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Journal of Neurochemistry, 2005, 92, 375–387
doi:10.1111/j.1471-4159.2004.02867.x
Mutagenesis analysis of the serotonin 5-HT2C receptor
and a Caenorhabditis elegans 5-HT2 homologue: conserved
residues of helix 4 and helix 7 contribute to agonist-dependent
activation of 5-HT2 receptors
Jinling Xie,1 Serghei Dernovici and Paula Ribeiro
Institute of Parasitology, McGill University, Ste Anne de Bellevue, Quebec, Canada
Abstract
An alignment of serotonin [5-hydroxytryptamine (5-HT)] G protein-coupled receptors identified a lysine at position 4.45 (helix
4) and a small polar residue (serine or cysteine) at 7.45 (helix 7)
that occur exclusively in the 5-HT2 receptor family. Other
serotonin receptors have a hydrophobic amino acid, typically a
methionine, at 4.45 and an invariant asparagine at 7.45. The
functional significance of these class-specific substitutions was
tested by site-directed mutagenesis of two distantly related
5-HT2 receptors, Caenorhabditis elegans 5-HT2ce and rat
5-HT2C. Residues 4.45 and 7.45 were each mutated to a
methionine and asparagine, respectively, or an alanine and the
resulting constructs were tested for activity. A K4.45M mutation
decreased serotonin-dependent activity (Emax) of the rat
5-HT2C receptor by 60% and that of the C. elegans homologue
by 40%, as determined by a fluorometric plate-based calcium
assay. The rat mutant also exhibited nearly sixfold higher
agonist binding affinity and significantly lower constitutive
activity compared with wildtype. Mutagenesis of S7.45 in the
C. elegans receptor increased serotonin binding affinity by up to
25-fold and decreased Emax by up to 65%. The same mutations
of the cognate C7.45 in rat 5-HT2C produced a smaller fourfold
change in the affinity for serotonin and decreased agonist
efficacy by up to 50%. Substitutions of S/C7.45 did not produce
a significant change in the basal activity of either receptor. All
mutants tested exhibited levels of receptor expression similar
to the corresponding wildtype based on measurements of
specific [3H]-mesulergine binding or flow cytometry analyses.
Taken together, these results suggest that K4.45 and S/C7.45
play an important role in the conformational rearrangements
leading to agonist-induced activation of 5-HT2 receptors.
Keywords: Caenorhabditis elegans, G protein-coupled
receptor, 5-HT2C, mutagenesis, serotonin.
J. Neurochem. (2004) 92, 375–387.
Serotonin [5-hydroxytryptamine (5-HT)] is a ubiquitous
neuroactive agent of both vertebrates and invertebrates. In
mammals, 5-HT regulates a variety of physiological phenomena in the CNS and periphery, including cognition,
sleep, pain perception, mood, feeding behavior, sexual
behavior, temperature regulation and gastrointestinal function (Weiger 1997). Among invertebrates, 5-HT acts as both
a neurotransmitter and hormone and mediates feeding,
locomotion, circadian rhythm, defense behavior and metabolic activity across various invertebrate phyla (Walker et al.
1996). This diversity of effects is mediated by multiple 5-HT
receptors, a total of seven structurally distinct receptor
classes (5-HT1–7), each of which is further divided into
several subtypes (Boess and Martin 1994). With the exception of the mammalian 5-HT3 ionotropic receptor and a
recently identified nematode (roundworm) 5-HT-gated chloride channel (Ranganathan et al. 2000) all other known
classes of 5-HT receptors belong to the large superfamily of
seven transmembrane-spanning G protein-coupled receptors
(GPCR).
As a group, 5-HT2 receptors are characterized by having a
relatively lower affinity for indolealkylamines, including
Received May 19, 2004; revised manuscript received September 3,
2004; accepted September 10, 2004.
Address correspondence and reprint requests to Paula Ribeiro, Institute of Parasitology, McGill University, 21 111 Lakeshore Road, Ste
Anne de Bellevue, Quebec, Canada H9X 3V9.
E-mail: [email protected]
1
The present address of Jinling Xie is Vanderbilt University Medical
Center, Department of Pharmacology, 452 Preston Research Building,
23rd Avenue South at Pierce, Nashville, TN 37232-6600, USA.
Abbreviations used: FACS, fluorescence-activated cell sorting; GPCR,
G protein-coupled receptor; 5-HT, 5-hydroxytryptamine; 5-HT2ce, Caenorhabditis elegans 5-HT2 receptor; TM, transmembrane domain.
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
375
376 J. Xie et al.
serotonin itself, and are preferentially linked to the Gq/phospholipase C-b pathway of signal transduction. Three 5-HT2
subtypes have been identified in mammals, 5-HT2A, 2B and
2C, which differ on the basis of primary structure and
pharmacological profiles (Roth et al. 1998). In addition,
5-HT2 receptors have been cloned from invertebrates, including
Drosophila (Colas et al. 1995), the snail Lymnaea (Gerhardt
et al. 1996) and two nematodes (roundworms), Caenorhabditis
elegans (Hamdan et al. 1999) and the pig parasite Ascaris suum
(Huang et al. 1999, 2002). The Drosophila and Lymnaea
receptors show some binding characteristics of the mammalian
5-HT2B prototype, whereas the nematode receptors have a
distinctive pharmacological profile and may constitute a
separate subtype of 5-HT2 receptor (Hamdan et al. 1999).
The finding of 5-HT2 receptors in the lower invertebrates
supports the notion that the 5-HT2 class diverged early in
evolution, at least before the separation of nematodes.
An impressive amount of research over the last few years has
been aimed at unveiling the structural organization of the
5-HT2 receptor, particularly the 5-HT2A and 2C subtypes. In
the absence of high-resolution crystallographic data, which are
still lacking for all biogenic amine GPCRs, most of the
information available on 5-HT2 structure is derived from
mutagenesis analyses and comparisons with bovine rhodopsin,
the class I GPCR prototype (Palczewski et al. 2000; Teller
et al. 2001). This research has identified a number of primary
agonist binding residues located mainly on transmembrane
domains (TM) 3, 5 and 6, which are believed to constitute a core
of the receptor’s binding pocket (Choudhary et al. 1995;
Almaula et al. 1996; Roth et al. 1997; Kristiansen et al. 2000;
Shapiro et al. 2000; Visier et al. 2002a; Ebersole et al. 2003).
A number of other residues identified on TM2, 3, 6 and 7 and
intracellular loops have been implicated in receptor activation
and G protein coupling (Sealfon et al. 1995; Herrick-Davis
et al. 1997, 1999; Roth et al. 1997; Prioleau et al. 2002;
Shapiro et al. 2002; Visiers et al. 2002a,b) Despite these
advances, however, a great deal remains to be learned about the
structural organization of serotonergic GPCRs, in particular the
subtle differences between the various receptor subtypes. In
this study, we have used site-directed mutagenesis to test a
number of TM4 and 7 residues which were found to occur
exclusively in 5-HT2 receptors, both vertebrate and invertebrate, and thus were postulated to have functional significance. A
mutagenesis analysis of two distantly related 5-HT2 receptors,
rat 5-HT2C and C. elegans 5-HT2ce, implicated a conserved
lysine of TM4 (K4.45) and a small polar residue of TM7 (S or
C7.45) in the conformational activation of both receptors.
Experimental procedures
Site-directed mutagenesis
A C. elegans 5-HT2ce expression construct was used as a template for
site-directed mutagenesis. The construct was made previously in
pCIneo and includes the complete coding sequence of 5-HT2ce fused
at the C-terminal end to a FLAG epitope (Hamdan et al. 1999) For
studies of rat 5-HT2C, a cDNA encoding the full-length unedited
receptor was obtained from the American Type Culture Collection
(ATCC, Manassas, VA, USA) and modified by PCR to introduce an
N-terminal FLAG epitope. The resulting construct was subcloned
between the NheI/NotI sites of pCEP4 mammalian expression vector
(Invitrogen, Burlington, Canada), confirmed by DNA sequencing and
used for site-directed mutagenesis. All point mutations were generated
with the QuickChange mutagenesis kit (Stratagene, La Jolla, CA,
USA), according to the recommendations of the manufacturer. The
mutations were verified by sequencing the full-length cDNAs.
Cell culture and transfection
COS7 were grown in Dulbecco’s modified Eagle’s medium
supplemented with 10% bovine fetal serum (Invitrogen) and
20 mM HEPES buffer at 37C in a humidified environment
containing 5% CO2. HEK293(EBNA1) cells were cultured in
Dulbecco’s modified Eagle’s medium containing L-glutamine and
supplemented with 10% fetal bovine serum (Invitrogen), 1 mM
sodium pyruvate, 250 lg/mL G418 and 20 mM HEPES buffer
(Invitrogen). For transfection, cells were seeded in HEPES-buffered
Dulbecco’s modified Eagle’s medium containing 10% dialysed fetal
bovine serum and cultured overnight to approximately 80%
confluency. Unless indicated otherwise, cells were transfected in
100-mm culture dishes (1.5–2.5 · 106 cells and 3 lg plasmid DNA/
dish) using FuGENE 6 (Roche Diagnostics, Laval, Canada),
according to the specifications of the manufacturer. Transfection
efficiency was monitored routinely by using a green fluorescence
protein-encoding plasmid (pTracer) and was typically 40–50%.
Binding assays
Binding assays of 5-HT2ce wildtype and mutants were performed
on crude membrane preparations of transiently transfected COS7
cells. Rat 5-HT2C wildtype and mutants were transiently expressed
in HEK293(EBNA1) cells. In both cases, transiently transfected
cells were harvested 48 h post-transfection and lysed by brief
sonication in ice-cold TEM buffer (50 mM Tris-HCl, pH 7.4,
0.5 mM EDTA, 10 mM MgCl2). Binding assays were performed
with aliquots (5–10 lg protein/reaction) of a 28 000 g crude
membrane preparation in a total volume of 200 lL TEM buffer
containing [3H]-mesulergine (75–86 Ci/mmol; Amersham, Baie
d’Urfé, Canada) as the radiolabeled ligand. Saturation curves were
generated from a minimum of seven different labeled ligand
concentrations in the presence and absence of 10 lM mianserin
(Sigma, Oakville, Canada) for measurements of non-specific
binding. Competition studies were performed by testing seven to
eight concentrations of unlabeled competitor in the presence of a
constant amount of [3H]-mesulergine. All test ligands were prepared
in 0.1% ascorbic acid. Reaction mixtures were incubated at room
temperature for 90 min. Reactions were then terminated by rapid
filtration over 1.0 lm Filtermat (Molecular Devices, Sunnyvale,
CA, USA) pre-soaked in 0.3% polyethylenimine and subsequently
washed three times with ice-cold buffer (50 mM Tri-HCl, pH 7.4)
using a Skatron ClassicCell Harvester (Molecular Devices). All
saturation and competition kinetic parameters were determined by
computer-assisted non-linear regression analysis using the Prism
software package (GraphPad Software Inc., San Diego, CA, USA).
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
Mutagenesis analysis of the serotonin 5-HT2 receptor 377
Ca2+ assays
Receptor signaling activity was measured in intact cells using a
fluorometric plate-based assay for detecting changes in intracellular
calcium. A preliminary test determined that the rat 5-HT2C receptor
produced robust calcium responses in both transfected HEK293(EBNA1) and COS7 cells upon agonist stimulation. However, the
HEK293(EBNA1) cells were easily removed from the plate during
the washing procedure, which increased experimental variability.
COS7 cells produced considerably less well-to-well variation and
thus were selected for the study. COS7 cells were cultured overnight
in HEPES-buffered Dulbecco’s modified Eagle’s medium containing 10% dialysed fetal bovine serum and transiently transfected in
100-mm dishes as described above (1.5–2.5 · 106 cells and 3 lg
plasmid/dish). Approximately 24 h post-transfection, cells were
harvested, seeded in black-walled, clear-base 96-well plates at a
density of 80 000 cells/well and cultured for another 24 h before the
calcium assay. Cell density was optimized in preliminary experiments within a range of 10 000–100 000 cells/well. In some
experiments, cells were transfected directly into 96-well plates (1.5–
2 · 104 cells and 50 ng of plasmid DNA/well), using FuGENE6,
and assayed in the same plates 48 h post-transfection. The assay was
performed with the use of a FLIPR calcium assay kit, according to
the recommendations of the manufacturer (Molecular Devices).
Briefly, cells were washed once in Hanks balanced saline supplemented with 2.5 mM probenecid (pH 7.4) and incubated with
100 lL of calcium dye reagent for 60 min at 37C. After incubation,
cells were placed immediately in a FlexStation plate fluorometer
equipped with a multichannel injector (Molecular Devices) and set
at kex ¼ 485 nm, kem ¼ 520 nm. Agonist or a vehicle was added in
a volume of 50 lL/well at an injection speed of 80 lL/ s.
Fluorescence measurements were taken at 1.52 s intervals before
and after agonist addition for a total of 60 s per well. Each assay
plate included a mock-transfected control, wildtype receptor and a
test mutant all transfected at the same time and seeded at the same
cell density. The raw data were analysed with the SoftmaxPro
software package (Molecular Devices). The baseline was defined as
the average of the first five recordings on each curve. Functional
responses were measured as peak fluorescence levels after subtraction of the baseline. Basal (spontaneous) receptor activity was
determined by subtracting the baseline in the mock-transfected
control from that of the test sample also measured on the same assay
plate. All measurements were derived from at least three separate
experiments each performed in sets of three to four replicates.
Three-dimensional modeling
Theoretical three-dimensional models of the C. elegans 5-HT2ce
receptor and rat 5-HT2C were generated with the homology
modeling program Composer of the Biopolymer module of Sybyl
6.9 (Tripos Inc., St Louis, MO, USA). The models were produced by
using the coordinates of bovine rhodopsin (1hzx) (Teller et al. 2001)
as a structural template. The primary sequence of each receptor was
first aligned with the sequence of rhodopsin and the alignment was
inspected to insure that seed residues matched perfectly, including
the reference residues designated at position 50 of each helix
(Ballesteros and Weinstein 1995) and conserved motifs, such as the
E/DRY and NPxxY motifs. The structural alignment was subsequently performed using default parameters. Structurally conserved
regions corresponding to the seven transmembrane helices and
cytoplasmic helix 8 were identified and their boundaries were
adjusted manually to coincide with those of the rhodopsin template.
Variable loop regions were built by searching the Sybyl protein
databank with Composer using default parameters. The N-terminus
(5-HT2C residues 1–53; C. elegans 5-HT2ce residues 1–54), third
intracellular loop (5-HT2C residues 239–305; C. elegans 5-HT2ce
residues 238–364) and C-terminus (5-HT2C residues 388–460;
C. elegans 5-HT2ce residues 445–683) were not built due to lack of
structural information. In addition, a fragment of the predicted
extracellular loop 2 of 5-HT2C (5-HT2C residues 208–211) could
not be built and was omitted. The two model structures were refined
by energy minimization in the subroutine Powell using the KollmanAll Atom force field with a non-bonded cut-off of 8 Å and a
dielectric constant of 1. Minimizations were performed first with the
carbon backbone fixed, after which the entire model was minimized
until a convergence gradient value of 0.1 kcal/mol.Å was reached. To
assess the effects of the mutations of interest, the appropriate residues
were modified with the Biopolymer module and the models were
reminimized to convergence.
Numbering of G protein-coupled receptor amino acid residues
Amino acids of rat 5-HT2C and C. elegans 5-HT2ce are identified
according to the system of Ballesteros and Weinstein (1995). Each
amino acid within a TM region is identified by the TM number
(1–7) followed by the position in the TM helix relative to an
invariant reference residue, which is arbitrarily assigned the number
50. Residues of interest to this study are identified as K4.45
(5-HT2C K175 and C. elegans 5-HT2ce K175) and S/C7.45 (5-HT2C
C362 and C. elegans 5-HT2ce S419).
Other methods
Expression levels of the various receptors in COS7 and HEK293(EBNA1) were monitored by in situ immunofluorescence, as described
previously (Hamdan et al. 2002), using a monoclonal antibody
directed against the FLAG epitope (anti-FLAGM2; Sigma; 5 lg/mL)
and a secondary FITC-conjugated antibody (goat anti-mouse IgG;
Sigma; 1 : 200 dilution). The level of receptor expression on the cell
surface was measured by fluorescence-activated cell sorting (FACS)
analysis on a FACScan flow cytometer (Becton-Dickinson, Oakville,
Canada) according to standard procedures. FACS analyses were
performed on both COS7 and HEK293(EBNA1) cells transiently
transfected with FLAG-tagged 5-HT2C-expressing constructs and
cell viability was assessed based on exclusion of propidium iodide.
Secreted alkaline phosphatase reporter gene assays were performed in
293CRE-SEAP cells (Durocher et al. 2000) transiently transfected
with 5-HT2C wildtype or mutant expression constructs, as described
previously (Hamdan et al. 2002). Protein was determined according
to the method of Bradford (1976) using a Protein Assay Kit (Bio-Rad,
Mississauga, Canada). Statistical tests for significance were conducted using unpaired two-tailed Student’s t-tests.
Results
Identification of 5-HT2-specific residues
A total of 38 vertebrate and invertebrate 5-HT receptor
sequences (Table 1), belonging to all six major classes of
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
378 J. Xie et al.
Receptor
Accession no.
Receptor
Accession no.
5-HT1A_RAT
5-HT2A_RAT
5-HT2B_RAT
5-HT2C_RAT
5-HT4_RAT
5-HT6_RAT
5-HT7_RAT
5-HT1A_HUMAN
5-HT-1Da_HUMAN
5-HT-1Db_HUMAN
5-HT1E_HUMAN
5-HT2A_HUMAN
5-HT2B_HUMAN
5-HT2C_HUMAN
5-HT4_HUMAN
5-HT5A_HUMAN
5-HT6_HUMAN
5-HT7_HUMAN
5-HT5A_MOUSE
J05276
P14842
P30994
P08909
U20907
L03202
L22558
P08908
P28221
M81590
P28566
P28223
P41595
P28335
Q13639
P47898
P50406
P34969
Z18278
5-HT5B_MOUSE
5-HT1A_FUGRU
5-HT2A_CIRGR
5-HT_Balanus
5-HTdroA_Drosophila
5-HTdroB_Drosophila
5-HT2Dro_Drosophila
5-HT_Spisula
5-HTap1_Aplysia
Ap5-HTB1_Aplysia
Ap5-HTB2_Aplysia
5-HTLym_Lymnaea
5-HT2Lym_Lymnaea
5-HT1_Dugesia
5-HT4_Dugesia
5-HT2Asc_Ascaris
5-HTce_C. elegans
5-HT2ce_C. elegans
5-HT_Haemonchus
X69867
O42385
P18599
D83547
Z11489
Z11490
X81835
AAL23575
AF041039
L43557
L43558
L06803
U50080
BAA22404
BAA22403
AF005486
U15167
AF031414
AAO45883
Table 1 5-Hydroxytryptamine (5-HT) receptor sequences
List of vertebrate and invertebrate 5-HT receptor sequences analysed in this study. Details of the
multisequence alignment are provided in the text.
serotonergic GPCRs, were aligned with the program MacVector (version 7.0) using the ClustalW method. The analysis
identified several amino acids that are unique to 5-HT2
receptors (Fig. 1). Among these residues is a TM4 lysine at
position 4.45 (K4.45). The cognate amino acid in other
receptor subtypes is hydrophobic and almost always a
methionine. In TM7, the 5-HT2 sequences are characterized
by having a small polar residue, a serine or cysteine, at
position 7.45 of the helix. The 5-HT2C subtype has a
cysteine at this position whereas other 5-HT2 receptors,
including C. elegans 5-HT2ce, have a serine. Most serotonergic GPCRs and other amine receptors have a highly
conserved asparagine at 7.45 (Fig. 1). K4.45, S7.45 and
C7.45 were designated as 5-HT2-specific residues and were
targeted for site-directed mutagenesis.
Mutagenesis of the rat 5-HT2C receptor
Four mutant forms of 5-HT2C were generated, each
carrying a single point mutation. The mutagenesis was
designed to change the native amino acid to that normally
found at the same position in other serotonin receptors.
K4.45 was mutagenized to a methionine (K4.45M) and
C7.45 to an asparagine (C7.45N). An alanine substitution
of C7.45 (C7.45A) was also produced. Wildtype and
mutant receptors were tested for binding activity using the
selective 5-HT2 ligand, [3H]-mesulergine. All constructs
exhibited saturable [3H]-mesulergine binding and similar
kinetics. The Bmax values for [3H]-mesulergine varied by
about twofold (Fig. 2), suggesting that the density of
functional binding sites was not significantly altered by the
mutagenesis.
Agonist and antagonist binding affinities were determined
by competition against [3H]-mesulergine. A number of
5-HT2-selective ligands were tested. In addition, we tested
ligands that bind preferentially to other 5-HT receptors to
determine whether the mutagenesis altered the pharmacological profile of 5-HT2C. The analysis revealed that the
binding of serotonin was influenced by mutations of K4.45
and C7.45 (Fig. 2). Compared with the wildtype, the
K4.45M mutant showed a significant sixfold increase in
serotonin binding affinity (p £ 0.0005). The C7.45N substitution produced a fourfold increase in affinity (p £ 0.005)
whereas an alanine mutation decreased binding affinity
fourfold (p £ 0.05). In addition to serotonin, the mutagenesis
produced modest but significant changes in the binding
affinities of other serotonergic ligands (Table 2). The
K4.45M mutant exhibited approximately fivefold higher
affinity for the serotonin derivative, 5-methoxytryptamine,
and threefold higher affinity for the 5-HT2 agonist, 1-(4iodo-2,5-dimethoxylphenyl)-2-aminopropane (DOI). This
mutation had no effect on any of the 5-HT2 antagonists
tested, including mesulergine, metergoline, ketanserin and
mianserin, or selective ligands of other 5-HT receptor
subtypes [methiothepin, 8-hydroxydipropylaminotetralin
(8-OH-DPAT)]. In the case of the C7.45N mutant, the most
pronounced effects were a significant fivefold decrease in the
affinity for lisuride and a smaller two- to threefold decrease
in the affinity for the antagonists mianserin and ketanserin.
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
Mutagenesis analysis of the serotonin 5-HT2 receptor 379
Fig. 1 Multiple sequence alignment of all six major classes of serotonergic G protein-coupled receptors, including mammalian and
invertebrate 5-HT2 sequences. The 5-HT2 class consists of three
mammalian subtypes (5-HT2A, 2B and 2C) and four invertebrate
sequences, including the Caenorhabditis elegans 5-HT2ce receptor.
The rat and C. elegans sequences used in this study are marked. The
regions shown are representative of an alignment of 38 sequences
and include the conserved E/DRY motif, transmembrane (TM) 4 and a
portion of TM7. Accession numbers for all the sequences used in the
analysis are provided in Table 1. The positions of 5-HT2-specific
residues tested in this study are indicated by arrows.
Activity assays of the rat 5-HT2C receptor
Wildtype and mutant 5-HT2C constructs were transiently
expressed in COS7 and tested for calcium activity using a
fluorometric plate-based assay. Stimulation of the wildtype
receptor with serotonin produced a rapid, transient increase
in the level of intracellular calcium (Fig. 3). The agonist
response peaked within 10 s of stimulation and returned to
near basal level after 60 s. Control cells transfected with
empty vector did not respond to serotonin. All three mutants
tested caused a significant decrease in the magnitude of the
agonist response. The most pronounced effect was observed
with the K4.45M mutant. A kinetic analysis of the various
receptor species revealed that the K4.45M mutant decreased
the Emax for serotonin by nearly 60% compared with the
wildtype receptor (Fig. 4). Substitutions of C7.45 also
decreased agonist efficacy significantly but to a lesser extent;
asparagine and alanine mutations of C7.45 decreased Emax by
42 and 54%, respectively.
In addition to agonist-stimulated activity, the rat 5-HT2C
receptor is known to have spontaneous (ligand-independent)
activity, which is sensitive to inhibition by inverse agonists
(Herrick-Davis et al. 1999). In this study, we observed that
the transiently expressed wildtype receptor exhibited an
average basal activity of 4034 ± 638 relative fluorescence
units above the corresponding mock-transfected control
assayed on the same plate. This basal activity could be
inhibited by the inverse agonist, mianserin (not shown),
consistent with the notion that the receptor was spontaneously activated. Compared with the wildtype, the C7.45N
and C7.45A mutants had slightly elevated baselines but the
difference was not statistically significant. In contrast, the
K4.45M mutation reduced the receptor’s basal activity to less
than 10% of the wildtype level (Fig. 4). The average basal
activity of the K4.45M mutant was 300 ± 94 relative
fluorescence units.
In other experiments, we tested whether residues at
positions 4.45 or 7.45 contributed to the selectivity of
5-HT2 receptors for Gq and the inositol triphosphate/Ca2+
signaling pathway. Cells transiently expressing 5-HT2C
wildtype or a mutant were assayed for cAMP-mediated
signaling by using a reporter CRE-SEAP assay (Durocher
et al. 2000) in both the presence and absence of forskolin.
The results showed no evidence of cAMP signaling in the
wildtype or any of the three mutants (data not shown).
Measurements of wildtype and mutant 5-HT2C
expression
There is increasing evidence that some loss-of-function
mutations of GPCRs are associated with receptor desensitization and the loss of receptor molecules from the cell
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
380 J. Xie et al.
Fig. 2 Saturation kinetics and inhibition of specific [3H]-mesulergine
binding to rat 5-HT2C. Kinetic parameters Kd and Bmax were obtained
from saturation binding curves, using at least seven concentrations of
radioligand. The Ki values for serotonin (5-HT) were obtained from
competition assays against [3H]-mesulergine. Serotonin competition
curves for the wildtype (WT) receptor and three test mutants are
shown. Each curve is the average of duplicate determinations from a
typical experiment repeated at least three times. The data were normalized relative to the level of maximum binding obtained in the
absence of competitor. Kd, Bmax and Ki values were calculated by nonlinear curve-fitting analyses with the Prism (GraphPad Software Inc.)
software package. All kinetic parameters are the means and SEM of at
least three independent determinations, each in duplicate. *Statistically different from WT at p £ 0.05.
Table 2 Inhibition of [3H]-mesulergine binding to wildtype (WT) rat
5-HT2C and mutants
Ki (nM)
Compound
WT
K4.45M
C7.45N
Methoxy-5-HT
DOI
Mianserin
Ketanserin
Lisuride
Metergoline
Methiothepin
8-OH-DPAT
51.3 ± 6.5
134 ± 13
1.35 ± 0.25
38.9 ± 8.9
12.7 ± 1.3
0.29 ± 0.11
0.73 ± 0.10
> 10 000
9.38 ± 0.31a
40.2 ± 2.4a
1.78 ± 0.21
39.3 ± 3.6
28.9 ± 2.4
0.14 ± 0.08
1.25 ± 0.40
> 10 000
42.0 ± 4.0
94.6 ± 11.9
4.45 ± 0.28a
128 ± 14.5a
59.4 ± 14.5a
0.23 ± 0.02
1.6 ± 0.38
> 10 000
Ki values were obtained from competition assays against [3H]-mesulergine using seven to eight concentrations of each drug. Data are the
means and SEM from two to three independent experiments. Drugs
that produced ‡ twofold differences between the WT and mutants
were tested three separate times, each in duplicate.
DOI, 1-(4-iodo-2,5-dimethoxylphenyl)-2-aminopropane.
a
Statistically different from WT at p £ 0.05.
Fig. 3 Time course of serotonin-induced elevation of intracellular
calcium in cells expressing 5-HT2C wildtype (WT) or the K4.45M
mutant. A control transfected with vector only is also shown. Transfected cells were pre-loaded with a calcium fluorescent indicator and
then placed in a FlexStation (Molecular Devices) plate fluorometer set
at kex ¼ 485 nm, kem ¼ 520 nm. The baseline was recorded for 17 s
at which point serotonin (10 lM) was injected into each well at a speed
of 80 lL/ s. Fluorescence was monitored at 1.52-s intervals for up to
60 s. Data were normalized by subtracting the baseline in each test
sample. The representative results shown are from a single experiment that was repeated at least three times.
surface (Kristiansen et al. 2000; Wilbanks et al. 2002). To
test whether the mutations described here had similar effects
on the expression of the 5-HT2C receptor we performed
in situ immunofluorescence and flow cytometry analyses of
the various populations of transfected cells. Each of the rat
5-HT2C constructs was engineered with an N-terminal
(extracellular) FLAG epitope, which was targeted for the
study. A first in situ immunofluorescence study of nonpermeabilized transfected COS7 revealed essentially the same
pattern of surface expression in all test cells examined (not
shown). This was subsequently confirmed by fluorescenceactivated cell sorting (FACS) analysis using live transfected
cells (Figs 5a and b). The results showed similar levels of cell
surface fluorescence in all mutant and wildtype-expressing
cells but not in the mock-transfected controls or negative
controls lacking primary antibody. A slightly higher level of
expression was observed in cells transfected with the C7.45A
mutant, consistent with the elevated Bmax value of this mutant
in the [3H]-mesulergine binding assays (see Fig. 2). However,
this small difference in expression levels was not statistically
significant at p £ 0.05. The assays were repeated with
HEK293(EBNA1) transfected with the same 5-HT2C-expressing constructs and, again, no difference could be detected
between the various mutants and wildtype (not shown). Thus,
the results suggest that the effects of the mutations on calcium
activity were due to changes in the activation of the receptor
rather than changes in the level of protein expression or the
loss of receptor molecules from the surface.
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
Mutagenesis analysis of the serotonin 5-HT2 receptor 381
(a)
(b)
Fig. 4 Serotonin (5-HT)-induced stimulation of intracellular calcium in
cells expressing rat 5-HT2C wildtype (WT) or mutants. The data on
each curve are the means and SEM of three to four replicates from a
typical experiment repeated at least three times. The mock-transfected control (vector) did not respond to serotonin. The results were
standardized relative to the maximum response produced by the WT
assayed at the same time and on the same plate. EC50 and Emax
values were obtained by computer-assisted non-linear curve-fitting
analyses of dose–response curves. Basal activity was determined by
subtracting the baseline of the mock-transfected control assayed on
the same plate from that of each test sample. Under the conditions
tested, the basal activity of the WT receptor was 4034 ± 638 relative
fluorescence units. EC50, Emax and basal activity values are the means
and SEM of at least three independent experiments, each in sets of
three to four replicates. *Statistically different from WT at p £ 0.05.
Mutagenesis and characterization of the Caenorhabditis
elegans 5-HT2ce receptor
The analysis of the rat 5-HT2C receptor suggested that
positions 4.45 and 7.45 influenced serotonin binding and
efficacy. To test whether this was true in other 5-HT2
receptors, we repeated the analysis with a C. elegans 5-HT2
homologue, 5-HT2ce, which has exceptionally low affinity
for the natural ligand (Hamdan et al. 1999). The C. elegans
sequence was mutagenized at the same sites and three
mutants (K4.45M, S7.45N and S7.45A) were generated and
assayed for [3H]-mesulergine binding. [3H]-mesulergine had
not been previously tested on this C. elegans receptor but
was shown here to bind saturably and specifically to both the
wildtype and mutant species. Figure 6 shows a typical
Fig. 5 Flow cytometry analysis of COS7 cells expressing 5-HT2C
wildtype (WT) or mutants. Cells were transfected with vector only
(mock) or a construct designed to express 5-HT2C fused to an
N-terminal (extracellular) FLAG epitope. (a) Cell surface expression
was estimated by flow cytometry, using a monoclonal anti-FLAG
antibody followed by a FITC-conjugated secondary antibody. Mocktransfected cells and cells incubated with secondary antibody only
(blk) were used as controls. Data were normalized relative to the WT
level and are shown as the means and SEM of three independent
experiments. (b) Typical histogram plots produced by flow cytometry
analysis of cells expressing WT 5-HT2C (solid line), mutant 5-HT2C
(broken lines) or the mock-transfected control (shaded). Only two
mutants (K4.45M and C7.45A) are shown.
saturation curve for [3H]-mesulergine along with a summary
of binding kinetics. Non-specific binding was measured in
the presence of 10 lM mianserin and represented approximately 10% of total binding. As in the case of the rat
receptor, mutagenesis of C. elegans 5-HT2ce did not alter
expression of functional binding sites in a significant manner.
Bmax values of mutants and wildtype were similar within a
range of 1.0–2.4 pmol/mg.
Competition assays against [3H]-mesulergine confirmed
the low affinity of the C. elegans receptor for serotonin
(Fig. 6). The Ki values for serotonin and the related
derivative 5-methoxytryptamine were 9.7 and 7.5 lM,
respectively, nearly two orders of magnitude above the
values obtained with the rat receptor. The K4.45M mutation
did not change the Ki values significantly. In contrast, the
S7.45N mutation markedly increased the affinity for both
ligands. Compared with the wildtype, the S7.45N mutant
exhibited 25-fold higher affinity for serotonin (Ki, 0.39 lM)
and nearly 11-fold higher affinity for 5-methoxytryptamine
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
382 J. Xie et al.
Fig. 6 Binding studies of the Caenorhabditis elegans 5-HT2ce
receptor. (a) Typical [3H]-mesulergine saturation curve of the wildtype
(WT) receptor. Specific binding (squares) was calculated after subtraction of the non-specific component (triangles). Non-specific binding
was determined in the presence of 10 lM mianserin. The data are the
average of duplicate determinations from a typical experiment
repeated three times. (b) Inhibition of specific [3H]-mesulergine binding
by serotonin (5-HT). Results were normalized relative to the level of
maximal binding measured in the absence of competitor. Data shown
are typical competition curves for the 5-HT2ce WT and S7.45 mutant
and were repeated at least three times. Ki values were estimated from
competition curves for serotonin and the derivative, 5-methoxytryptamine. All kinetic parameters are shown as the means and SEM of at
least three independent experiments, each performed in duplicate.
*Values significantly different from WT at p £ 0.05.
(Ki, 0.69 lM). The alanine substitution (S7.45A) had no
significant effect on the binding affinity of serotonin at
p £ 0.05.
Mutant and wildtype forms of the C. elegans 5-HT2ce
receptor were transiently transfected in COS7 cells and
tested for calcium activity. As in the case of the rat receptor,
all three mutations tested produced a decrease in the
magnitude of the serotonin-induced response compared with
the wildtype (Fig. 7). Substitutions of S7.45 had the
greatest impact on agonist activity. The S7.45A mutant
had 55% less agonist-induced activity, whereas the S7.45N
mutant exhibited both a decrease in EC50 (from 1340 to
90 nM) and a decrease in Emax of about 65%. The K4.45M
mutant also exhibited diminished activity but to a lesser
extent; the efficacy of the agonist response in this mutant
Fig. 7 Calcium assays of the Caenorhabditis elegans 5-HT2ce
receptor. Cells expressing wildtype (WT) or mutant receptor were
stimulated with serotonin and assayed for intracellular calcium using a
fluorometric method, as described above (see Fig. 3). Typical serotonin dose–response curves for the WT, S7.45N mutant or a mocktransfected control (vector) are shown. Each curve is the average and
SEM of three to four replicates from a single experiment repeated
three times. The corresponding EC50 and relative Emax values are the
means and SEM of three independent experiments performed in sets
of three to four replicates. The basal activity was calculated as in Fig. 3
and is presented as the mean and SEM of three to five independent
experiments. The basal activity of the WT C. elegans receptor was
1280 ± 606 relative fluorescence units. *Statistically different from WT
at p £ 0.05.
was decreased by about 40% compared with the wildtype.
The C. elegans receptor expressed in COS7 cells exhibits a
basal activity of 1280 ± 606 relative fluorescence units
above that of the corresponding mock-transfected control.
None of the substitutions tested had a significant effect on
this basal activity.
Three-dimensional modeling of K4.45 and S/C7.45
Theoretical three-dimensional structures of the rat and
C. elegans receptors were produced by homology modeling, using the coordinates of the 2.8 Å crystal structure of
bovine rhodopsin as a template. The two receptors share
only about 66% overall homology at the level of primary
sequence (Hamdan et al. 1999). However, the regions
surrounding 4.45 and 7.45 within the helical bundle are
similar in the two models. K4.45 is located within the
transmembrane portion of TM4, nearly two turns of the
helix above the predicted cytoplasmic boundary (position
4.38). The lysine resides in a predominantly hydrophobic
region formed by non-polar residues of TM4 (positions
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
Mutagenesis analysis of the serotonin 5-HT2 receptor 383
Fig. 8 Computer-generated models of (a)
the rat 5-HT2C receptor and (b) the Caenorhabditis elegans 5-HT2ce receptor
show proximity of the K4.45 side chain to
aspartate 3.49 of the E/DRY motif and a
neighboring residue at 3.45. (c) Predicted
orientation of K4.45 relative to residues of
the E/DRY motif in the 5-HT2C model.
Arginine 3.50 is held by interactions with
both aspartate 3.49 and a conserved TM6
glutamate (6.30). K4.45 interacts with the
side chain carboxylate of D3.49 and the
peptide carbonyl of A3.45. For clarity, only
the cytoplasmic ends of helices 3, 4 and 6
are shown.
4.42–4.50) and neighboring helices, in particular TM3 and
TM2. Several of these non-polar amino acids are conserved in
the two receptors, for example F2.42 in TM2, I3.46 and L3.48
in TM3. The predicted orientation of the lysine side chain is
shown in Fig. 8. In both models the side chain of K4.45 is
oriented towards the cytoplasmic end of TM3 and comes
within a relatively short distance (< 5 Å) of aspartate 3.49,
which is conserved in both receptors. The rat 5-HT2C model
predicts a direct contact between K4.45 and D3.49. The
estimated distance between the e amino nitrogen of K4.45 and
the side chain carboxylate of D3.49 in 5-HT2C is 2.6 Å. In
addition, the lysine side chain forms a direct contact with the
peptide carbonyl of an adjacent residue at 3.45 in both
5-HT2C (N–O distance of 2.3 Å) and C. elegans models
(N–O distance of 2.7 Å). Aspartate 3.49 belongs to the
invariant E/DRY motif of rhodopsin-like GPCRs. A number
of models have proposed that the motif’s arginine, R3.50,
forms a network of electrostatic interactions with the
neighboring D3.49 and a conserved glutamate (E6.30) at
the cytoplasmic end of TM6 (Visiers et al. 2002a). These
interactions are also predicted in our two models (Fig. 8).
Position 7.45 occurs approximately midway in helix 7.
Our rat and C. elegans models suggest that 7.45 resides
within a short stretch of amino acids that also includes a
conserved glycine (G7.42) and is predicted to disrupt the
alpha helical nature of the TM7 segment (Fig. 9). The
5-HT2C model shows the side chain of its cysteine 7.45
turned towards the interior of the helical bundle. In contrast,
the side chain hydroxyl of serine 7.45 in the C. elegans
model is turned upwards and may form an intrahelical
H-bond with the peptide carbonyl of G7.42. In both models,
7.45 occurs within relative spatial proximity of the TM6
aromatic cluster, in particular tryptophan 6.48 and the
neighboring phenylalanine 6.44, which are conserved in all
amine GPCRs. The indole ring of W6.48 is shown nearly
perpendicular to the plane of the membrane, as predicted by
the rhodopsin structure and several recent models of amine
GPCRs (Visiers et al. 2002a). Immediately below 7.45 on
Fig. 9 Spatial orientation of C7.45 in the rat
5-HT2C model (right panel) and S7.45 in
the Caenorhabditis elegans 5-HT2ce model
(left panel) relative to residues of the aromatic cluster motif. Only helices 6 and 7 are
shown.
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
384 J. Xie et al.
the same side of the TM7 helix is residue N7.49 of the
NPxxY motif. The side chain of N7.49 is turned towards
conserved TM2 aspartate 2.50 in both models (not shown),
consistent with existing mutagenesis and modeling evidence
(Sealfon et al. 1995; Visiers et al. 2002a).
Discussion
The growing numbers of invertebrate GPCR sequences
available in the database provide a new wealth of information
for receptor structure–function studies. Multisequence comparisons of mammalian receptors with lower vertebrate and
invertebrate orthologues allow for identification of residues
that are conserved across phylogeny and thus are likely to have
structural or functional significance. In this study, we have
taken advantage of recently cloned invertebrate 5-HT receptor
sequences to identify a few conserved residues that distinguish
the entire 5-HT2 group from other serotonergic GPCRs.
The lysine at position 4.45 is conserved only in the 5-HT2
receptors. The majority of amine GPCRs, not only serotonergic but also adrenergic, dopaminergic and muscarinic, have
a hydrophobic residue at this site, usually a Ile, Leu or Met.
A Met substitution of K4.45 significantly decreased agonist
efficacy of the rat 5-HT2C receptor by 60% and that of the C.
elegans homologue to a lesser extent, by about 40%. In
addition, the mutation increased serotonin binding affinity of
5-HT2C and decreased this receptor’s basal activity without
affecting the level of receptor protein expression on the cell
surface. How a mutation at 4.45 could have such a
pronounced effect on activity is difficult to explain at
present. Serotonergic 5-HT2 receptors have been the subject
of extensive research but the majority of these studies have
focused on helices 3, 5, 6 and 7, which comprise the key
functional domains of GPCRs. Considerably less is known
about other helices, in particular helix 4. There is evidence
that the extracellular (upper) half of TM4, beginning at the
invariant tryptophan 4.50, plays a role in ligand binding and
may contribute to the receptor’s binding pocket (Roth et al.
1997; Javitch et al. 2000). In contrast, the cytoplasmic half
of TM4, where 4.45 resides, has not been widely investigated. Two recent studies of the dopamine D2 and muscarinic M1 receptors reported that position 4.45 had no effect on
either ligand binding or receptor activation (Javitch et al.
2000; Lu et al. 2001). However, these receptors both have a
methionine at 4.45. A lysine may function differently in the
5-HT2 receptors. Recent GPCR models have proposed that
basic residues located near the cytoplasmic interface of TM
segments may interact with charged headgroups of membrane phospholipids, thereby anchoring the receptor onto the
membrane (Visiers et al. 2002a). If K4.45 also functions in
this manner, the decrease in activity associated with the
mutagenesis may be related to changes in the interaction
between the receptor and the membrane. However, the
anchoring Arg/Lys residues of the TM4 helix are clustered
closer to the cytoplasmic boundary, between positions 4.38
and 4.41, and are not typically conserved (Javitch et al.
2000) whereas K4.45 is located within the predicted
transmembrane region and is type specific. Although the
possibility of a structural role cannot be ruled out, we
postulated that K4.45 was more likely to be involved in a
particular aspect of receptor activation.
To interpret the results of the mutagenesis, we generated
three-dimensional models for both the rat 5-HT2C receptor
and the C. elegans homologue, using the coordinates of the
2.8 Å crystal structure of bovine rhodopsin as a template.
There are a number of 5-HT2A and 5-HT2C models in the
literature (Chambers and Nichols 2002; Prioleau et al. 2002;
Shapiro et al. 2002; Visiers et al. 2002a,b) but the orientation of K4.45 relative to the surrounding helices has not been
discussed. Our models suggest at least two explanations for
the results of the K4.45M mutagenesis. One possibility is
that the loss of activity in the mutants was an indirect effect
caused by the introduction of a methionine in this predominantly non-polar environment. Replacing the native lysine
with a methionine in the 5-HT2C model moved the side
chain away from D3.49 and towards hydrophobic residues of
TM2, in particular A2.38 and F2.42 (not shown). A closer
packing of these helices in the mutant may hinder the ability
of the receptor to become activated. A second explanation for
the results of the mutagenesis is that the native lysine is
required for receptor activation, possibly due to its apparent
proximity to the E/DRY motif. The motif contributes to a
network of electrostatic interactions that stabilize the inactive
state of rhodopsin-like GPCRs by holding the cytoplasmic
ends of TM3 and 6 close together (Visiers et al. 2002a,b). A
large body of modeling and experimental evidence suggests
that the activation of GPCRs is associated with a weakening
of these interactions and the separation of the two helices
(Ballesteros et al. 2001; Angelova et al. 2002; Shapiro et al.
2002; Visiers et al. 2002a,b). Among the 5-HT2 receptors, in
particular, mutations of D3.49 and the motif’s invariant
arginine 3.50 have been shown to cause significant inhibition
of agonist-stimulated activity (Shapiro et al. 2002; Visiers
et al. 2002b). Ala and Glu substitutions of D3.49 in the 5HT2A receptor both decreased agonist efficacy (Visiers et al.
2002b), suggesting that the precise length and positioning of
the acidic side chain at this site is critical for receptor
activation. Our models suggest that K4.45 may be sufficiently close to this region to influence the orientation of
D3.49 within the motif. This could facilitate the transition to
an active state by destabilizing the motif and/or stabilizing
active forms of the receptor during these conformational
rearrangements. Moreover, the direct contact between K4.45
and D3.49 predicted by the 5-HT2C model would be
expected to facilitate spontaneous activation of this receptor,
which could explain the loss of constitutive activity observed
in the rat mutant. It should be emphasized that the rhodopsin
structural template has a glycine at 4.45 and there are no
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
Mutagenesis analysis of the serotonin 5-HT2 receptor 385
interactions between this site and any of the surrounding
helices (Palczewski et al. 2000; Teller et al. 2001). Thus, the
predicted orientation of the lysine side chain in our two
models must be viewed with caution. Nevertheless, together
with the mutagenesis data, this study suggests that the
cytoplasmic end of TM4 may be more important for the
activation of the 5-HT2 receptors than is currently believed.
Whereas the M4.45 of other receptors appears to have no
direct functional role (Javitch et al. 2000; Lu et al. 2001), the
5-HT2-specific lysine may facilitate conformational activation and may also contribute to the natural propensity of
5-HT2C towards spontaneous activity. This has important
implications for the study and modeling of 5-HT2 receptors
and deserves further investigation.
In addition to K4.45, 5-HT2 receptors can be distinguished
by the lack of a highly conserved TM7 asparagine at position
7.45. This asparagine is part of a NSxxNP(7.50)xxY motif,
which is common to a majority of amine GPCRs, including
other 5-HT receptors, as well as muscarinic and gonadotropin
receptors (Konvicka et al. 1998; Lu et al. 2001; Angelova
et al. 2002; Prioleau et al. 2002; Visiers et al. 2002a).
Whereas the NP(7.50)xxY portion of this motif is also
conserved in the 5-HT2 sequences, the asparagine at 7.45 is
replaced with a smaller polar residue, typically a serine or, in
the case of 5-HT2C, a cysteine. Residues of this motif,
notably N7.49 and Y7.53, form intramolecular contacts with
neighboring helices that contribute both to the stability of the
inactive state and the transition to an active conformation
(Kristiansen et al. 2000; Sealfon et al. 1995; Rosendorff
et al. 2000; Prioleau et al. 2002; Visiers et al. 2002a).
Position 7.45 has not been tested in any of the 5-HT
receptors. However, in the glycoprotein and muscarinic
GPCRs the cognate N7.45 is required for receptor activation
and may contribute directly to the binding crevice (Angelova
et al. 2000, 2002; Lu et al. 2001). In this study, we have
found that the smaller polar residue of the 5-HT2 sequences
also plays an important role in GPCR activity. Ala and Asn
substitutions of C7.45 in the rat 5-HT2C receptor both
decreased efficacy by about 50%, suggesting that this residue
is required for full activation of the receptor. Interestingly, the
mutations had no apparent effect on basal activity, in contrast
to mutations of the neighboring NPxxY motif, which either
decreased or increased basal activity (Kristiansen et al. 2000;
Prioleau et al. 2002). Thus, C7.45 appears to be involved
mainly in the process of agonist-induced activation and does
not contribute to the stability of the inactive state. C7.45 may
facilitate the transition to an active conformation, in part, by
contributing to the binding of serotonin as an Ala mutation
decreased serotonin binding affinity about fourfold. Other
residues of the mid-TM7 helix, in particular F7.38 and Y7.43,
have also been implicated in both agonist binding and
activation (Roth et al. 1997). In addition, the effect of C7.45
may stem from its spatial proximity to W6.48 and F6.44 of the
TM6 aromatic cluster. The conserved aromatic residues of
TM6 have been widely implicated in GPCR activity (Visiers
et al. 2002a). Rhodopsin studies and several recent models
suggest that the indole ring of W6.48 shifts from a
perpendicular to a parallel plane when the receptor is
activated by an agonist (Lin and Sakmar 1996; Visiers et al.
2002a). This is thought to mediate a conformational switch
that serves to relay the signal from one end of the receptor to
the other (Visiers et al. 2002a). A possible explanation for the
results of the mutagenesis is that C7.45 is required to facilitate
this rearrangement of the neighboring tryptophan, either by
stabilizing a favorable conformation in the surrounding region
or by contributing directly to the conformational shift. Our
models do not show any intramolecular contacts that might
suggest a direct involvement in this process. However, there
may be new contacts formed following agonist activation that
help the transition to the active state.
An inspection of the C. elegans model suggests that the
orientation of S7.45 in this receptor may be different from
that of the cognate cysteine in 5-HT2C. The side chain
hydroxyl of S7.45 is turned upwards and is linked to the
peptide carbonyl of G7.42 by an intrahelical H-bond. As in
the case of 5-HT2C, an Ala substitution of 7.45 decreased
agonist efficacy by about 50% and therefore the serine is also
required for full activation of this receptor. On the other
hand, the asparagine mutagenesis had a more pronounced
effect on the C. elegans receptor than 5-HT2C, decreasing
agonist efficacy by as much as 65%. Replacing the native
serine with an asparagine in the C. elegans model removed
the additional intrahelical bond with G7.42 and changed the
orientation of H-bond-forming groups in the asparagine side
chain towards the interior of the helical bundle (not shown).
It is possible that the interaction between S7.45 and G7.42 is
needed to stabilize the active conformation of this receptor.
In addition, the bulkier side chain of N7.45 may hinder the
repositioning of the indole ring of W6.48, which could
explain the decrease in efficacy seen in both the rat C7.45N
and C. elegans S7.45N mutants.
One unexpected effect of the S7.45N mutation in the
C. elegans receptor was that the loss of efficacy was
associated with a significant increase in the affinity for
serotonin. S7.45 does not contribute to serotonin binding in
the C. elegans receptor as an alanine substitution produced
no significant change in binding affinity. In contrast, the
asparagine mutation increased binding affinity 25-fold and
also increased potency (i.e. decreased EC50) 15-fold, despite
the loss of agonist efficacy. A similar trend, although less
pronounced, was observed in the rat 5-HT2C receptor; the
asparagine substitution of C7.45 decreased Emax but
increased serotonin binding affinity approximately fourfold
and potency about twofold. There is general consensus that
the core of the binding pocket in the 5-HT receptors is
formed mainly by residues near the extracellular ends of
TM6, 3 and 5 (Almaula et al. 1996; Roth et al. 1997;
Shapiro et al. 2000; Ebersole et al. 2003). However, there
2004 International Society for Neurochemistry, J. Neurochem. (2005) 92, 375–387
386 J. Xie et al.
are additional interactions with neighboring helices, TM7 in
particular, that can influence the positioning of the ligand
within the pocket. Mutations of conserved TM7 aromatic
residues, F7.38 and Y7.43, increased the Ki for agonists
several fold (Roth et al. 1997), an indication that the
extracellular (upper) half of the helix may contribute directly
to the binding site. Our mutagenesis data suggest that 7.45
may also play a role in binding and, moreover, the type of
polar residue at 7.45 may influence the affinity for the natural
ligand. The smaller serine is not required for binding and
appears to be associated with a lower-affinity state. This may
be one of the reasons why some 5-HT2 receptors, notably
5-HT2A and this C. elegans receptor, have typically lower
affinities for serotonin. On the other hand, a cysteine and, to a
greater extent, an asparagine at 7.45 may reach further into
the receptor’s binding crevice and contribute interactions that
increase both affinity and potency. We postulate that the
naturally occurring N7.45 of most 5-HT receptors is an
important contributor to the serotonin binding site and is thus
worthy of further investigation.
In summary, this study has determined that type-specific
TM4 and 7 residues are required for activity of two
evolutionarily distant 5-HT2 receptors. That the mutagenesis
should produce similar effects in such different microenvironments reinforces the functional importance of these
residues for the entire 5-HT2 group. Our sequence alignments have identified a number of other residues that are
conserved across the 5-HT2 sequences and have been
tentatively designated as type specific. Examples include
I4.60, G5.42, T5.61, V6.40 and F6.41. Also noteworthy is
the absence of a highly conserved tyrosine (Y4.33) of most
5-HT GPCRs, which is replaced with a variable residue in
the 5-HT2 sequences. Some of these amino acids, for
example Y4.33 and T5.61, occur within predicted intracellular loop regions and thus may play a role in the interactions
between the receptor and its G protein. These residues are
currently under investigation in our laboratory.
Acknowledgement
This work was supported by a grant from the Canadian Institutes of
Health Research (CIHR) to PR.
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