From the Departments of Pharmacology, ** Molecular
Physiology and Biophysics, and
Psychiatry
and the § Center for Molecular Neuroscience, Vanderbilt
University, Nashville, Tennessee 37232-6600 and the
Departments
of Pharmacology and Neuroscience, Albany Medical College,
Albany, New York 12208-3479
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ABSTRACT |
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RNA transcripts encoding the serotonin
5-hydroxytryptamine 2C (5-HT2C) receptor
(5-HT2CR) undergo adenosine-to-inosine RNA editing events
at up to five specific sites. Compared with rat brain, human brain
samples expressed higher levels of RNA transcripts encoding the amino
acids valine-serine-valine (5-HT2C-VSV) and valine-glycine-valine (5-HT2C-VGV) at positions 156, 158, and 160, respectively. Agonist stimulation of the nonedited human receptor (5-HT2C-INI) and the edited 5-HT2C-VSV
and 5-HT2C-VGV receptor variants stably expressed in
NIH-3T3 fibroblasts demonstrated that serotonergic agonists were less
potent at the edited receptors. Competition binding experiments
revealed a guanine nucleotide-sensitive serotonin high affinity state
only for the 5-HT2C-INI receptor; the loss of high affinity
agonist binding to the edited receptor demonstrates that RNA editing
generates unique 5-HT2CRs that couple less efficiently to G
proteins. This reduced G protein coupling for the edited isoforms is
primarily due to silencing of the constitutive activity of the
nonedited 5-HT2CR. The distinctions in agonist potency and
constitutive activity suggest that different edited 5-HT2CRs exhibit distinct responses to serotonergic ligands
and further imply that RNA editing represents a novel mechanism for controlling physiological signaling at serotonergic synapses.
RNA editing is a post-transcriptional modification resulting in an
alteration of the primary nucleotide sequence of RNA transcripts by
mechanisms other than splicing. The enzymatic conversion of adenosine
to inosine by RNA editing has been identified within an increasing
number of RNA transcripts, indicating that this modification represents
an important mechanism for the generation of molecular diversity.
Several of these editing events have been shown to have significant
consequences for cellular function. Transcripts encoding the B-subunit
(GluR-B) of the The monoamine 5-hydroxytryptamine (serotonin;
5-HT)1 interacts with a large
family of receptors to induce signal transduction events important in
the modulation of neurotransmission (5). The 2C subtype of serotonin
receptor (5-HT2CR) is a member of the G protein-coupled
receptor superfamily and stimulates phospholipase C, resulting in the
production of inositol phosphates and diacylglycerol (6). We have
recently shown that RNA transcripts encoding the rat, mouse, and human
5-HT2CR undergo adenosine-to-inosine RNA editing events at
five positions, termed A, B, C, D, and E (previously termed C') (7, 8),
resulting in an alteration of amino acid coding potential within the
putative second intracellular loop of the encoded protein. Editing at
the A site, or at the A and B sites concurrently, converts an
isoleucine to a valine at amino acid 156 of the human receptor, while
editing at the B position alone generates a methionine codon at this
site (Fig. 1A). Editing at C converts asparagine 158 to a
serine; editing at E generates an aspartate at this site, and
conversion at both C and E generates a glycine triplet. Editing at D
results in the substitution of a valine for an isoleucine at position 160.
We have previously demonstrated a decrease in 5-HT potency
when interacting with the rat 5-HT2CR isoform
5-HT2C-VSV, which is simultaneously edited at the A,
B, C, and D positions encoding valine, serine, and valine at positions
157, 159, 161, respectively. This decrease in potency was reflected as
a rightward shift in the dose-response curve for
[3H]inositol monophosphate accumulation (7). We proposed
that the decreased potency resulted from a reduced G protein coupling efficiency induced by the introduction of novel amino acids into the
second intracellular loop, a region known to be important for G protein
coupling (9-16). In the present study, we show that the patterns of
5-HT2CR editing differ in human versus rat
brain, resulting in the generation of novel receptor isoforms in human tissue. Radioligand binding and functional studies were designed to
more fully test the hypothesis that the decreased agonist potency of
edited isoforms reflects alterations in G protein coupling. The results
show that differences in the ability to spontaneously isomerize to the
active R* conformation play a role in the altered coupling properties
of the fully edited receptor, suggesting that RNA editing of the
5-HT2CR represents a novel mechanism for regulating neuronal excitability by stabilizing receptor signaling and enhancing the signal:noise ratio at serotonergic synapses.
Preparation of RNA, Reverse Transcription-PCR, and Editing
Efficiency Analyses--
Total RNA isolated from whole human brain was
obtained from CLONTECH (Palo Alto, CA). Reverse
transcription-PCR amplification of 5-HT2CR messenger
mRNA was performed as described previously (7) with the antisense
oligonucleotide 5'-GCAGTAACATCAAAGCTTGTCGGCG-3' (coordinates 723-747
relative to the translation start site of the human 5-HT2CR
cDNA; Ref. 17) employed for cDNA synthesis. The sense
oligonucleotide primer
5'-CCAGGGAATTCAAACTTTGGTTGCTTAAGACTGAAGC-3' (coordinates
Preparation of Human 5-HT2CR Isoform DNA and
Expression in Cell Culture--
Human 5-HT2CR variants
were prepared by oligonucleotide-directed mutagenesis of human INI
receptor DNA (a gift of Dr. Alan Saltzman) as described previously (10,
19). These constructs were unidirectionally cloned into the eukaryotic
expression vector pCMV2 (a gift of Dr. David Russell) at the
EcoRI and XbaI sites. Transient experiments in
NIH-3T3 cells were performed as described previously (7). For transient
expression into the COS-7 cell line, cells were plated in 24-well
plates at a density of 1 × 105 cells/well 24 h
prior to transfection. Two µl of Lipofectamine reagent®
(Life Technologies, Inc.) and 0.5 µg of plasmid were combined in 400 µl of serum-free DMEM and added to each well for 5 h. Cells were
washed with phosphate-buffered saline, and complete medium (DMEM, 10%
fetal bovine serum, 100 units/ml of penicillin, and 100 µg/ml of
streptomycin) was added for 24 h. Stable cell lines were generated
by co-electroporation of human isoforms in pCMV2 (60 µg) and pRC/CMV
(Invitrogen, Carlsbad, CA) (6 µg) using electroporation conditions
described previously (7). Single clones were selected using 1 mg/ml
G418 (Geneticin®) in DMEM containing 10% dialyzed calf
serum, 100 units/ml penicillin, 100 µg/ml streptomycin (Life
Technologies), and 1% calf serum (Hyclone®; Hyclone
Laboratories, Logan UT). Cells were maintained in DMEM with 10% calf
serum (Hyclone®), 100 units/ml penicillin, 100 µg/ml
streptomycin, 500 µg/ml G418, and 1 µM 2-bromolysergic
acid diethylamide.
Phosphoinositide Hydrolysis Assay--
Transiently transfected
cells were prepared for inositol monophosphate analysis as described
previously (NIH-3T3 cells, 7; COS-7 cells, 21). For stable cell lines,
cells were washed three or four times with Hanks' buffer prior to
plating in a 24-well plate (Falcon 3047, Becton-Dickinson Laboratories,
Lincoln Park, NJ) in DMEM containing 10% dialyzed calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. 24 h later,
cells were labeled for 16-20 h with 1 µCi of
myo-[3H]inositol/ml (20-25 Ci/mmol, NEN Life
Science Products) in serum-free, inositol-free DMEM containing 100 units/ml penicillin and 100 µg/ml streptomycin. For isolation of
inositol monophosphates from both transient and stable transfectants,
agonists were added in the presence of 10 mM lithium
chloride and 10 µM pargyline. Incubations continued for
30 min unless otherwise noted. [3H]Inositol
monophosphates were isolated as described previously (22).
Concentration response curves were analyzed using GraphPad Prism® software (GraphPad Software Inc., San Diego, CA).
Radioligand Binding--
Radioligand binding analyses were
performed in transiently transfected cells as described previously (7,
21). Stable cell lines were prepared by washing three or four times in
Hanks' buffer and replacing the medium with DMEM containing 10%
dialyzed calf serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin for 24 h. Subsequently, cells were placed into
serum-free DMEM containing 100 units/ml penicillin and 100 µg/ml
streptomycin for 16-18 h prior to analyses.
[3H]Mesulergine binding was assayed in crude membrane
preparations in 50 mM Tris-HCl, 10 mM
MgCl2, pH 7.5, as described previously (23). Competition
binding was performed with 0.5 nM
[3H]mesulergine in the presence of increasing
concentrations of nonlabeled competitor at 37 °C for 30 min. In some
experiments, 100 µM guanosine
5'-( The 5-HT2CR Editing Pattern in Human Brain Is Distinct
from Rat Brain--
To determine the editing pattern for
5-HT2CR transcripts in human brain, whole brain total RNA
was amplified by reverse transcription-PCR. The extent of editing at
specific sites and the editing patterns within individual RNA species
were determined by primer extension analysis and by subcloning and
sequencing individual cDNA isolates, respectively. These studies
revealed that the extent of editing in human brain at the A and D
positions was similar to the levels previously identified within rat
brain RNA, while editing at position B was lower in human brain (Ref.
7; Fig. 1B). By contrast, the
editing efficiency at the C position was much higher in
5-HT2CR transcripts isolated from human compared with rat
brain (60 versus 35%). Editing at the E position
(previously referred to as C') was barely detectable in rat brain
mRNAs from a variety of brain regions (<5%; Ref. 7), but this
position was edited with much higher efficiency in human brain
transcripts (Fig. 1B).
Due to increased levels of editing at the C and E positions, the
pattern of editing observed within individual human brain 5-HT2CR transcripts differed from that found in the rat
(Fig. 1C). For example, the 5-HT2C-VSV isoform,
which comprised approximately 10% of the 5-HT2CR
transcripts isolated from whole rat brain, represented almost
40% of the messages derived from whole human brain (Fig.
1C). The enhanced editing at the E position in humans was
also reflected by the appearance of variant RNAs encoding the novel
5-HT2C-VGV, 5-HT2C-VGI, and
5-HT2C-IGI protein isoforms (Fig. 1C). RNA
encoding the 5-HT2C-VNV variant, which represented approximately 50% of the transcripts isolated from rat brain (7), only
comprised 8% of the total 5-HT2CR RNAs found in human
brain (Fig. 1C). Together, these results indicated that
editing of the 5-HT2CR RNA differs considerably between
rodent and human species and further suggested that the isoforms
specific to human brain may have distinct functional roles.
Differential Signaling Profiles of the Human Edited Receptor
Isoforms--
To assess the functional consequences of editing within
human 5-HT2CR RNAs, we transiently expressed six of the
major human isoforms in NIH-3T3 fibroblasts and examined the ability of
these receptors to stimulate the phospholipase C signal transduction cascade by measuring the accumulation of [3H]inositol
monophosphates. Results from these studies revealed that the human
5-HT2C-VSV receptor exhibited a 5-fold shift in potency for
5-HT compared with the 5-HT2C-INI receptor (Fig.
2). The 5-HT2C-VGV receptor
exhibited a more substantial rightward shift in the dose-response curve
for 5-HT, with an EC50 value of 59 versus 2.3 nM for the 5-HT2C-INI receptor variant. The
EC50 values for all other isoforms were not significantly
different from the EC50 observed for the
5-HT2C-INI receptor.
To more carefully examine the properties of the human edited receptors,
stable cell lines of the human 5-HT2C-INI,
5-HT2C-VSV, and 5-HT2C-VGV receptor isoforms
were generated in NIH-3T3 cells. The density of receptors in these cell
lines was 258 ± 87 for 5-HT2C-INI, 173 ± 28 for
5-HT2C-VSV, and 1054 ± 260 fmol/mg protein for the
5-HT2C-VGV isoform. Similar to the phenotype observed in
the transient transfections, 5-HT exhibited a lower potency when
interacting with either the 5-HT2C-VSV or
5-HT2C-VGV isoforms as compared with the
5-HT2C-INI receptor (Fig.
3A). The
5-HT2C-VGV receptor-expressing cells, however, did not
exhibit the substantial EC50 shift seen in the transient
experiments. It is possible that the higher receptor expression in the
5-HT2C-VGV receptor line resulted in a significant receptor
reserve for the 5-HT response, causing the EC50 value to
appear inappropriately low. To address this possibility,
5-HT2C-VGV receptor cells were treated with the alkylating
agent phenoxybenzamine (25). This treatment inactivated approximately
60% of the 5-HT2C-VGV receptors, resulting in a statistically equivalent density between the three cell lines, thereby
allowing a more direct comparison of EC50 values.
Subsequent stimulation of this partially inactivated population with
5-HT revealed that stably expressed 5-HT2C-VGV receptors
now exhibited a decreased maximum response (20% of untreated cells)
and a 29-fold lower EC50 for 5-HT when compared with the
5-HT2C-INI isoform (Fig. 3A). This potency shift
agrees closely with the 26-fold difference determined in transient
transfection analyses (Fig. 2).
Stimulation with other 5-HT2CR agonists revealed similar
discrepancies in EC50 values between different edited
isoforms. (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane (DOI), a
hallucinogenic agonist with structural similarity to amphetamine, was
9- and 43-fold less potent at the 5-HT2C-VSV and
5-HT2C-VGV receptors, respectively, when compared with its ability to activate signaling of the 5-HT2C-INI isoform
(Fig. 3B). N,N-dimethyltryptamine
(DMT), a hallucinogenic indoleamine, was 40-fold less potent at the
5-HT2C-VSV receptor and 91-fold less potent at the
5-HT2C-VGV variant compared with the response generated by
interaction of this ligand with the 5-HT2C-INI receptor (Fig. 3C).
Two Agonist Affinity States Are Observable Only for the
5-HT2C-INI Isoform--
According to current theory of
receptor/G protein interaction, the coupling of receptor with G protein
results in an increase in the affinity of agonists for the receptor.
Competition binding analyses were performed to evaluate differences in
agonist affinity states for the 5-HT2C-INI and
5-HT2C-VGV receptors (Fig.
4). Competition binding regularly
revealed a low and high affinity state upon interaction of 5-HT with
the 5-HT2C-INI receptor (Fig. 4A); competition binding curves could never be fit to a two-site model for
5-HT2C-VGV receptors (Fig. 4B; Table
I). Data with the 5-HT2C-VSV
receptor was variable, sometimes revealing two sites, but more often
best fit by a one-site model (data not shown). The 5-HT high affinity population of 5-HT2C-INI receptors could be shifted to low
affinity when binding analyses were performed in the presence of 100 µM Gpp(NH)p, while the curves for the edited
5-HT2C-VGV receptor remained unchanged. In the presence of
Gpp(NH)p, significantly higher affinities were consistently observed
for agonists when the 5-HT2C-INI receptor was compared with
the 5-HT2C-VGV variant (Table I). This was not the case,
however, when competition analyses were performed with mianserin, an
inverse agonist at the 5-HT2CR and a ligand that is
predicted to preferentially interact with uncoupled receptors. These
results again support the hypothesis that the 5-HT2C-INI
receptor couples more efficiently to G proteins, even in situations
where no agonist is present to induce the coupled state.
Constitutive Activity Differences between Human 5-HT2CR
Isoforms--
Rat 5-HT2CRs have been shown to exhibit
constitutive activity (22, 23), defined as the ability of a receptor to
stimulate downstream signaling events in the absence of agonist
occupation (reviewed in Ref. 26). The binding studies described
previously suggested that the edited receptors might exhibit
differential abilities to stimulate basal levels of inositol phosphate
hydrolysis. Studies of constitutive receptor activity were performed in
COS-7 cells, where transient expression gave reproducibly high levels of receptor expression, which is needed to accurately evaluate agonist-independent activity. An analysis of transient transfection experiments in which the nonedited and edited receptors were expressed at similar densities indicated that the 5-HT2C-INI isoform
elicited 5-fold greater levels of basal [3H]inositol
monophosphate generation compared with the 5-HT2C-VGV receptor (Fig. 5). The addition of 1 µM methysergide, a neutral antagonist, blocked
5-HT-stimulated inositol phosphate formation but had no effect on the
basal activity of the 5-HT2C-INI receptor (data not shown).
The disparity in basal activity mirrors the differences in
"precoupling" ability reflected in the competition binding
experiments, again suggesting that the 5-HT2C-INI receptor interacts more efficiently with the G protein linked to phospholipase C. Importantly, these analyses also revealed that similar maximal responses were obtained for both receptor isoforms (Fig. 5), indicating that the primary effect of editing may be to alter agonist-independent activity rather than affecting the intrinsic ability of receptor isoforms to promote G protein coupling.
RNA editing is a post-transcriptional process that contributes to
molecular diversity within cells. 5-HT2CR RNA transcripts undergo adenosine-to-inosine RNA editing events resulting in the generation of distinct amino acids within the second intracellular loop
of the protein (7), a region known to be important for G protein
coupling (9-16). Editing within rat 5-HT2CR RNA is
mediated by the coordinated actions of a family of adenosine deaminases termed ADARs (adenosine deaminases that
act on RNA; Refs. 7 and 27). These editing
events are conserved among rodent and human species (8), suggesting
that they serve an important role in receptor function.
Editing in the human brain creates a different complement of receptor
isoforms due to an increase in the percentage of isoforms edited at
position 158, most notably the 5-HT2C-VSV and
5-HT2C-VGV isoforms. Transient transfection of the
principal human isoforms revealed that 5-HT exhibited the lowest
potency at the novel 5-HT2C-VGV receptor. The
5-HT2C-VSV receptor was the only other isoform to show a
significant difference in 5-HT potency relative to the nonedited
isoform. In cell lines stably expressing the 5-HT2C-INI, 5-HT2C-VSV, or 5-HT2C-VGV receptors, agonists
exhibited a decreased potency when interacting with the edited receptor
isoforms with the effect again being most dramatic upon
interaction with the 5-HT2C-VGV receptor. The potency
difference between 5-HT2C-VGV and 5-HT2C-INI
receptors was augmented when a majority of the 5-HT2C-VGV
receptors were inactivated with phenoxybenzamine, indicating that the
observed potency phenotypes are not due to variations in receptor
reserve between different receptor populations. Instead, these results
are consistent with the interpretation that certain edited receptors
exhibit a reduced G protein coupling capacity.
As a first step in defining the mechanism responsible for the reduced
potency of agonists at edited 5-HT2CRs, competition binding
profiles were examined to exploit the differential affinities of an
agonist for G protein-coupled versus G protein-uncoupled receptor populations. The 5-HT competition binding curves for 5-HT2C-INI receptors were shallow and best fit by a
two-site competitive binding model. The high affinity state of 5-HT,
representing approximately 50% of the binding, was eliminated by the
addition of the GTP analog, Gpp(NH)p. In contrast, the 5-HT competition
binding curves with 5-HT2C-VGV receptors were best fit by a
one-site competitive binding model, and the affinity approximated the
low affinity site found with the 5-HT2C-INI isoform. For
the 5-HT2C-VSV receptor, the curves sometimes fit two sites
(data not shown), but most often the one-site competitive binding model
gave the best fit. These results demonstrate that, compared with the
5-HT2C-VSV and 5-HT2C-VGV receptor populations,
a relatively large proportion of 5-HT2C-INI receptors
exists in a G protein coupled state, providing the first direct
evidence that RNA editing generates 5-HT2CRs that differ in
the efficiency of coupling to G proteins. Thus, there appear to be
graded states of precoupling ability for the edited
5-HT2CRs, with 5-HT2C-INI receptor being most
efficacious at G protein coupling, the 5-HT2C-VSV variant
being intermediate, and 5-HT2C-VGV receptors existing
predominantly in the uncoupled state.
The ternary complex model of receptor/G protein interaction has
recently undergone revision to accommodate the finding that some
receptors have the ability to induce effective G protein coupling in
the absence of an agonist, termed constitutive activity (28). The model
predicts that receptors have the capacity to spontaneously isomerize
from an inactive conformation, termed R, to an active state, R*, with
the R* version of the receptor having the ability to interact with G
proteins. In experimental paradigms studying the properties of
constitutively active mutant receptors, it has been observed that these
receptors exhibit both a higher level of basal activity and a greater
potency for agonists (28, 29). Since the nonedited 5-HT2CR
exhibits constitutive activity (22, 30), we considered the possibility
that the agonist potency differences generated by RNA editing may
reflect differential abilities to isomerize to the R* state, with the 5-HT2C-INI receptor existing more readily in the R* form
compared with the edited 5-HT2C-VSV and
5-HT2C-VGV isoforms. To evaluate alterations in
constitutive activity, the basal activity of the nonedited
5-HT2C-INI receptor was compared with the fully edited 5-HT2C-VGV isoform, which exhibited the greatest shift in
5-HT potency. In COS-7 cells transiently expressing
5-HT2C-INI receptors, basal phosphoinositide hydrolysis
was substantially increased in comparison with mock-transfected cells.
In contrast, the 5-HT2C-VGV receptor isoform supported only
a modest degree of basal [3H]inositol phosphate
formation, about 20% of that produced by 5-HT2C-INI
receptors expressed at a comparable density. These results indicate
that the fully edited 5-HT2C-VGV receptor has a much lower
intrinsic ability to support agonist-independent phosphoinositide
hydrolysis than does the nonedited receptor isoform. Two alternative
possibilities could explain the differences in 5-HT2C-INI
and 5-HT2C-VGV receptor constitutive activity. One hypothesis is that large differences in the fraction of receptors residing in the R* conformation might underlie the distinction in
constitutive activities. An alternative explanation is that both
receptor isoforms exhibit the same ability to convert to R*, but the
combination of amino acids within the 5-HT2C-INI receptor promotes more efficient interaction with the G protein, due to either
the favorable conformation of the second intracellular loop or actual
contact of the nonedited amino acids with the G protein. In experiments
designed to test the latter hypothesis, we found that 5-HT treatment
elicited an increase in [3H]inositol phosphate formation
of the same magnitude in cells expressing equal densities of
5-HT2C-INI or 5-HT2C-VGV receptors, strongly
suggesting that the two receptor isoforms have equivalent ability to
couple to G proteins when bound by agonists. Thus, these results
indicate that 5-HT2C-INI receptors have a greater propensity to spontaneously isomerize into or maintain the active R*
conformation than 5-HT2C-VGV receptors. Thus, we propose
that one consequence of RNA editing of the 5-HT2CR is to
silence its constitutive activity, thus increasing the signal:noise
ratio at sites where editing efficiency is high.
The extended ternary complex model predicts that receptor isoforms with
high levels of constitutive activity would exhibit higher affinity for
agonists, even in the absence of G protein coupling (28, 29). As a
first step at evaluating agonist affinities in the absence of G
proteins, we compared the binding affinities in the presence of
Gpp(NH)p, assuming that this reflects the affinity of the G
protein-uncoupled receptor. The affinity of 5-HT for the
5-HT2C-VGV receptor was 5-fold lower than for the
5-HT2C-INI isoform; significant differences were also found
for the other agonists tested but not for the inverse agonist
mianserin. These observations suggest that some structural perturbation
allows the edited receptors to assume an inactive receptor conformation with lower affinity for agonists. Studies of agonist affinities using
purified receptor protein are needed to definitively show that the
agonist affinity differences do not depend on G protein interactions.
Recent structure-activity studies of other G protein-coupled receptors
point to the importance of amino acid residues in positions analogous
to the edited sites in the dynamics of receptor/G protein interactions.
Amino acid residues that reside at positions analogous to
Ile/Val156 and Ile/Val160 in other G
protein-coupled receptors are important in terms of general G protein
coupling ability and receptor regulation (10, 12, 16). For example,
within the m1 and m3 muscarinic receptors and the The consequences of RNA editing within 5-HT2CR RNA are
summarized in the model presented in Fig.
6, with A representing the 5-HT2C-INI receptor and B representing the
5-HT2C-VGV receptor isoform. In this model, the
5-HT2C-INI receptor has a much greater capacity to
isomerize to the active R* conformation (represented in
boldface type and described by the constant
J). The R* form of the receptor interacts
efficiently with G proteins in the absence of agonist (H);
upon agonist binding, the response is enhanced. For edited
5-HT2C-VGV receptors, however, the J constant is
much smaller, reflecting a decreased spontaneous ability of these
receptors to convert to the coupling-competent state. It is only upon
binding of agonist that these receptors achieve the ability to
significantly convert to a form able to elicit efficient G protein
interaction. Our current working hypothesis is that the edited receptor
has a lower value for the J constant, while the M constant, an index of
the intrinsic ability of an isoform to couple to G proteins, remains
relatively constant.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
subtype of glutamate receptor undergo RNA editing events that modulate
both the ion permeation and electrophysiological characteristics of
this glutamate-gated ion channel (1-3). Mice that are deficient in
their ability to edit GluR-B transcripts die at 3 weeks of age due to
epileptic seizures, suggesting that editing of GluR-B RNA is important
in the modulation of normal glutamatergic neurotransmission (4). These
results suggest that the consequences of editing events within other,
diverse RNA molecules might also have important ramifications for
cellular function.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
30 to
5; Ref. 18) and the original cDNA synthesis primer or, in
some cases, 5'-ATTAGAATTCTATTTGTGCCCCGTCTGG-3' (coordinates 372-389; Ref. 17) and the cDNA priming
oligonucleotide (boldface sequence indicates introduced
EcoRI restriction sites) were used for PCR amplification of
human 5-HT2CR sequences. Amplification proceeded for
75 s at 94 °C, for 75 s at 50 °C, and for 150 s at
72 °C for 35 cycles. Products were purified on a 2% agarose gel,
and primer extension analyses at the A and C/D sites were performed as
described (7). For sequencing analysis, PCR fragments were digested
with EcoRI and HindIII and unidirectionally
subcloned into pBKSII
(Stratagene). Individual cDNA
isolates were sequenced using the fmole sequencing system (Promega) as
described previously (7).
,
-imido)triphosphate (Gpp(NH)p) was added during the
incubation. IC50 values were determined by fitting data to
a sigmoidal curve with variable slope using GraphPad
Prism® (GraphPad Software, Inc., San Diego, CA); one-site
and two-site binding curves were compared using the F ratio.
IC50 values were converted to Ki using
the transformation of Cheng-Prusoff (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
5-HT2CR editing efficiencies in
human versus rat brain. A, the
positions of the editing sites within human 5-HT2CR RNA are
shown with the nonedited RNA sequence at the top and the
fully edited sequence indicated at the bottom. The positions
of the five editing sites, encompassing amino acids 156-160 of the
human 5-HT2CR sequence are shown. B, the editing
efficiency at each editing site for whole human (black
bars) and whole rat (white bars) brain
is shown. Editing efficiencies at the A, C, and D sites were determined
by a combination of primer extension (n = 2
independent analyses) and sequencing analyses (n = 50 independent cDNA clones for human samples); values represent the
mean ± S.E. Editing efficiencies at the B and E sites were
determined by sequencing of 50 independent cDNA clones from human
brain. C, the relative expression level, presented as a
percentage of total isoform expression derived from a sequencing
analysis of 50 (human) or 100 (rat) brain cDNA clones, is compared
for 5-HT2CR RNA derived from human (black
bars) versus rat (white
bars) brain. Rat data are from Burns et al.
(7).
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Fig. 2.
5-HT potency to activate phosphoinositide
hydrolysis in NIH-3T3 fibroblasts transiently expressed with human
edited 5-HT2CRs. The human 5-HT2C-INI,
5-HT2C-VSI, 5-HT2C-INV, 5-HT2C-VNV,
5-HT2C-ISV, 5-HT2C-VSV, and
5-HT2C-VGV isoforms were transiently expressed in NIH-3T3
cells, and the EC50 of 5-HT for activation of
phosphoinositide hydrolysis was determined as described under
"Experimental Procedures." Values represent the mean ± S.E.
of 5-11 separate determinations for each isoform.
Bmax values, determined by binding analyses with
a single 8 nM concentration of
[3H]mesulergine, were as follows: 5-HT2C-INI,
900 ± 259 fmol/mg; 5-HT2C-VSI, 1399 ± 626 fmol/mg; 5-HT2C-ISV, 627 ± 133 fmol/mg;
5-HT2C-VNV, 1617 ± 339 fmol/mg;
5-HT2C-INV, 732 ± 280 fmol/mg;
5-HT2C-VSV, 1669 ± 384 fmol/mg; and
5-HT2C-VGV, 422 ± 36 fmol/mg. These values were
determined not to be statistically different based upon one-way
analysis of variance. *, the EC50 value of 5-HT was
significantly different from 5-HT2C-INI,
5-HT2C-VSI, and 5-HT2C-INV as determined by
individual unpaired Student's t tests (p = 0.005, 0.0197, and 0.0347, respectively). **, the
EC50 value of 5-HT was significantly different from
all other examined isoforms as determined by individual unpaired
Student's t tests (p < 0.015 in all
cases).
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Fig. 3.
Differential responses of agonists to
activate phosphoinositide hydrolysis in human edited
5-HT2CR stable cell lines. The EC50 for
activation of phosphoinositide hydrolysis by 5-HT (A), DOI
(B), and DMT (C) in NIH-3T3 cells stably
expressing 5-HT2C-INI, 5-HT2C-VSV, and
5-HT2C-VGV receptors is shown. Values represent the
mean ± S.E. of 3-16 independent determinations performed in
triplicate for each drug. Receptor densities
(Bmax) were as follows: 5-HT2C-INI,
258 ± 87 fmol/mg; 5-HT2C-VSV, 173 ± 70 fmol/mg;
and 5-HT2C-VGV, 1054 ± 260 fmol/mg (n = 4-6). Inactivation of 5-HT2C-VGV receptor-expressing
cells with 3.2 µM phenoxybenzamine resulted in a decrease
in receptor density to 305 ± 141 fmol/mg (n = 3).
A, *, these values were significantly different from
5-HT2C-INI (p = 0.0141 for
5-HT2C-VSV, p = .0059 for
5-HT2C-VGV). **, this value was significantly different
from 5-HT2C-VGV alone (p < 0.0001).
5-HT2C-VSV and 5-HT2C-VGV were not
significantly different from each other (p = 0.4550).
B, *, a significant difference from 5-HT2C-INI
(p = 0.0160). **, a significant difference from
5-HT2C-INI (p = 0.0201) and
5-HT2C-VSV (p = 0.0476). C, *, a
significant difference from 5-HT2C-INI (p = 0.0388). **, a significant difference from 5-HT2C-INI
(p = 0.0006) and 5-HT2C-VSV
(p = 0.0444). All statistical analyses were performed
using individual unpaired Student's t tests.
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Fig. 4.
5-HT affinity for human edited
5-HT2CRs and modulation by the GTP analogue Gpp(NH)p.
Competition binding analyses are shown for 5-HT2C-INI
(A) and 5-HT2C-VGV receptor cell lines
(B) in the presence (closed symbols)
and absence (open symbols) of 100 µM Gpp(NH)p. Increasing concentrations of 5-HT were
incubated with [3H]mesulergine as described under
"Experimental Procedures." Data were fitted to both one- and
two-site models using GraphPad Prism software. The mean
Ki values for 5-HT, determined by the method of
Cheng and Prusoff, for 5-HT2C-INI were 0.68 ± 0.13 nM (high affinity) and 85.7 ± 17.2 nM
(low affinity) in the absence of Gpp(NH)p and 57.4 ± 12.0 nM in the presence of 100 µM Gpp(NH)p. For
5-HT2C-VGV, the mean Ki values for 5-HT
were 167.0 ± 16.1 nM in the absence of Gpp(NH)p and
302.4 ± 46.4 nM in the presence of 100 µM Gpp(NH)p, respectively. Kd values
for [3H]mesulergine, determined in the presence of 100 µM Gpp(NH)p, were as follows: 0.99 ± 0.07 for
5-HT2C-INI and 1.09 ± 0.21 for 5-HT2C-VGV
(p = .601). Data represent the mean ± S.E. of
four or five independent determinations performed in duplicate.
Relative affinities of agonists for 5-HT2C-INI and
5-HT2C-VGV receptors
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Fig. 5.
Constitutive activity of transiently
expressed 5-HT2C-INI and 5-HT2C-VGV
receptors. cDNAs for edited receptors were transiently
expressed in COS-7 cells, and basal [3H]inositol
monophosphate formation was measured after a 35-min incubation. #,
p < 0.05 versus vector alone; *,
p < 0.01 compared with vector and the
5-HT2C-VGV receptor cell line. The densities of the
receptors, estimated using a single concentration of
[3H]mesulergine (0.5 nM), were 2.7 ± 0.2 pmol/mg for 5-HT2C-INI and 2.9 ± 0.2 pmol/mg
protein for 5-HT2C-VGV.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 adrenergic
receptors, the amino acid at Ile/Val160 must be bulky and
hydrophobic to induce a productive G protein interaction (10),
consistent with our evidence that editing at position 160 contributes
to a low efficiency of G protein coupling. Molecular modeling of the
gonadotropin-releasing hormone receptor has shown that the Ile
corresponding to the 5-HT2CR Ile/Val156 is
critical for forming a "cage" around the arginine of the highly conserved DRY sequence in the second intracellular loop (16). It was
proposed that the "arginine cage" stabilizes interaction with the
adjacent aspartate residue, thereby enabling the receptor to remain in
an active state. Alteration of the Ile/Val156 position of
the 5-HT2C receptor in the context of other edited amino
acids might produce different conformations of this critical arginine,
allowing some edited isoforms to couple more efficiently than others.
The expression of more than 12 predominant isoforms in the human brain
further suggests that unique degrees of coupling ability might be
produced with each distinct edited receptor.
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Fig. 6.
Model of regulation of
5-HT2CRs by RNA editing. 5-HT2C-INI
(A) and 5-HT2C-VGV (B) receptors
exhibit differential abilities to isomerize to the productive R* state.
Conversion to this coupling-competent receptor version is represented
for 5-HT2C-INI by the large J constant and
long forward arrow.
5-HT2C-VGV receptors can convert to the productive R* state
but only when agonist is present.
In summary, the current results demonstrate that the
5-HT2C-INI receptor isoform exhibits a greater level of
constitutive activity than does the edited 5-HT2C-VGV
receptor isoform and therefore possesses a greater propensity to
spontaneously isomerize to the R* state. The differential degrees of
constitutive activity and the accompanying secondary alterations in
agonist potency and G protein coupling could have important
implications for the physiological effects of 5-HT. Brain regions that
contain the nonedited receptor would be more sensitive to 5-HT that may
be tonically released at distinct sites, possibly generating
considerable noise in the system. In addition, the magnitude of signal
produced by firing of presynaptic serotonergic terminals may be reduced because of a high basal tone at the constitutively active
5-HT2C-INI receptor. Region-specific generation of edited
isoforms, coupled with the possibility that individuals have different
editing patterns, suggests that the repertoire of expressed edited
5-HT2CRs might determine the response to endogenous
serotonin as well as control the signal:noise ratio at central
serotonergic synapses.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Antoinette Poindexter, Lori McGrew, and Ellinor Grinde.
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FOOTNOTES |
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* This work was supported by a postdoctoral fellowship from the Pharmaceutical Research and Manufacturers of America Foundation (to C. M. N.) and National Institutes of Health Grants MH 57019 (to K. H. D.), NS35891 (to R. B. E.), and MH 34007 (to E. S. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Present address: K-554 HSB, Department of Pharmacology, University of Washington, Seattle, WA 98195.
§§ To whom correspondence should be addressed. Tel.: 615-936-1685; Fax: 615-343-6532; E-mail: elaine.bush{at}mcmail.vanderbilt.edu.
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ABBREVIATIONS |
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The abbreviations used are: 5-HT, 5-hydroxytryptamine; 5-HT2CR, 5-HT2C receptor; PCR, polymerase chain reaction; DMEM, Dulbecco's modified Eagle's medium; DOI, (±)-1-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane; DMT, N,N-dimethyltryptamine; LSD, lysergic acid diethylamide.
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REFERENCES |
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