse agonist (clozapine)—
we show that different biochemical pathways are involved
in receptor trafficking. As ligand-specific pathways of receptor regulation arise perforce from ligand-specific signaling via the 5-HT2A receptor, we use events in receptor
trafficking as direct readouts of functional selectivity at
the receptor. These readouts include receptor internalization, recycling, and the involvement of PKC in receptor
trafficking.
43
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Raote et al.
Results
Some Ligands That Bind to 5-HT2A Bring about Its
Internalization. We looked at receptor endocytosis in response to the five ligands (Fig. 1) known to bind to the 5-HT2A
receptor. As we have previously shown, 5-HT and DA bring
about 5-HT2A internalization in SB1 cells (HEK293 cells
stably expressing 5-HT2A-EGFP) (Bhattacharyya et al., 2002,
2006); the inverse agonist clozapine also causes receptor
endocytosis (Willins et al., 1999). Further, we now show that
the synthetic agonist DOI also brings about receptor internalization. The antagonist ketanserin, however, does not
effect receptor internalization.
Representative images of receptor-associated EGFP fluorescence show the SB1 cells cleared of internal 5-HT2A-EGFP–
associated fluorescence by serum starvation for 24 hours and
treatment with cycloheximide (100 mg/ml) for 6 hours (Fig. 2A,
D, and G). The cells were then incubated with a ligand for 15
minutes. The extent to which different concentrations of the
four ligands brought about receptor internalization was
characterized by ELISA using an antibody targeted against
an extracellular portion of the 5-HT2A receptor (Supplemental
Fig. 1). For all subsequent experiments, the concentrations of
the ligands chosen were those that were well above those that
caused maximal receptor internalization. The concentration of
clozapine used was 10% that seen at steady-state levels in the
blood in patients being treated with the drug (Miller et al.,
1994; Perry et al., 1991; Potkin et al., 1994). 5-HT2A endocytosis
(Fig. 2) was assayed for using 10 mM 5-HT (Fig. 2B), 10 mM DA
(Fig. 2C), 100 nM DOI (Fig. 2E), 100 nM clozapine (Fig. 2F),
and 100 nM ketanserin (Fig. 2H). 5-HT, DA, DOI, and
clozapine all induced robust internalization, but ketanserin
brought about no detectable 5-HT2A endocytosis.
As we have described elsewhere (Bhattacharyya et al.,
2002), PKC-activation by a phorbol ester (PMA) is sufficient to
bring about 5-HT2A endocytosis (Supplemental Fig. 2).
After internalization, the receptor localized to transferrinlabeled endosomes. We used this as a method of quantification
of receptor internalization (Fig. 2I), measuring the ratio of
endosome-associated EGFP to the total EGFP and normalizing this to vehicle-treated (control) cells (see Materials and
Methods). The process of 5-HT2A endocytosis has been shown
to be clathrin dependent and arrestin independent in
HEK293 cells (Bhatnagar et al., 2001).
As confirmation that ketanserin did bind to the 5-HT2A
receptor, we assayed its ability to inhibit 5-HT-mediated
intracellular Ca21 changes. In SB1 cells, 5-HT increases
cytosolic Ca21 via an IP3-mediated pathway (Bhattacharyya
Fig. 1. 5-HT2A ligands used in this study, agonists or partial agonists 5HT, DOI, dopamine, neutral antagonist ketanserin, and inverse agonist
clozapine. (All images have been modified from Wikipedia.)
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 16, 2017
nonpermeabilizing conditions). After fixation, all dishes were stored
at 4°C in M1 buffer (20 mM HEPES, 1.3 mM CaCl2, 2 mM MgCl2, 150
mM NaCl, and 5 mM KCl, pH 7.4) and were processed together. The
cells were then blocked in 0.2% bovine serum albumin (BSA) in M1 on
ice for 1 hour, followed by application of 1:200 dilution of extracellularly directed anti-5-HT2A antibody from Calbiochem (San Diego, CA)
or Neuromics (Edina, MN) in M1, overnight at 4°C. Cells were washed
and incubated with 1:500 diluted goat-anti-rabbit horseradish
peroxidase (HRP) secondary antibody (Molecular Probes, Eugene,
OR) for 1 hour on ice. A colorimetric reaction was developed by the
addition of TMB/H2O2 for 90 seconds at room temperature, followed
by adding an equal volume of 1 N H2SO4. Colorimetric measurement
was done on an enzyme-linked immunosorbent assay (ELISA) plate
reader at 450 nm from Bio-Rad Laboratories (Hercules, CA). The
graph plotted represents the mean of at least three independent
experiments; in each, the ligand treatment was performed in 8 to 16
wells of the 96-well plate.
Epifluorescence Microscopy. Imaging was performed with
a Nikon Eclipse T2000E epifluorescence microscope (Tokyo, Japan)
using a 20/NA 0.75 for live imaging and 60/NA 1.4 oil immersion
objective. Fluorescence was visualized using bandpass filter sets from
Chroma Technologies (Rockingham, NC): exciter 500 nm/20, dichroic
535 nm/30, emitter 515 nm long pass, and exciter 545 nm/30, dichroic
620 nm/60, emitter 575 nm long pass for detecting GFP and Alexa
568–tagged human transferrin, respectively. Images were acquired
using a Photometrix Cascade II 512 EM-CCD camera (Photometrix,
Tuscon, AZ) with Image Pro-Plus AMS software (Media Cybernetics,
Bethesda, MD).
Ca21 Imaging. The SB1 cells were plated on poly-DL-ornithinecoated glass coverslip dishes at a density of 40,000–60,000 cells per
dish in DMEM supplemented with 10% FBS. The cells were incubated
in serum-free DMEM overnight to eliminate the extracellular 5-HT
and then were loaded with 2.5 mM Rhod-2, AM (Invitrogen) with
Pluronic F-127 (Invitrogen) (1:1) in Hanks’ balanced salt solution
(HBSS, with Ca21 and Mg21) on ice for 1 hour, washed with HBSS,
and left for an hour for Rhod-2, AM ester to be hydrolyzed in the cell.
The cells were washed three times with HBSS (without Ca21 and
Mg21).
To observe receptor activation, the changes in fluorescence before
and after the addition of 5-HT were monitored. To confirm that
ketanserin bound to the receptor, cells were pretreated with
ketanserin (10 nM) before adding 5-HT (10 mM). The addition of the
ligand and imaging of the changes in fluorescence associated with
Rhod-2, AM were performed at 37°C in a heated and humidified
chamber (model INU-G2-WELS; Tokai Hit, Shizuoka-ken, Japan).
Images were acquired at 5-second intervals for 3 minutes.
Data Analysis. The receptor internalization was quantified as
previous described elsewhere (Bhattacharya et al., 2010; Kalia et al.,
2006) using a modified version of a quantitative colocalization routine
implemented in MATLAB (MathWorks, Natick, MA). A set of two
corresponding images was taken of a field of cells to record EGFP
(receptor-associated) and Alexa Fluor 568 (transferrin) signals
separately. A top-hat algorithm was used to perform local background
subtraction on both images using a disk of radius 5 pixels. In-focus
endosomes were identified in the transferrin image using iterative
thresholding and intensity-dependent trimming. In brief, initial
thresholds were set by inspection. At every iterative step (going from
0.1 of maximal object intensity to 0.5, step size 5 0.1), connected
nonzero pixels were labeled as individual objects (putative endosomes). Objects smaller than 3 pixels or larger than 500 pixels were
discarded. The ratio of intensity of the EGFP signal colocalized with
transferrin endosomes to total EGFP signal was calculated (colocalization ratio). We analyzed 24 such pairs of images as a set. The
averaged values from each such set of images from an experimental
condition were normalized to values from the control cells that had
not been exposed to any ligand. This was repeated a total of three
times, resulting in 72 pairs of images. Statistical comparisons were
made using the Mann-Whitney U test.
Functional Selectivity in 5-HT2A Recycling
45
et al., 2002). Preincubating SB1 cells with 10 nM ketanserin
for 15 minutes completely abrogates any Ca21 changes
induced by 10 mM 5-HT (data not shown).
PKC Activation Is Required for Some, but Not All,
Ligands, To Bring about 5-HT2A Internalization. As
previously described elsewhere, PKC activation is required for
internalization mediated by 5-HT but not DA (Bhattacharyya
et al., 2006). We also have observed that DOI but not clozapine
requires PKC to bring about internalization of 5-HT2A. SB1
cells, cleared of internal fluorescence (Fig. 3I), were treated with
either ligand alone (Fig. 3, A–D) or first incubated with
sphingosine (10 mM), an inhibitor of PKC activation, for 15
minutes (Fig. 3, E–H). Afterward, 5-HT, DA, DOI, and clozapine
(100 nM) were added in the continued presence of sphingosine.
In accordance with previous observations, sphingosine
inhibited 5-HT-mediated receptor internalization almost entirely (Fig. 3E), but not DA-mediated internalization (Fig. 3F).
Further, DOI-mediated 5-HT2A internalization was also completely prevented (Fig. 3G). Inverse agonist clozapine, however,
effected receptor trafficking independent of PKC inhibition (Fig.
3H). We quantified the effects of PKC activation (using PMA)
and inhibition (Fig. 3J); the gray bars represent internalization
without sphingosine, and the black bars represent levels of
internalization when cells were preincubated in sphingosine.
Abrogation of a Putative PKC Phosphorylation Site
(S291) in the 5-HT2A Receptor Inhibits Receptor Internalization Mediated by 5-HT and DOI but Not
Clozapine or Dopamine. As PKC plays a role in receptor
trafficking, we hypothesized there could be a PKC-dependent
phosphorylation of the receptor. Using various prediction
algorithms online (Amanchy et al., 2007; Blom et al., 1999,
2004; Blom et al., 2004; Xue et al., 2006), we identified four
putative PKC phosphorylation sites on the receptor. Individually mutating each of these sites, we identified one site, serine
291, which when altered to an alanine (5-HT2A-S291A-EGFP)
abolished PKC-mediated internalization. This residue within
a peptide segment of the receptor was also shown to be phosphorylated by PKC in an in vitro assay (Turner and Raymond,
2005). HEK293 cells stably expressing the 5-HT2A-S291A-EGFP
receptor were cleared of internal fluorescence and treated with
each of the four ligands mentioned (Fig. 4, A–D). This singlepoint mutation completely inhibited 5-HT2A internalization by
5-HT (Fig. 4A) and DOI (Fig. 4C) but behaved exactly as the wild
type vis-à-vis its internalization by DA (Fig. 4B) and clozapine
(Fig. 4D).
These data were in accordance with our observations
regarding the effects of inhibition of PKC activation on
ligand-mediated receptor internalization. The endocytic process is unaffected in the case of those ligands that did not
require PKC activation to bring about receptor trafficking, but
ligands that did require PKC activation were unable to effect
internalization of the 5-HT2A-S291A.
Ligand-independent 5-HT2A internalization can be brought
about by activation of PKC (Bhattacharyya et al., 2002, 2006)
using PMA. If receptor phosphorylation by PKC was required to initiate 5-HT2A trafficking, we predicted that PKCphosphorylation-deficient 5-HT2A-S291A would be insensitive to
PMA-mediated internalization. Consistent with our prediction,
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Fig. 2. Ligand-mediated 5-HT2A endocytosis.
SB1 cells (HEK293 cells stably expressing the
5-HT2A-EGFP receptor) were cleared of internal
receptor-associated EGFP fluorescence (A, D,
and G) with serum starvation (24 hours) and
cycloheximide treatment (100 mg/ml for 6 hours)
and were incubated with 10 mM 5-HT (B), 10 mM
DA (C), 100 nM DOI (E), 100 nM clozapine (F),
and 100 nM ketanserin (H) for 15 minutes.
Receptor endocytosis was marked by the appearance of intracellular receptor-associated EGFP
fluorescence. This was quantified (described under Materials and Methods) and plotted (I). *P #
0.05 ligand versus control, Mann-Whitney U test.
46
Raote et al.
Fig. 3. Ligand-specific role of PKC in 5-HT2A internalization. All images
show EGFP fluorescence after the defined treatment. SB1 cells cleared of
internal fluorescence (I) were treated with ligand alone (A–D) or in the
presence of 10 mM sphingosine (E–H). Endocytosis is marked by the
appearance of receptor-associated EGFP in intracellular endosomes.
Inhibition of PKC by sphingosine prevents receptor endocytosis in
response to 5-HT (A versus E) and DOI (C versus G) but not DA (B versus
F) or clozapine (D versus H). Quantified and plotted (J), gray bars
represent relative levels of 5-HT2A internalization in the absence of
sphingosine, and black bars represent internalization in the presence of 10
mM sphingosine. Scale bar 20 mm.
when PKC was activated using 4 nM PMA, the modified
receptor showed no detectable internalization (Fig. 4E).
Time Course of 5-HT2A Recycling Is Ligand Dependent. We proceeded to follow 5-HT2A to determine its
postendocytic fate. SB1 cells were treated with ligand for 15
minutes and kept at 37°C for various times, then fixed and
imaged. As described earlier, receptors internalized in response to both 5-HT and DA were completely recycled in 2.5
hours (Bhattacharyya et al., 2006). The cells treated with 5HT (Fig. 5A) showed the presence of an intracellular pool of
receptors 2 hours after ligand washout (Fig. 5B). This
intracellular receptor is localized to transferrin-positive
endosomes (data not shown) but by 2.5 hours, all receptorassociated fluorescence was membrane localized (Fig. 5C).
Similar results were seen with DA (Fig. 5, D–F).
Receptor recycling was also observed when receptorexpressing cells were treated with the synthetic ligand agonist
DOI (Fig. 5, G–I). Again, SB1 cells were pulsed for 15 minutes
with the ligand and chased for different time points up to 8
hours. Instead of the 2.5 hours taken for receptors to recycle
when 5-HT or DA was used, receptors were resident in
transferrin-labeled endosomes for 5 hours extra (Fig. 5,
H and K). Recycling of DOI-internalized receptors was complete in 7.5 hours. Receptor recycling was quantified for 5HT, DA, DOI, and ketanserin by ELISA (Fig. 6M).
Synthetic inverse agonist clozapine also appeared to induce
5-HT2A recycling at the same rate as DOI (Fig. 5, J–L). At 7
hours after the addition and withdrawal of clozapine, 5-HT2AEGFP was localized to intracellular structures, but at 7.5
hours, all receptor-associated EGFP fluorescence was membrane localized.
A simplistic explanation that could account for shorter 5-HT2A
recycling times in response to 5-HT or DA is their relatively
quick degradation in comparison with clozapine or DOI. To rule
this out, we quantified internalization/recycling in the presence
of an antioxidant (ascorbic acid) to reduce DA oxidation. The 5HT2A recycling brought about by 5-HT and DA was unaffected
by the presence of 2 mM ascorbic acid (Supplemental Fig. 3). In
addition, the time taken for 5-HT2A recycling in response to
clozapine was independent of the concentration of ligand used.
All concentrations tested, from 4 nM to 10 mM clozapine,
brought about recycling in 7.5 hours (data not shown).
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Fig. 4. Mutation of a PKC phosphorylation site (serine 291) inhibits 5HT2A endocytosis in a ligand-specific manner. 5-HT2A-S291A expressing
human embryonic kidney 293 cells (HEK293) were treated with 5-HT (A),
DA (B), 4 nM PMA (C), DOI (D), or clozapine (E). Internalization mediated
by DA or clozapine was unimpeded, as can be seen by the appearance of
intracellular receptor-associated EGFP fluorescence (B and E). This was
quantified and plotted (F). *P # 0.05 ligand versus control by MannWhitney U test. Scale bar 20 mm.
Functional Selectivity in 5-HT2A Recycling
47
Protein Phosphatase 2A Is Required for Receptor
Recycling in the Case of Ligands That Bring about
PKC-Dependent Receptor Internalization. As PKC activation and hence potential receptor phosphorylation are
important steps in 5-HT2A internalization by 5-HT and DOI,
we checked for the involvement of enzymes active in the
endosome that would be required to return receptors to a form
that is competent to recycle by dephosphorylating them.
Inhibiting endosome acidification using ammonium chloride
(2 mM) inhibited recycling of receptors that had been
internalized by 5-HT (Supplemental Fig. 4, B–D) or by PKC
activation (PMA 4 nM, data not shown) but not for receptors
internalized by dopamine (Supplemental Fig. 4, E–G).
The pH-sensitive, endosome-localized enzymes that may
have a role in the process of receptor recycling internalized in
a phosphorylation-dependent manner, including protein
phosphatase 2A (PP2A) (Krueger et al., 1997). We inhibited
PP2A activity using the specific inhibitor okadaic acid at 10
nM. The cells were treated with 5-HT (Fig. 6A), DA (Fig. 6D),
DOI (Fig. 6G), or clozapine (Fig. 6J). Again, the cells were
observed for 2.5 hours (5-HT and DA) in the absence (Fig. 6, B
and E) or presence (Fig. 6, C and F) of okadaic acid. With DOI
treatment, the cells were observed for 7.5 hours in the absence
(Fig. 6H) or presence (Fig. 6I) of okadaic acid. In accordance
with our observations thus far, the receptors that internalized
via a PKC-dependent mechanism (in response to 5-HT and
DOI) also required PP2A activity to recycle, and those
internalized by DA and clozapine resisted treatment by the
PP2A inhibitor. A quantitative measure inhibition of recycling by PP2A is included in Fig. 6M.
Fig. 6. Ligand-specific PP2A involvement in receptor recycling. SB1 cells
were treated with 5-HT (A), DA (D), DOI (G), or clozapine (J). 5-HT2A
recycling was observed. As earlier, receptor recycling mediated by 5-HT
and DA was complete in 2.5 hours (B and E, respectively); and recycling
mediated by DOI and clozapine was complete in 7.5 hours (H and K,
respectively). However, when the PP2A inhibitor okadaic acid was
present, 5-HT2A recycling was inhibited in a ligand-specific manner. 5HT-endocytosed receptors were retained in intracellular endosomes
(arrowheads in C) as were DOI-internalized receptors (arrowheads in I),
while recycling in response to DA and clozapine was unimpeded (F and L).
Scale bar 20 mm. Quantification of this extent of internalized receptor was
performed by ELISA (E). **P , 0.01 by two tailed Student’s t test.
A qualitative description of 5-HT2A recycling in response to
clozapine seemed to show that PP2A has no role in receptor
recycling as recycling appears complete within 7.5 hours in
the absence (Fig. 6K) or presence (Fig. 6L) of okadaic acid.
As an estimate of the critical time period during which
PP2A might be acting to dephosphorylate the receptor, we
treated SB1 cells with 5-HT and added okadaic acid at
different times after this treatment. Up to 60 minutes after
addition of 5-HT, PP2A inhibition was able to block 5-HT2A
recycling. At 90 minutes after addition of the ligand, however,
okadaic acid treatment had no effect on recycling. It would
seem that receptor dephosphorylation is complete between 60
and 90 minutes after endocytosis (Supplemental Fig. 5)
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 16, 2017
Fig. 5. Ligand-specific time course of 5-HT2A recycling. SB1 cells were
treated with 5-HT (A), DA (D), DOI (G), or clozapine (J) for 15 minutes,
washed, and observed for varying times. At 2 hours after the 5-HT and DA
pulse (B and E, respectively), receptors were still retained in the
endosome, but after 2.5 hours, both 5-HT- and DA-internalized 5-HT2A
had recycled completely (C and F, respectively). At 7 hours after
internalization with DOI (H) or clozapine (K), 5-HT2A was still retained
in endosomes. At 7.5 hours, the receptors endocytosed by DOI (I) and
clozapine (L) were all localized to the cell surface again, as can be seen by
the concentration of all receptor-associated EGFP fluorescence. Scale bar
20 mm.
48
Raote et al.
Recycling of PMA-Internalized 5-HT2A Is Also PP2A
Dependent. When SB1 cells cleared of internal fluorescence
(Fig. 7A) were incubated in 4 nM PMA to activate PKC, wildtype receptor was internalized (Fig. 7B), and receptor
recycling was complete in 2.5 hours (Fig. 7C) in a manner
similar to that seen with activation of the receptor by 5-HT.
Again, we predicted receptor recycling after PMA-mediated
internalization would be PP2A dependent. In agreement with
this prediction, incubation of cells with 10 nM okadaic acid
inhibited receptor recycling after internalization using PMA
(Fig. 7D).
Discussion
Fig. 7. Receptor recycling mediated by PMA is dependent on PP2A. SB1 cells, cleared of internal fluorescence (A) were treated with 4 nM PMA for 15
minutes (B) and incubated for up to 2.5 hours in the absence (C) or presence (D) of the PP2A inhibitor okadaic acid 10 nM. The appearance of intracellular
receptor-associated fluorescence (arrowhead in B) showed internalization with recycling marked by the disappearance of these intracellular bodies (C).
Inhibition of PP2A prevented recycling, as seen by the continued residence of 5-HT2A in intracellular endosomes (arrowhead, D). These results have been
quantified and graphically represented (E). Scale bar 20 mm.
Downloaded from molpharm.aspetjournals.org at ASPET Journals on June 16, 2017
Although functional selectivity in receptor signaling has
been known for the better part of two decades (Berg et al.,
1998; Clarke and Bond, 1998; Ghanouni et al., 2001;
Gonzalez-Maeso et al., 2003; Urban et al., 2007), our
appreciation of how this feeds back to modulating the receptor
itself is limited. By use of 5-HT2A, we have studied the
intracellular sorting of a GPCR after the interaction of its
cognate ligand 5-HT and four other ligands: partial-efficacy
agonist DA, synthetic agonist DOI, antagonist ketanserin,
and inverse agonist clozapine. Ligand-specific regulation of
5-HT2A trafficking must arise as a direct result of functionally
selective signaling at the receptor. With the time course of
recycling and the involvement of PKC-mediated receptor
phosphorylation in trafficking as functional readouts, the
results described herein have been used to characterize
functional selectivity at the 5-HT2A receptor and to describe
the tightly controlled regulation of GPCR endocytosis and
recycling by multiple ligands.
In brief, using endogenous or therapeutically significant
GPCR-ligand interactions, we have shown that 1) every
ligand tested elicited a unique or “functionally selective” set
of behaviors at the 5-HT2A receptor, 2) GPCR functional
selectivity acts at both proximal and distal signaling events
and can feed back to the receptor by modulating its
spatiotemporal functioning, and 3) 5-HT2A may be phosphorylated in a ligand-dependent manner and that this PKCmediated phosphorylation acts as a regulatory step in 5-HT2A
trafficking.
Four of five ligands tested (5-HT, DA, DOI, and clozapine)
brought about receptor internalization and subsequent receptor recycling. As the inverse agonist clozapine caused
receptor endocytosis, it is clear that 5-HT2A endocytosis was
independent of receptor activation at its canonical intracellular transduction cascade: IP3-mediated Ca21 release from the
endoplasmic reticulum. The lack of 5-HT2A endocytosis in
response to ketanserin was in accordance with previous
observations in two cell lines where treatment of 5-HT2Aexpressing cells with a 10-minute pulse of ketanserin brought
about little to no receptor internalization (Berry et al., 1996;
Hanley and Hensler, 2002). Likewise, clozapine too has been
shown to induce 5-HT2A endocytosis in multiple cell lines and
even in vivo (Bhatnagar et al., 2001; Willins et al., 1998,
1999).
Studies have shown that rat 5-HT2A is not phosphorylated
by GRK as are many other GPCRs (Gray et al., 2001). Given
the lack of GRK involvement, it has been believed that 5-HT2A
endocytosis occurs independently of ligand-mediated phosphorylation. It has been difficult to probe for and identify 5HT2A phosphorylation (Sheffler et al., 2006), although it has
been identified that ribosomal S6 kinase phosphorylates the
receptor and modifies its signaling. PKC has also been shown
to phosphorylate 5-HT2A at two specific residues in vitro and
could regulate intracellular signaling via the receptor (Turner
and Raymond, 2005; Turner et al., 2007). We chose to better
characterize the effects of previously described PKC-mediated
phosphorylation of the 5-HT2A. We observed the effect of this
phosphorylation at one of these residues, serine 291, and its
effect on modulation of receptor trafficking. We have
demonstrated a ligand-dependent requirement for PKC
phosphorylation at this residue to bring about endocytosis
and concordantly for PP2A to allow for receptor dephosphorylation and recycling. Those ligands that require PKC
activation to bring about receptor internalization also require
PP2A-mediated dephosphorylation before receptor recycling
can occur. Mutation of the serine 291 to an alanine to prevent
phosphorylation by PKC mimicked the effect of PKCinhibition on internalization. Rat 5-HT2A is thus a useful
model GPCR to study GRK-independent, PKC-dependent
regulation of receptor endocytosis and recycling.
The physiologic functions of 5-HT2A trafficking/recycling
have not been identified, but it is clear that different ligands
display functional selectivity at the receptor in terms of events
proximal to receptor-ligand binding. Here, we show that distal
effects—recycling over 2.5 to 7.5 hours—are also differentially
modulated in a ligand-dependent manner.
Conventionally, GPCR recycling has been thought to be
a nonregulated process. It is only recently that some processes
involved specifically in the regulation of receptor recycling
Functional Selectivity in 5-HT2A Recycling
Acknowledgments
The authors thank Gautam Dey for creating the MATLAB routine
for quantifying internalization, the NCBS Central Imaging and Flow
Facility and Dr. Krishnamurthy for FACS sorting, the NCBS for
MATLAB licenses, and Dr. Aditi Bhattacharya for valuable input
and advice.
Authorship Contributions
Participated in research design: Raote, Bhattacharyya, Panicker.
Conducted experiments: Raote, Bhattacharyya.
Performed data analysis: Raote, Bhattacharyya, Panicker.
Contributed new reagents or analytic tools: Raote.
Wrote or contributed to the writing of the manuscript: Raote,
Bhattacharyya, Panicker.
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