Accelerat ing t he world's research.

Hallucinogenic 5-HT2AR agonists

LSD and DOI enhance dopamine
D2R protomer recognition and
signaling of D2-5-HT2A...
Dasiel O Borroto-Escuela, Luigi Agnati
Biochemical and Biophysical Research Communications

Cite this paper Downloaded from Academia.edu 

Get the citation in MLA, APA, or Chicago styles

Related papers Download a PDF Pack of t he best relat ed papers 

Dopamine D2 het erorecept or complexes and t heir recept or-recept or int eract ions in vent ral s…
Dasiel O Borrot o-Escuela, Luigi Agnat i, Sergio Tanganelli, Wilber Romero-Fernandez, Michael D…

Diversit y and Bias t hrough Recept or–Recept or Int eract ions in GPCR Het erorecept or Complexes. Fo…
Luigi Agnat i

Funct ional crosst alk and het eromerizat ion of serot onin 5-HT 2A and dopamine D2 recept ors
Terrell D Holloway
 Biochemical and Biophysical Research Communications 443 (2014) 278–284

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications

journal homepage: www.elsevier.com/locate/ybbrc

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine

D2R protomer recognition and signaling of D2-5-HT2A
heteroreceptor complexes
Dasiel O. Borroto-Escuela a, Wilber Romero-Fernandez a, Manuel Narvaez b, Julia Oflijan c, Luigi F. Agnati d,
Kjell Fuxe a,⇑
a
Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
b
Department of Physiology, School of Medicine, University of Málaga, Spain
c
Department of Physiology, Faculty of Medicine, University of Tartu, Estonia
d
IRCCS Lido, Venice, Italy

a r t i c l e i n f o a b s t r a c t

Article history: Dopamine D2LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection
Received 18 November 2013 of the two receptors and shown to have bidirectional receptor–receptor interactions. In the current study
Available online 2 December 2013 the existence of D2L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the
ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR ago-
Keywords: nists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the den-
G-protein-coupled receptors sity of D2like antagonist 3H-raclopride binding sites and significantly reduced the pKiH values of the high
Dopamine D2 receptor
affinity D2R agonist binding sites in 3H-raclopride/DA competition experiments. Similar results were
Serotonin 5-HT2A receptor
Lysergic acid diethylamide
obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists
2,5-Dimethoxy-4-iodoamphetamine on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced
Antipsychotic drugs CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells
Heterodimerization DOI and LSD but not TCB2 significantly enhanced the D2LR agonist quinpirole induced inhibition of
Heteroreceptor complexes CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing
Allosteric modulation actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mech-
Receptor–receptor interactions anism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely
mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of
the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and
the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic
5-HT2A agonists.
Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction Conversely, when the D2LR-mediated adenylyl cyclase inhibition

Biophysical methods demonstrated dopamine D2LR-serotonin
5-HT2AR heteromers in cellular models after cotransfection of
the two receptors with bidirectional receptor–receptor interac-
tions [1,2]. The 5-HT2AR-mediated phospholipase C activation by
5-HT was synergistically enhanced by the concomitant activation
of the D2LR protomer by the D2R agonist quinpirole as shown in
a NFAT-luciferase reporter gene assay. A specific and significant
elevation of the intracellular calcium levels was also observed
when both receptor protomers were simultaneously activated.
⇑ Corresponding author. Fax: +46 8 315721.
E-mail addresses: Dasiel.Borroto-Escuela@ki.se (D.O. Borroto-Escuela),
wromfdez@gmail.com (W. Romero-Fernandez), mnarvaez@uma.es (M. Narvaez),
juliaofli@gmail.com (J. Oflijan), luigiagnati@tin.it (L.F. Agnati), Kjell.Fuxe@ki.se
(K. Fuxe).
by the D2R agonist quinpirole was assayed coagonist stimulation
of D2LR and 5-HT2AR protomers by quinpirole and 5-HT, respec-
tively, led to a reduction of the D2LR protomer signaling. These
results suggested the existence of a 5-HT2AR-mediated D2LR
trans-inhibition phenomenon [1]. The D2LR-5-HT2AR heteromer
represents a novel target for antipsychotic drugs since such drugs
are well known to exert their therapeutic actions at least with
regard to positive symptoms via blockade of D2Rs [3].
Albizu et al. [4] recently observed that activation of D2Rs by
quinpirole increases the affinity of the hallucinogenic 5-HT2AR
agonist DOI ((±)-2,5-dimethoxy-4-iodoamphetamine) for the
5-HT2AR and reduces the DOI induced Gq/11 coupling to the 5-HT2AR
as seen from the diminished production of inositol phosphate by
DOI. This is different from the case when the endogenous ligand
5-HT was used to activate the 5-HT2AR [1]. These findings have
led us to study if the observed ability of 5-HT to produce an

0006-291X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bbrc.2013.11.104
 D.O. Borroto-Escuela et al. / Biochemical and Biophysical Research Communications 443 (2014) 278–284
allosteric counteraction of D2R agonist induced D2R signaling via
the 5-HT2AR protomer [1] is altered when using the known hallu-
cinogenic high affinity 5-HT2AR agonists lysergic acid diethyl-
amide (LSD) and DOI [5,6]. The 5-HT receptor agonist activity of
LSD in the CNS was first described in 1968 [7]. Such an alteration
if enhancing D2R protomer function may contribute to their psy-
chotic actions. The current study tested this hypothesis in
HEK293 cells and ventral striatum studying 5-HT2AR-D2R interac-
tions using LSD and DOI and a standard 5-HT2AR agonist TCB2 [8]
to modulate D2R binding and signaling. Indications were obtained
for the existence of D2R-5-HT2AR heteroreceptor complexes in
ventral and dorsal striatum with proximity ligation assays (PLA)
[9].
2. Materials and methods
Detailed descriptions are available in Supplementary Material
and Methods on: chemicals, reagents and drugs; antibodies; cell
culture and transfection; membrane preparation and immunofluo-
rescence microscopy.
2.1. Animals
All studies involving animals were performed in accordance
with the Swedish National Board for Laboratory Animal and Euro-
pean Communities Council Directive (Cons 123/2006/3) guidelines
for accommodation and care of Laboratory Animals. Male Sprague–
Dawley rats, 10 weeks old, weighing 310–350 g were obtained
from Charles River Laboratories (Germany). The animals were
housed one week before experiments under a 12-h light/dark cy-
cle, with ambient temperature of 21 ± 2 °C, relative humidity of
50 ± 5%. Food and water available ad libitum.
2.2. Proximity ligation in situ assay
HEK293T cells transiently co-expressing D2LR and 5-HT2AR or
rat striatal floating sections (30 lm) were employed for
in situ proximity ligation assay (PLA) [10–12]. In situ PLA was per-
formed according to manufacturer’s instructions (Duo-
link in situ PLA detection kit, Olink, Sweden). The primary
antibodies of different species, monoclonal anti-D2R produced in
mouse (WH00018113M1, 5 lg/ml, Sigma–Aldrich, Sweden) and
polyclonal anti-5-HT2AR produced in rabbit (ab16028, 5 lg/ml,
Abcam, Sweden), were used. Control experiments employed only
one primary antibody or HEK293T cells transfected with cDNAs
encoding only one type of receptor. The products were visualized
using a Leica SP2 confocal microscope (Leica, USA). Each image
represents a single Z-scan. and was taken using the following
acquisition parameters: Nuclei: DAPI, ex.405 em.windows 440/
500 (35% intensity, gain 468, offset 1); PLA clusters/blobs: Texa
Red ex.543 em.windows 600/670 (50% intensity, gain 650, offset
1).
2.3. Luciferase reporter gene assay
We used a dual luciferase reporter assay to indirectly detect
variations of cAMP levels in transiently transfected cell lines trea-
ted with different compounds in a range of concentrations (typi-
cally 25 nM to 1 lM). Stimulation of Gi/o-coupled GPCRs
decreases intracellular cAMP reducing CRE-linked reporter-gene
transcription. In order to study this inhibitory effect, assays are
usually performed in the presence of forskolin, a stimulator of
adenylyl cyclase, to raise the cAMP level so that the inhibition is
easier to detect [13,14]. For luciferase assays, 24 h before transfec-
tion cells were seeded at a density of 1  106 cells/well in 75 cm2
279
flasks and transfected with FugeneÒ HD transfection reagent
(Promega, Sweden). Cells were co-transfected with plasmids
corresponding to three constructs as follows: 2 lg firefly lucifer-
ase-encoding experimental plasmid (pGL4-CRE-luc2p; Promega,
Sweden), 2 lg of wild type 5-HT2A or D2LR expression vectors
and 1 lg Renilla luciferase-encoding internal control plasmid
(phRG-B; Promega). Approximately 36 h post transfection, cells
were treated for 30 min with appropriate ligands, including
forskolin, washed and incubated for 4 h before harvested with
passive lysis buffer (Promega). Then the luciferase activity of cell
extracts was determined using a luciferase assay system according
to the manufacturer’s protocol in a POLARstar Optima plate reader
(BMG Labtech) using a 30-nm bandwidth excitation filter at
535 nm. Firefly luciferase was measured as firefly luciferase
luminescence over a 15 s reaction period. The luciferase values
were normalized against Renilla luciferase luminescence values.
Transfection experiments were performed in quadruplicate and
repeated at least three times.
2.4. [3H]-raclopride binding experiments
Saturation binding experiments with the D2-likeR antagonist
[3H]-raclopride (specific activity 82.8 Ci/mmol, PerkinElmer Life
Sciences, USA) was performed essentially as described earlier
[15,16]. Briefly, membrane homogenates from HEK cells expressing
5-HT2AR and D2R or ventral striatal rat membrane preparations
(100 lg protein/ml) were incubated with increasing concentra-
tions of [3H]-raclopride (ranging from 0.1 to 10 nM) in 250 ll of
incubation buffer (IB; 50 mM Tris–HCl pH 7.4, containing
100 mM NaCl, 7 mM MgCl2, 1 mM EDTA, 0.05% BSA and 1 mM
dithiothreitol) for 60 min at 30 °C, in the presence or absence of
different concentrations of hallucinogenic 5-HT2AR agonists LSD
and DOI, a standard 5-HT2AR agonist TCB2 or 5-HT. Nonspecific
binding was defined as the binding in the presence of 1 mM dopa-
mine hydrochloride (Sigma–Aldrich, Sweden). The incubation was
terminated by rapid filtration through Whatman GF/B filters
(Maidstone, Kent, UK) using a MultiScreen™ Vacuum Manifold
96-well (Millipore Corp, Sweden), followed by five washes
(250 ll per wash) with ice-cold washing buffer (50 mM Tris–
HCl pH 7.4). The filters were dried, 5 ml of scintillation cocktail
was added and the bound ligand was determined after 12 h in each
sample by liquid scintillation spectrometry (4 min). [3H]-raclo-
pride (2.0–3.0 nM) binding was displaced by dopamine in compe-
tition experiments to determine pKiH and pKiL values from the
competition curves obtained. Briefly, membrane homogenates
from HEK cells expressing 5-HT2A and D2 receptors or ventral stri-
atal rat membrane preparations (100 lg protein/ml) were incu-
bated with increasing concentrations of dopamine (ranging from
0.1 to 1 mM) in 250 ll of IB for 60 min at 30 °C, in the presence
or absence of different nanomolar concentrations of the hallucino-
genic high affinity 5-HT2A agonists LSD and DOI [5,6], a standard
5-HT2AR antagonist TCB2 or 5-HT. The incubation was terminated
as described above and the radioactivity content of the filters was
detected by liquid scintillation spectrometry. Nonspecific binding
was defined by radioligand binding in the presence of 1 mM dopa-
mine hydrochloride (Sigma Aldrich, Sweden).
2.5. Statistical analysis
The number of samples (n) in each experimental condition is
indicated in figure legends. Data from saturation and competition
experiments were analyzed by nonlinear regression analysis using
GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA). The
density of the binding sites (Bmax), dissociation constant (KD) and
inhibition constants of the high and low affinity state of the recep-
tor (pKiH, pKiL) from several independent replications were aver-
280 D.O. Borroto-Escuela et al. / Biochemical and Biophysical Research Communications 443 (2014) 278–284

aged allowing statistical comparisons using a one way analysis of
variance (ANOVA). Group differences after ANOVAs were measured
by post hoc Turkey’s Multiple Comparison Test. The P value 0.05
and lower was considered significant.
3. Results
3.1. PLA and immunofluorescence experiments
PLA demonstrated the presence of red clusters (blobs) of
D2R and 5-HT2AR, representing D2-5-HT2A heteroreceptor
complexes in discrete regions of the neuropil of the dorsal
striatum and nucleus accumbens core but not in the corpus
callosum and the anterior limb of the anterior commissure
(Fig. 1). Also no PLA positive clusters were observed in the
cortical regions analized at the Bregma level 1.7. These
results were validated in HEK293 cells transiently cotransfected
with human D2LR and 5-HT2AR as seen from colocalization
and generation of PLA positive signals (red blobs) found
after cotransfection but not after single D2LR transfections
(Supplementary Fig. S1).
Using double immunofluorescence procedures with green and
red immunofluorescence for D2Rs and 5-HT2ARs, respectively, it
was possible to observe a widespread appearance of punctate yel-
lowish fluorescence in the dorsal striatum and the nucleus accum-
bens. It likely mainly represents a dendritic colocalization of the D2
and 5-HT2A receptors (Supplementary Fig. S2) since the main part
of the D2 and 5-HT2A receptors shows a postjunctional location in
the forebrain [17,18]. Yellowish immunofluorescence was also ob-
served in discrete nerve cell body populations in the striatal
regions.
3.2. CRE-luciferase reporter gene experiments
A forskolin-induced CRE-luciferase reporter gene assay in
cotransfected HEK293 cells was used to study the modulation of
the D2LR protomer responses by the hallucinogenic 5-HT2AR ago-
nists DOI and LSD and the standard 5-HT2AR agonist TCB2. As seen
in Fig. 2, 60 and 10 nM of DOI and LSD, respectively (three times
the Ki values) significantly enhanced the quinpirole induced inhibi-
tion of CRE-luciferase activity. These effects could not be observed
in cells singly transfected with D2LR (Fig. 2B). The D2R antagonist

Fig. 1. D2-5-HT2A heteroreceptor complexes in striatal sections of rat detected by PLA. In situ PLA was performed using primary antibodies of different species directed to
D2R and 5-HT2AR (see Section 2). The detected heteroreceptor complexes are seen as red clusters indicated by arrows. Specific D2R-5-HT2AR clusters are visualized within
discrete regions of the dorsal striatum (A: CPu) and nucleus accumbens, especially the core (B: AcbC). They were almost absent in cortical regions, the corpus callosum (cc)
and the anterior limb of the anterior commissure (aca). In ‘‘C’’ the relative densities of PLA cluster distributions are schematically illustrated by the density of red puncta.
Nuclei are shown in blue (DAPI). Scale bars are shown in the right low part of each panel. Three independent experiments (three rats) were performed. Bregma level 1.7. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
 D.O. Borroto-Escuela et al. / Biochemical and Biophysical Research Communications 443 (2014) 278–284 281

A 120

(% forskolin stimulated)
+++

CREB-luc induction
*** ••• ΦΦΦ
90

∅∅∅
60

∆
∆∆
30

M
M

nM

nM

M
M

nM

nM

nM

nM
nM

nM

nM
3µ

0n

0n
0n
60

00
10

60

60

00
50

10

60

10
I6

10
K

t1
t1
op

op

p
Q

D
B2

lp
lF

et
o

t
LS

Ke

Ke

Ke
D

l
al

al
TC
ro

H
a

K
H

H
+
nt

+
+

+
+
+

+
nM
co

nM

M
nM

nM
nM

nM
nM

M
nM

nM

0n
50

50
0n

50

10
10
10
50

I6
50

10

I6
Q

D
Q

B2
O
Q
Q

B2

LS

LS
O

TC
D
TC

+
+

+
+

+
nM
nM

nM
+

nM

nM
nM

50
50

50
50

50
50

Q
Q

Q
Q

Q
Q

B 150
(% forskolin stimulated)
CREB-luc induction

120
•••
+++
*** ΦΦΦ
90

∅∅∅
60

30

0
M

nM

nM

nM
M

nM

nM

nM

nM

M
nM

nM

nM
3µ

0n
0n

0n

0n
60

10

60

00

00
50

60

10

60

10
I6

10
I6

1
K

op

t1

t1
op
Q

B2

D
op

D
p

B2
lF

t
al
LS

LS

Ke
al

Ke
Ke
D

D
al
al

TC

TC
ro

H
H
H

H
+
nt

+
+

+
+
+
+

+
nM
co

nM

M
nM

nM

nM
nM
nM

M
nM

0n
50

50
0n

10

10
10

10
50
50

I6
I6
Q

B2
B2

O
D
Q
Q

LS
O

LS

D
TC
TC

+
+

nM
+

nM
nM

50
50
50

Q
Q
Q

Fig. 2. (A) Demonstration that LSD and DOI but not TCB2 can enhance the D2LR agonist inhibition of the forskolin-induced CRE-luciferase reporter gene expression. The
counteraction exerted by haloperidol and ketanserin on these actions is also shown. HEK293T cells were transiently co-transfected with 1 lg firefly luciferase-encoding
experimental plasmid (pGL4-CRE-luc2p), 1 lg of both (5-HT2AR and D2LR) expression vectors and 50 ng Renilla luciferase-encoding internal control plasmid (phRG-B). After
cell incubation with respective agonists and/or antagonists (in presence of 3 lM forskolin, sub-maximal concentration value) luciferase activity was measured. Light emission
is expressed as a percentage of the control forskolin-induced value. The data represent the means ± S.E.M. of three independent experiments performed in triplicate.
Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison post-test. The P value 0.05 and lower was considered
significant. ØØØ: Significantly different compared to control (P < 0.001); ⁄⁄⁄: Significantly different compared to quinpirole 50 nM (P < 0.001); +++: Significantly different
compared to quinpirole 50 nM + TCB2 10 nM (P < 0.001); : Significantly different compared to quinpirole 50 nM + DOI 60 nM (P < 0.001); DD and D: Significantly different
compared to quinpirole 50 nM, quinpirole 50 nM + Ket 100 nM, quinpirole 50 nM + DOI 60 nM + Ket 100 nM and quinpirole 50 nM + LSD 10 nM + Ket 100 nM (P < 0.01 and
P < 0.05); UUU: Significantly different compared to quinpirole 50 nM + LSD 10 nM (P < 0.001). (B) Demonstration that LSD, DOI and TCB2 cannot enhance the D2LR agonist
inhibition of the forskolin-induced CRE-luciferase reporter gene expression in HEK293 cells singly transfected with D2LR. The experimental conditions are the same as
described above (A) with the only difference being that HEK293T cells were transiently transfected only with 1 lg firefly luciferase-encoding experimental plasmid (pGL4-
CRE-luc2p), 1 lg of D2LR expression vector and 50 ng Renilla luciferase-encoding internal control plasmid (phRG-B). The data represent the means ± S.E.M. of three
independent experiments performed in triplicate. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison
post-test. The P value 0.05 and lower was considered significant. ØØØ: Significantly different compared to control (P < 0.001); ⁄⁄⁄: Significantly different compared to
quinpirole 50 nM (P < 0.001); +++: Significantly different compared to quinpirole 50 nM + TCB2 10 nM (P < 0.001); UUU: Significantly different compared to quinpirole
50 nM + DOI 60 nM (P < 0.001); : Significantly different compared to quinpirole 50 nM + LSD 10 nM (P < 0.001). Forskolin (3 lM); Q, quinpirole (50 nM); halop, haloperidol
(60 nM), TCB2, 4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl)methylamine hydrobromide (10 nM), DOI, 1-(2,5-dimethoxy-4-iodophenyl)-propan-2-amine (60 nM), LSD,
(6aR,9R)-N,N-diethyl-7-methyl-4,6,6a,7,8,9-hexahydroindolo-[4,3-fg]quinoline-9-carboxamide (Lysergic acid diethylamide) (10 nM) and Ket, ketanserin (100 nM).

haloperidol blocked not only the inhibitory action of the D2R like 5-HT2AR antagonist ketanserin (100 nM) only blocked the
agonist quinpirole alone but also the enhancement by DOI and enhancement of D2LR receptor signaling produced by DOI and
LSD of the inhibition of CRE-luciferase activity (Fig. 2A). The LSD but not the action of quinpirole (Fig. 2A).
282 D.O. Borroto-Escuela et al. / Biochemical and Biophysical Research Communications 443 (2014) 278–284

Table 1
Effects of the hallucinogenic 5-HT2A agonists LSD and DOI, the standard 5-HT2A agonist TCB2 and 5-HT on the binding characteristics of D2like receptors in membrane
preparations from the ventral striatum.3H-raclopride binding saturation and 3H-raclopride/DA competition experiments. Rat membrane preparations from the ventral striatum
were incubated with increasing concentrations of [3H]-raclopride for 60 min at 30 °C in the presence or absence of the hallucinogenic 5-HT2AR agonists LSD and DOI, a standard
5-HT2AR agonist TCB-2 and 5-HT as indicated. The 5-HT2AR antagonist ketanserin in the concentration indicated blocked the action of LSD and DOI. Nonspecific binding was
defined as the binding in the presence of dopamine hydrochloride (1 mM). Nonlinear curve-fitting using GraphPad PRISM 5.0 determined maximal binding capacities (Bmax) and
dissociation constants (KD). Data are presented as means ± S.E.M. of three independent experiments, each one performed in triplicate. Statistical analysis was performed by one-
way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison post-test. The P value 0.05 and lower was considered significant. ⁄⁄(P < 0.01): Significant difference
compared with respective control Bmax value; #(p < 0.05) and &&(p < 0.01): Significant difference compared with LSD and DOI Bmax value, respectively.

Receptors 5-HT2AR-D2R [3H]-raclopride binding saturation [3H]-raclopride competition with dopamine

Bmax (fmol/mg protein) KD (nM) pKiH pKiL
Control 182 ± 13 1.29 ± 0.31 7.10 ± 0.18 5.30 ± 0.16
5-HT (0.5 lM) 189 ± 12 1.36 ± 0.19 7.36 ± 0.19 5.50 ± 0.12
5-HT (0.5 lM) + Ketanserin (1 lM) 196 ± 14 1.57 ± 0.35 7.22 ± 0.11 5.17 ± 0.10
Control 179 ± 10 1.21 ± 0.23 7.73 ± 0.18 5.33 ± 0.11
DOI (50 nM) 235 ± 11⁄⁄ 1.54 ± 0.24 8.31 ± 0.24⁄ 5.38 ± 0.14
DOI (50 nM) + Ketanserin (50 nM) 174 ± 10 && 1.51 ± 0.26 7.95 ± 0.11# 5.32 ± 0.06
Control 192 ± 11 1.33 ± 0.26 7.85 ± 0.25 5.76 ± 0.13
LSD (10 nM) 258 ± 19⁄⁄ 1.74 ± 0.39 8.50 ± 0.26⁄⁄ 5.58 ± 0.12
LSD(10 nM) + Ketanserin (50 nM) 216 ± 10# 1.59 ± 0.21 7.95 ± 0.15# 5.66 ± 0.17
Control 190 ± 12 1.37 ± 0.30 7.56 ± 0.16 5.56 ± 0.10
TCB-2 (10 nM) 194 ± 11 1.53 ± 0.27 7.41 ± 0.15 5.50 ± 0.11
TCB-2 (10 nM) + Ketanserin (50 nM) 194 ± 12 1.27 ± 0.28 7.24 ± 0.14 5.38 ± 0.12

Competition experiments with the dopamine D2-like receptor antagonist [3H] raclopride (2–3 nM) versus increasing concentrations of dopamine in ventral striatum were
also performed in the same four groups as above. Means ± S.E.M are given for the negative logarithm of the Ki values (Ki is the inhibition constant) of the high affinity DA
binding (pKiH) and low affinity DA binding (pKiL) sites from three independent experiments, each one performed in triplicate. The higher the value the higher the affinity of
DA. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey’s Multiple Comparison post-test. The P value 0.05 and lower was
considered significant. ⁄(P < 0.05) and ⁄⁄(P < 0.01): Significantly different compared to the respective control pKiH value; #(P < 0.05): Significantly different compared to the
LSD pKiH value and the DOI pKiH value, respectively.

Fig. 3. Effects of LSD, DOI, TCB2 and 5-HT on D2R [3H]-raclopride binding in saturation experiments in ventral striatal membrane preparations. Ventral striatal rat membrane
preparations were incubated with increasing concentrations of [3H]-raclopride for 60 min at 30 °C in the presence or absence of the two hallucinogenic 5-HT2AR agonists LSD
and DOI, the standard 5-HT2A receptor agonist TCB2 and 5-HT as indicated. Nonspecific binding was defined as the binding in the presence of dopamine hydrochloride
(1 mM). Maximal binding capacities (Bmax) and dissociation constants (KD) were determined fitting a nonlinear curve using GraphPad PRISM 5.0 software. Data are presented
as means ± S.E.M of three independent experiments, each one performed in triplicate. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed
by Tukey’s Multiple Comparison post-test. The P value 0.05 and lower was considered significant. ⁄⁄(P < 0.01): Significant difference in Bmax values compared to respective
control group. #(P < 0.05): Significantly different compared to LSD Bmax value. ##(P < 0.01): Significantly different compared to DOI Bmax value.

3.3. 3H-raclopride binding experiments experiments in ventral striatal membranes and in membranes
from cotransfected HEK293 cells. Both DOI and LSD but not TCB-
The modulation by DOI, LSD and TCB2 of the [3H]-raclopride 2 in equivalent nanomolar concentrations significantly increased
binding characteristics was studied in saturation and competition the Bmax values of [3H]-raclopride binding sites in several
 D.O. Borroto-Escuela et al. / Biochemical and Biophysical Research Communications 443 (2014) 278–284
Fig. 4. Displacement of D2-likeR antagonist [3H]-raclopride binding by LSD.
Competition experiments with the dopamine D2-like receptor antagonist [3H]
raclopride (2–3 nM) versus increasing concentrations of LSD: (A) in ventral striatal
and (B): HEK cell membrane preparations from transiently cotransfected 5-HT2AR-
D2R cells or singly transfected D2R cells. Nonspecific binding was defined as the
binding in the presence of dopamine hydrochloride (1 mM). Data are presented as
means ± S.E.M of three independent experiments, each one performed in triplicate.
The negative logarithm of the Ki value is given (pKi).
concentrations without affecting the KD values in ventral striatal
membranes (Table 1; Fig. 3 and Supplementary Table S1). The ef-
fects were blocked by the 5-HT2AR antagonist ketanserin
(50 nM). In [3H]-raclopride/DA competition experiments DOI and
LSD but not TCB-2 in concentrations from 10 to 100 nM) produced
a significant reduction in the pKiH values for high affinity D2R ago-
nist binding sites but not in their pKiL values (Table 1, Supplemen-
tary Table S2). These effects were all blocked by ketanserin
treatment. The binding results obtained in ventral striatal mem-
branes were validated in cotransfected but not in singly transfec-
ted D2LR HEK293 cells in both saturation and competition
experiments (Supplementary Tables S3 and S4). Only in higher
concentrations, with Ki values in the range of 500–1000 nM, did
the LSD directly effect the orthosteric site of the D2R protomer as
seen by the displacement of [3H]-raclopride in competition exper-
iments in ventral striatal and in HEK293 membrane preparations
(Fig. 4).
4. Discussion
Experimental evidence was obtained that the hallucinogenic 5-
HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist
TCB2 in the nanomolar range produced an enhancement of the D2R
agonist induced D2R protomer recognition and signaling. In con-
trast, the endogenous ligand 5-HT was previously shown to exert
an allosteric antagonistic action on D2R signalling in D2LR-5-
HT2AR cotransfected HEK293 cells [1]. TCB2 in the range of 10–
100 nM which can fully activate the 5-HT2A signaling pathways
[19] lacked the ability to modulate the D2R protomer recognition
and signaling in 3H-raclopride binding and CRE reporter gene as-
says, respectively. Therefore, the enhancement of D2R signaling
over the AC-PKA-CRE pathway produced by LSD and DOI cannot
be produced by crosstalk in the signaling cascades of the D2 and
5-HT2A receptors. The LSD and DOI enhancement of the agonist-
induced D2R protomer signalling observed and blocked by ketan-
serin is instead mediated by a biased agonist action of DOI and
LSD at the orthosteric site of the 5-HT2AR protomer. This leads
to an allosteric facilitatory receptor–receptor interaction in the
D2-5-HT2A heteroreceptor complex enhancing the D2 protomer
signaling over Gi/o.
This allosteric mechanism probably also underlies the ability of
DOI and LSD to increase the density of the D2like receptors in
membranes from D2R and 5-HT2AR cotransfected HEK cells and
from the ventral striatum, an action blocked by ketanserin. Thus,
there may exist cryptic D2R protomers of the D2-5-HT2A
283
heteroreceptor complex in the membranes which cannot bind
the D2like receptor antagonist. However, after the biased agonist
action of DOI and LSD at the 5-HT2AR protomer an allosteric
change develops in the cryptic D2R protomers and their orthosteric
binding sites become available to binding by 3H-raclopride. In the
3
H-raclopride/DA competition experiments LSD and DOI but not
TCB2 also significantly enhanced the affinity of the high affinity
but not of the low affinity D2R agonist binding sites which was
blocked by ketanserin. Thus, the biased 5-HT2AR actions of LSD and
DOI at the 5-HT2AR leads both to increased D2R density and
increased D2 agonist affinity at the high affinity agonist binding site.
Taken together, the hallucinogenic 5-HT2AR agonists LSD and
DOI induce pathological facilitatory allosteric receptor–receptor
interactions enhancing D2R protomer recognition and signaling
in D2-5-HT2A heteroreceptor complexes demonstrated in discrete
regions of the nucleus accumbens core and the dorsal striatum.
Thus, the psychotic actions of the 5-HT2AR hallucinogens [20]
can involve enhancement of D2R protomer signaling in the D2-5-
HT2A heteroreceptor complex in the ventral striatum. This gives
also a novel understanding of the molecular mechanism for anti-
psychotic actions of atypical antipsychotic drugs. Thus, risperidone
and clozapine are inter alia characterized by their higher affinity
for 5-HT2A than for D2 receptors resulting in their higher potency
to block 5-HT2AR than D2Rs [20–22]. One advantage of many atyp-
ical antipsychotics with such properties may be that they can
counteract the D2 receptor signaling at low doses in the D2-5-
HT2A heteroreceptor complex through their combined blockade
of the D2 and 5-HT2A protomers. This may lead to antipsychotic
effects against positive symptoms of schizophrenia in doses that
will not fully block several other D2 receptor populations in the
CNS which can be involved in producing cognitive and extrapyra-
midal side-effects of antipychotics [21,23].
Acknowledgments
This work has been supported by the Swedish Medical
Research Council (04X-715), Telethon TV3’s La Marató Foundation
2008 and Hjärnfonden to KF; by Grants from the Swedish
Royal Academy of Sciences (Stiftelsen B. von Beskows Fond and
Stiftelsen Hierta-Retzius stipendiefond) and Karolinska Institutets
Forskningsstiftelser 2011 and 2012 to D.O.B-E. D.O.B-E. and
W.R-F. belong to ‘‘Academia de Biólogos Cubanos’’ group. George
Milicevic is acknowledged for his practical help.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bbrc.2013.11.104.
References
[1] D.O. Borroto-Escuela, W. Romero-Fernandez, A.O. Tarakanov, D. Marcellino, F.
Ciruela, L.F. Agnati, K. Fuxe, Dopamine D2 and 5-hydroxytryptamine 5-
HT((2)A) receptors assemble into functionally interacting heteromers,
Biochem. Biophys. Res. Commun. 401 (2010) 605–610.
[2] S. Lukasiewicz, A. Polit, S. Kedracka-Krok, K. Wedzony, M. Mackowiak, M.
Dziedzicka-Wasylewska, Hetero-dimerization of serotonin 5-HT(2A) and
dopamine D(2) receptors, Biochim. Biophys. Acta 2010 (1803) 1347–1358.
[3] A. Carlsson, M.L. Carlsson, Adaptive properties and heterogeneity of dopamine
D(2) receptors – pharmacological implications, Brain Res. Rev. 58 (2008) 374–
378.
[4] L. Albizu, T. Holloway, J. Gonzalez-Maeso, S.C. Sealfon, Functional crosstalk and
heteromerization of serotonin 5-HT2A and dopamine D2 receptors,
Neuropharmacology 61 (2011) 770–777.
[5] R.A. Glennon, M. Titeler, M.R. Seggel, R.A. Lyon, N-methyl derivatives of the 5-
HT2 agonist 1-(4-bromo-2,5-dimethoxyphenyl)-2-aminopropane, J. Med.
Chem. 30 (1987) 930–932.
[6] M. Titeler, R.A. Lyon, R.A. Glennon, Radioligand binding evidence implicates
the brain 5-HT2 receptor as a site of action for LSD and phenylisopropylamine
hallucinogens, Psychopharmacology 94 (1988) 213–216.
284 D.O. Borroto-Escuela et al. / Biochemical and Biophysical Research Communications 443 (2014) 278–284
[7] N.E. Anden, H. Corrodi, K. Fuxe, T. Hokfelt, Evidence for a central 5-
hydroxytryptamine receptor stimulation by lysergic acid diethylamide, Br. J.
Pharmacol. 34 (1968) 1–7.
[8] M.A. Fox, H.T. French, J.L. Laporte, A.R. Blackler, D.L. Murphy, The serotonin 5-
HT(2A) receptor agonist TCB-2: a behavioral and neurophysiological analysis,
Psychopharmacology 212 (2010) 13–23.
[9] D.O. Borroto-Escuela, W. Romero-Fernandez, P. Garriga, F. Ciruela, M. Narvaez,
A.O. Tarakanov, M. Palkovits, L.F. Agnati, K. Fuxe, G protein-coupled receptor
heterodimerization in the brain, Methods Enzymol. 521 (2013) 281–294.
[10] D.O. Borroto-Escuela, K. Van Craenenbroeck, W. Romero-Fernandez, D.
Guidolin, A.S. Woods, A. Rivera, G. Haegeman, L.F. Agnati, A.O. Tarakanov, K.
Fuxe, Dopamine D2 and D4 receptor heteromerization and its allosteric
receptor–receptor interactions, Biochem. Biophys. Res. Commun. 404 (2011)
928–934.
[11] P. Trifilieff, M.L. Rives, E. Urizar, R.A. Piskorowski, H.D. Vishwasrao, J. Castrillon,
C. Schmauss, M. Slattman, M. Gullberg, J.A. Javitch, Detection of antigen
interactions ex vivo by proximity ligation assay: endogenous dopamine D2-
adenosine A2A receptor complexes in the striatum, Biotechniques 51 (2011)
111–118.
[12] D.O. Borroto-Escuela, W. Romero-Fernandez, G. Mudo, M. Perez-Alea, F.
Ciruela, A.O. Tarakanov, M. Narvaez, V. Di Liberto, L.F. Agnati, N. Belluardo,
K. Fuxe, Fibroblast growth factor receptor 1–5-hydroxytryptamine 1A
heteroreceptor complexes and their enhancement of hippocampal plasticity,
Biol. Psychiatry 71 (2012) 84–91.
[13] S.J. Hill, J.G. Baker, S. Rees, Reporter-gene systems for the study of G-protein-
coupled receptors, Curr. Opin. Pharmacol. 1 (2001) 526–532.
[14] S.J. Hill, C. Williams, L.T. May, Insights into GPCR pharmacology from the
measurement of changes in intracellular cyclic AMP; advantages and pitfalls of
differing methodologies, Br. J. Pharmacol. 161 (2010) 1266–1275.
[15] M. Torvinen, L.B. Kozell, K.A. Neve, L.F. Agnati, K. Fuxe, Biochemical
identification of the dopamine D2 receptor domains interacting with the
adenosine A2A receptor, J. Mol. Neurosci. 24 (2004) 173–180.
[16] S. Dasgupta, S. Ferre, B. Kull, P.B. Hedlund, U.B. Finnman, S. Ahlberg, E. Arenas,
B.B. Fredholm, K. Fuxe, Adenosine A2A receptors modulate the binding
characteristics of dopamine D2 receptors in stably cotransfected fibroblast
cells, Eur. J. Pharmacol. 316 (1996) 325–331.
[17] A. Jansson, B. Tinner, M. Bancila, D. Verge, H.W. Steinbusch, L.F. Agnati, K. Fuxe,
Relationships of 5-hydroxytryptamine immunoreactive terminal-like
varicosities to 5-hydroxytryptamine-2A receptor-immunoreactive neuronal
processes in the rat forebrain, J. Chem. Neuroanat. 22 (2001) 185–203.
[18] R.L. Jakab, P.S. Goldman-Rakic, 5-Hydroxytryptamine2A serotonin receptors in
the primate cerebral cortex: possible site of action of hallucinogenic and
antipsychotic drugs in pyramidal cell apical dendrites, Proc. Natl. Acad Sci.
U.S.A. 95 (1998) 735–740.
[19] T.H. Mclean, J.C. Parrish, M.R. Braden, D. Marona-Lewicka, A. Gallardo-Godoy,
D.E. Nichols, 1-Aminomethylbenzocycloalkanes: conformationally restricted
hallucinogenic phenethylamine analogues as functionally selective 5-HT2A
receptor agonists, J. Med. Chem. 49 (2006) 5794–5803.
[20] F.C. Colpaert, Discovering risperidone: the LSD model of psychopathology, Nat.
Rev. Drug Discovery 2 (2003) 315–320.
[21] S. Miyamoto, G.E. Duncan, C.E. Marx, J.A. Lieberman, Treatments for
schizophrenia: a critical review of pharmacology and mechanisms of action
of antipsychotic drugs, Mol. Psychiatry 10 (2005) 79–104.
[22] H.Y. Meltzer, S. Matsubara, J.C. Lee, Classification of typical and atypical
antipsychotic drugs on the basis of dopamine D-1, D-2 and serotonin2 pKi
values, J. Pharmacol. Exp. Ther. 251 (1989) 238–246.
[23] H.Y. Meltzer, S.M. Stahl, The dopamine hypothesis of schizophrenia: a review,
Schizophr. Bull. 2 (1976) 19–76.
Borroto-Escuela et al. D2-5HT2A heteroreceptor complexes

SUPPLEMENTARY MATERIAL

Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer

recognition and signaling of D2-5-HT2A heteroreceptor complexes

Dasiel O. Borroto-Escuela, Wilber Romero-Fernandez, Manuel Narvaez, Julia Oflijan,

Luigi F. Agnati and Kjell Fuxe

MATERIALS AND METHODS

Chemicals and reagents

Forskolin, quinpirole, haloperidol, 4-Bromo-3,6-dimethoxybenzocyclobuten-1-

yl)methylamine hydrobromide (TCB2), 1-(2,5-dimethoxy-4-iodophenyl)-propan-2-

amine (DOI), and ketanserin were purchased from Tocris Cookson Inc and Sigma

Aldrich. [3H] raclopride was purchased from PerkinElmer Life Inc. Dulbecco's modified

Eagle's medium, penicillin/streptomycin, and fetal bovine serum was purchased from

Invitrogen.

Cell culture and transfection

HEK293T cells (American Type Culture Collection, USA) were grown in Dulbecco’s

modified Eagle’s medium supplemented with 2mM L-glutamine, 100U/ml

penicillin/streptomycin, and 10% (v/v) fetal bovine serum (FBS) at 37°C and in an

atmosphere of 5% CO2. For transfection, cells were grown at a concentration of 1×106

cells/plate and cultured overnight prior transfection. Cells were transiently transfected

using Fugene® HD transfection reagent (Promega, Sweden).

Membrane Preparations

Ventral striatal membrane preparations were prepared for their use in radioligand

binding experiments. Rats were sacrificed by decapitation, and the brains were quickly

removed and chilled in ice-cold phosphate buffered saline. The ventral striatum

-1-
Borroto-Escuela et al. D2-5HT2A heteroreceptor complexes

including nucleus accumbens from each animal was dissected out (Paxinos & Watson

1998). Samples were immediately frozen on dry ice and later stored at -80°C until use.

Frozen tissue samples were placed in 10ml of 50mM Tris-HCl, pH 7.4, containing 100

mM NaCl, 7mM MgCl2, 1mM EDTA and a cocktail of protease inhibitors (Roche

Diagnostics, Mannheim, Germany). The tissue was homogenized using an Ultra-Turrax

homogenizer (Staufen, Germany) and samples were subjected to centrifugation at

40,000xg for 30min at 4°C. The pelleted membranes were resuspended and

homogenized in the same buffer and centrifuged an additional three times. The protein

concentration was determined for the membrane homogenates by means of BCA Protein

Assay (Pierce, Sweden) using bovine serum albumin as standard. Pelleted membranes

were resuspended in 50mM Tris-HCl, pH 7.4, containing 100mM NaCl, 7mM MgCl2,

1mM EDTA and 1mM DTT, immediately used or stored at -80°C until required.

Double-Immunolabeling

Rat striatum slide sections (10µm) were washed twice with Tris buffer saline (TBS), pH

7.4 containing 20 mM glycine (buffer A) to quench the aldehyde groups. Then, after

permeabilization with buffer A containing 0,5 % Triton X-100 for 30 min, sections were

treated with 10% FBS and 0.1% Triton X-100 in TBS (buffer B). After 1 h at room

temperature, receptors were double immunostained with the indicated primary

antibodies at 4°C overnight, extensively washed, and stained with the indicated

fluorescence labelled secondary antibodies conjugated with Alexa dyes for 1 h at room

temperature. The slide sections were rinsed in TBS (0.01% Tween-20), mounted in a

Vectashield immunofluorescence medium (Vector Laboratories, UK) and visualized

with a Leica SP2 confocal microscope (Leica, USA). The primary antibodies used were

as follows: monoclonal anti-D2R produced in mouse (WH00018113M1, 5 µg/ml,

Sigma-Aldrich, Sweden) and polyclonal anti-5-HT2AR produced in rabbit (ab16028, 5

-2-
Borroto-Escuela et al. D2-5HT2A heteroreceptor complexes

µg/ml, Abcam, Sweden). The secondary antibodies used were as follows: Alexa Fluor

546-conjugated goat anti-mouse IgG (dilution 1:500, Invitrogen, Sweden), Alexa Fluor

488-conjugated donkey anti-rabbit IgG (dilution 1:500, Invitrogen, Sweden). D2R-5-

HT2AR sequential colocalization is shown as a single z-scan image.

FIGURE LEGEND

Supplementary Figure S1.

PLA-detected D2LR-5-HT2AR heteromers in transiently transfected HEK293 cells.

Specific in situ PLA was performed in HEK293 cells transiently transfected with D2LR

or co-transfected with D2LR-5-HT2AR (cDNA molar ratio 1:1). After fixation and

permeabilization, in situ PLA was performed. In "A" is demonstrated the presence of

PLA clusters after D2LR and 5HT2AR co-transfections. In "B" absence of PLA cluster

is found after single D2LR transfection. Nuclei are shown in blue (DAPI). Scale bars are

shown in the right low part of each panel.

Supplementary Figure S2.

D2R and 5-HT2AR double immunolabeling in striatal sections of rat.

Immunohistochemistry was performed with D2R and 5-HT2AR specific antibodies,

followed by secondary antibodies conjugated with Alexa dyes. D2R (in red) and 5-

HT2AR (in green) immunoreactivities are shown to colocalize as seen from the yellow-

orange color developed (upon merger of the two images) in dorsal (A) and ventral

striatal (B) regions. Three independent experiments were carried out. Scale bars are

shown in the right low part of each panel. CPU: caudate putamen; ec: external capsule;

Acb: accumbens nucleus; aca: anterior commissure anterior. Bregma level -1.7.

-3-
Borroto-Escuela et al. D2-5HT2A heteroreceptor complexes

Supplementary Table S1.

D2-likeR antagonist [3H]-raclopride saturation binding was performed in ventral striatal

membranes in the presence or absence of different concentrations of the hallucinogenic

5-HT2A receptor agonists LSD (10-100nM) and DOI (10-100nM), the standard 5-

HT2A receptor agonist TCB2 (10-100nM) and 5-HT(0.5µM). Data are presented as

means±sem of three independent experiments, each one performed in triplicate.

Statistical analysis was performed by one-way analysis of variance (ANOVA) followed

by Tukey's Multiple Comparison post-test. The P value 0.05 and lower was considered

significant. *(P<0.05) and **(P<0.01) are significantly different compared to respective

control Bmax value; #(P<0.05) is significantly different compared to LSD Bmax value

(10nM); &&(P<0.01) is significantly different compared to DOI Bmax value (50nM).

Supplementary Table S2.

[3H]-raclopride/dopamine competition experiments studying the modulatory effects of

LSD (10-100nM), DOI (10-100nM), TCB2 (10-100nM) and 5-HT (0.5µM) on the pKi

values for high and low affinity DA binding sites in ventral striatal membrane

preparations. Competition experiments of the dopamine D2-like receptor antagonist [3H]

raclopride versus increasing concentrations of dopamine were performed in ventral

striatum in the presence or absence of the hallucinogenic 5-HT2AR agonists LSD, DOI ,

the standard 5-HT2AR agonist TCB2 and 5-HT as indicated. Nonspecific binding was

defined as the binding in the presence of dopamine hydrochloride (1mM). Data are

presented as means±sem of the -Log values of the ki values of the high affinity DA

binding sites (pKiH) and of the low affinity DA binding sites (pKiL) from three

independent experiments, each one performed in triplicate. Statistical analysis was

performed by one-way analysis of variance (ANOVA) followed by Tukey's Multiple

-4-
Borroto-Escuela et al. D2-5HT2A heteroreceptor complexes

Comparison post-test. The P value 0.05 and lower was considered significant. *(P<0.05)

and **(P<0.01) are significantly different compared to respective control pKiH value;

#(P<0.05) is significantly different compared to LSD (10nM) pKiH value; &(P<0.05) is

significantly different compared to DOI (50nM) pKiH value.

Supplementary Table S3.

D2-likeR antagonist [3H]-raclopride saturation binding was performed in HEK293

membrane preparation from cotransfected D2R-5-HT2AR or singly transfected D2R in

the presence or absence of the hallucinogenic 5-HT2A receptor agonists LSD (10nM)

and DOI (50nM), the standard 5-HT2AR agonist TCB2 (10nM) and 5-HT (0.5µM) as

indicated. Data are presented as means ± S.E.M of three independent experiments, each

one performed in triplicate. Statistical analysis was performed by one-way analysis of

variance (ANOVA) followed by Tukey's Multiple Comparison post-test. The P value

0.05 and lower was considered significant. *(P<0.05) are significantly different

compared to respective control Bmax value; #(P<0.05) is significantly different

compared to LSD(10nM) Bmax value ; &(P<0.05) is significantly different compared to

DOI (50nM) Bmax value. No significant actions were found in the four groups in the

singly D2R transfected cells.

Supplementary Table S4

[3H]-raclopride/dopamine competition experiments were performed studying the

modulatory effects of LSD (10nM), DOI (50nM), TCB2 (10nM) and 5-HT (0.5µM) on

the pKi values for high and low affinity DA binding sites in HEK293 membrane

preparations from cotransfected D2R-5-HT2AR or singly transfected D2R cells.

Competition experiments of the dopamine D2-like receptor antagonist [3H] raclopride

-5-
Borroto-Escuela et al. D2-5HT2A heteroreceptor complexes

versus increasing concentrations of dopamine in HEK293 cells in the presence or

absence of hallucinogenic 5-HT2AR agonists LSD, DOI, a standard agonist TCB2 and

5-HT as indicated. Nonspecific binding was defined as the binding in the presence of

dopamine hydrochloride (1mM). Data are presented as means ± S.E.M of the -Log

values of the Ki values of the high affinity DA binding sites (pKiH) and of the low

affinity DA binding sites (pKiL) from three independent experiments, each one

performed in triplicate. Statistical analysis was performed by one-way analysis of

variance (ANOVA) followed by Tukey's Multiple Comparison post-test. The P value

0.05 and lower was considered significant. **(P<0.01) are significantly different

compared to control pKi value; #(P<0.05) is significantly different compared to LSD

pKi value(10nM); &(P<0.05) is significantly different compared to DOI pKi value

(50nM). No significant actions were found in the four groups in the singly D2R

transfected cells.

-6-
SUPPLEMENTARY FIGURE S1. Borroto-Escuela et al.

B
SUPPLEMENTARY FIGURE S2. Borroto-Escuela et al.

B
SUPPLEMENTARY TABLE S1. Borroto-Escuela et al.

[3H]-raclopride
Receptors binding saturation
5-HT2AR-D2R Bmax KD
(ventral striatum) (fmol/mg protein) (nM)

Control 182 ± 13 1.29 ± 0.31

5-HT(0.5µM) 189 ± 12 1.36 ± 0.19
5-HT(0.5µM) + Ketanserin(1µM) 196 ± 14 1.57 ± 0.35

Control 192 ± 11 1.33 ± 0.26

LSD(10nM) 258 ±19** 1.74 ± 0.39
LSD(50nM) 234 ± 14* 1.72 ± 0.31
LSD(100nM) 242 ± 12** 1.65 ± 0.24
LSD(10nM) + Ketanserin(50nM) 216 ± 10# 1.59 ± 0.21

Control 179 ± 10 1.21 ± 0.23

DOI(10nM) 235 ± 11** 1.54 ± 0.24
DOI(50nM) 227 ± 13* 1.38 ± 0.23
DOI(100nM) 228 ± 12* 1.46 ± 0.24
DOI(50nM) + Ketanserin(50nM) 174 ± 10&& 1.51 ± 0.26

Control 190 ± 12 1.37 ± 0.30

TCB2 (10nM) 194 ± 11 1.53 ± 0.27

TCB2(50nM) 189 ± 17 1.16 ± 0.22
TCB2(100nM) 198 ± 10 1.23 ± 0.20
TCB2 (10nM) + Ketanserin(50nM) 194 ± 12 1.27 ± 0.28
SUPPLEMENTARY TABLE S2. Borroto-Escuela et al.

[3H]-raclopride
Receptors competition with dopamine
5-HT2AR-D2R pKiH pKiL
(ventral striatum)
Control -7.10 ± 0.18 -5.30 ± 0.16
5-HT(0.5µM) -7.36 ± 0.19 -5.50 ± 0.12
5-HT(0.5µM) + Ketanserin(1µM) -7.22 ± 0.11 -5.17 ± 0.10

Control -7.85 ± 0.25 -5.76 ± 0.13

LSD(10nM) -8.50 ± 0.26** -5.58 ± 0.12
LSD(50nM) -8.34± 0.20* -5.45 ± 0.10
LSD(100nM) -8.36± 0.18* -5.58 ± 0.11
LSD(10nM) + Ketanserin(50nM) -7.95 ± 0.15# -5.66 ± 0.17

Control -7.73 ± 0.18 -5.33 ± 0.11

DOI(10nM) -8.31 ± 0.24* -5.38 ± 0.14
DOI(50nM) -8.32 ± 0.16* -5.25 ± 0.10
DOI(100nM) -8.33 ± 0.10* -5.29 ± 0.05
DOI(50nM) + Ketanserin(50nM) -7.95 ± 0.11& -5.32 ± 0.06

Control -7.56 ± 0.16 -5.56 ± 0.10

TCB2 (10nM) -7.41 ± 0.15 -5.50 ± 0.11
TCB2(50nM) -7.29 ± 0.12 -5.39 ± 0.11
TCB2(100nM) -7.33 ± 0.15 -5.64 ± 0.10
TCB2 (10nM) + Ketanserin(50nM) -7.24 ± 0.14 -5.38 ± 0.12
SUPPLEMENTARY TABLE S3. Borroto-Escuela et al.

[3H]-raclopride
Receptors binding saturation
5-HT2AR-D2R Bmax KD
(HEK cells) (fmol/mg protein) (nM)

Control 818 ± 47 1.71 ± 0.29

LSD(10nM) 930 ± 51* 1.50 ± 0.27
LSD(10nM) + Ketanserin(50nM) 821 ± 38# 1.08 ± 0.18
DOI(50nM) 931 ± 44* 1.30 ± 0.21
DOI(50nM) + Ketanserin(50nM) 804 ± 41& 1.76 ± 0.28
TCB2(10nM) 834 ± 45 1.51 ± 0.26
TCB2(10nM) + Ketanserin(50nM) 899 ± 43 1.42 ± 0.25
Receptor
D2R
(HEK cells)
Control 698 ± 37 1.46 ± 0.25
LSD(10nM) 707± 60 1.88 ± 0.47
LSD(10nM) + Ketanserin(50nM) 682 ± 42 1.56 ± 0.30
DOI(50nM) 711 ± 30 1.55 ± 0.21
DOI(50nM) + Ketanserin(50nM) 674 ± 29 1.50 ± 0.22
TCB2(10nM) 718 ± 35 1.29 ± 0.21
TCB2(10nM) + Ketanserin(50nM) 669 ± 43 1.23 ± 0.27
SUPPLEMENTARY TABLE S4. Borroto-Escuela et al.

[3H]-raclopride
Receptors competition with dopamine
5-HT2AR-D2R pKiH pKiL
(HEK cells)

Control -7.47 ± 0.16 -5.59 ± 0.12

LSD(10nM) -8.11 ± 0.23** -5.66 ± 0.12
LSD(10nM) + Ketanserin(50nM) -7.64 ± 0.13# -5.52 ± 0.10
DOI(50nM) -8.03 ± 0.13** -5.50 ± 0.08
DOI(50nM) + Ketanserin(50nM) -7.61 ± 0.14& -5.47 ± 0.10
TCB2(10nM) -7.32 ± 0.10 -5.53 ± 0.09
TCB2(10nM) + Ketanserin(50nM) -7.39 ± 0.12 -5.42 ± 0.10
Receptor
D2R
(HEK cells)
Control -7.26 ± 0.14 -5.58 ± 0.13
LSD(10nM) -7.24 ± 0.12 -5.53 ± 0.10
LSD(10nM) + Ketanserin(50nM) -7.05 ± 0.14 -5.46 ± 0.13
DOI(50nM) -7.32 ± 0.16 -5.65 ± 0.10
DOI(50nM) + Ketanserin(50nM) -7.21 ± 0.15 -5.56 ± 0.16
TCB2(10nM) -7.08 ± 0.12 -5.40 ± 0.11
TCB2(10nM) + Ketanserin(50nM) -7.18 ± 0.11 -5.39 ± 0.13