Journal of Neurochemistry

Lippincott—Raven Publishers, Philadelphia

© 1996 International Society for Neurochemistry

Cloning, Characterization, and Chromosomal Localization of

a Human 5-HT6 Serotonin Receptor

*~RuthKohen, tMark A. Metcalf, lNaseem Khan, §Teresa Druck, §Kay Huebner,

“Jean E. Lachowicz, tHerbert Y. Meltzer, ‘David R. Sibley,
~Bryan L. Roth, and *~MarkW. Hamblin
* Department of Psychiatry and Behavioral Sciences, University of Washington; tGeriatric Research, Education, and
Clinical Center, Seattle Veterans Affairs Medical Center, Seattle, Washington; ~Departments of Biochemistry and
Psychiatry, Case Western Reserve University, Cleveland, Ohio; § Jefferson Cancer Institute, Jefferson
Medical College, Philadelphia, Pennsylvania; and National Institute of Neurological Disorders and Stroke,
National Institutes of Health, Bethesda, Maryland, U.S.A.

Abstract: We describe the cloning and characterization
of a human 5-HT6 serotonin receptor. The open reading
frame is interrupted by two introns in positions corre-
sponding to the third cytoplasmic loop and the third ex-
tracellular loop. The human 5-HT6 cDNA encodes a 440-
amino-acid polypeptide whose sequence diverges sig-
nificantly from that published for the rat 5-HT6 receptor.
Resequencing of the rat cDNA revealed a sequencing
error producing a frame shift within the open reading
frame. The human 5-HT6 amino acid sequence is 89%
similar to the corrected rat sequence. The recombinant
human 5-HT6 receptor is positively coupled to adenylyl
cyclase and has pharmacological properties similar to
the rat receptor with high affinity for several typical and
atypical antipsychotics, including clozapine. The receptor
is expressed in several human brain regions, most promi-
nently in the caudate nucleus. The gene for the receptor
maps to the human chromosome region 1 p35—p36. This
localization overlaps that established for the serotonin
5-HTlD~ receptor, suggesting that these may be closely
linked. Comparison of genomic and cDNA clones for the
human 5-HT6 receptor also reveals an Rsal restriction
fragment length polymorphism within the coding region.
Key Words: Serotonin receptors—5-HT6 receptors—G
protein-coupled receptors—Atypical antipsychotics—
Clozapine—Chromosomal localization.
J. Neurochem. 66, 47—56 (1996).
cumbens, and hippocampus. Moderate levels of ex-
pression are found in the olfactory bulb, anterior olfac-
tory nucleus, neocortex, cerebellum, amygdala, and
hypothalamus, but expression is weak in the thalamus
(Ward eta!., 1995). Although little is known regarding
its function, when expressed in mammalian cells, this
receptor shows high affinity for several therapeutically
important antidepressant and antipsychotic drugs, in
particular the atypical antipsychotic clozapine and re-
lated compounds (Monsma et al., 1993; Roth et al.,
1994).
Because of its potential interest to psychopharma-
cology (Schoeffter and Waeber, 1994) and the history
of profound species differences in drug affinities of 5-
HT receptors (Adham et al., 1992; Hamblin et al.,
1992a,b), we have now cloned and characterized the
human homologue. Its gene structure, pharmacology,
and tissue distribution are very similar to those of the
rat receptor. We resequenced part of the rat S-HT6
receptor clone, St-B 17, investigated by Monsma et al.
(1993) and are now reporting a corrected version of
the previously described sequence. We have mapped
the chromosomal location of the human 5-HT6 gene
to lp35—p36 and tentatively identified a silent RsaI
restriction fragment length polymorphism within the
coding region of the human gene.

Currently 14 different mammalian serotonin [5-hy-

droxytryptamine (5-HT)j receptors have been cloned,
all but one (5-HT3, a ligand-gated ion channel) of
them members of the 0 protein-coupled receptor su-
perfamily (Humphrey et al., 1993; Hoyer et al., 1994).
Among them, three (5-HT4, 5-HT6, and 5-HT7) stimu-
late adenylyl cyclase via G. coupling. The first of these
to be cloned, rat 5-HT6 (Monsma et al., 1993), is
widely expressed in rat brain, particularly in the olfac-
tory tubercle, islands of Cajella, striatum, nucleus ac-
Resubmitted manuscript received August 16, 1995; accepted Sep-
tember 5, 1995.
Address correspondence and reprint requests to Dr. M. W. Ham-
bun at Geriatric Research, Education, and Clinical Center (182B),
Seattle V.A. Medical Center, 1660 South Columbian Way, Seattle,
WA 98108, U.S.A.
Abbreviations used: cAMP, cyclic AMP; 5-HT, 5-hydroxytrypta-
mine or serotonin; LSD, lysergic acid diethylamide; 8-OH-DPAT,
8-hydroxy-2-(di-n-propylamino)tetralin; ORF, open reading frame;
TFMPP, trifluoromethylphenylpiperazine; UTR, untranslated region.

47
48 R. KOHEN ET AL.
EXPERIMENTAL PROCEDURES
cDNA library screening
We labeled an ~ I-kb BstXl to XmaI restriction fragment
32P]dCTP nick translation. The
of the rat 5-HT
probe was used6 gene
to screen
by [cr-
a commercially obtained human
caudate eDNA library in the vector ~gtl0 (Clontech) and a
commercially available human genomic DNA library in the
vector Lambda Fix II (Stratagene) with hybridization (6
x SSPE at 55°C; 1 x SSPE = 150 mM NaCl, 10 mM
NaH
2PO4, and 1 mM Na2 EDTA, pH 7.4) and washing (2
x SSPE at 55—60°C)at moderate stringency. The X phages
found to hybridize to the probe were subsequently plaque-
purified, subcloned into pBluescript KS (—), and sequenced
(see below). Other standard nucleic acid and bacteriological
manipulations were as described by Sambrook et al. (1989).
PCR amplification
PCR amplification from the human caudate eDNA library
described above was done in two steps, using nested primers.
First, a i-,uI aliquot of the library was amplified with 2.5
U of Pfu DNA polymerase (Stratagene) according to the
manufacturer’s directions. We used! ,uM each of oligonucle-
otide primers corresponding to bp 362—383 (5 ‘-GCCTGG-
ACCGCTACCTGCTCAT-3’) and the reverse complement
of bp 1,292-1,312(5 ‘-GGATGCCAAGTGGATGCGGCC-
3’) of the human 5-HT5 sequence. Thirty-three cycles at an
annealing temperature of 62°C yielded a 951 -bp product.
Five microliters of I % agarose gel containing the desired
fragment was then used in a second PCR procedure, per-
formed as above but using primers corresponding to bp 397—
416 (5 ‘-CGCTACAAGCTGCGCATGAC-3’) and the re-
verse complement of bp 1,251 —1,271 (5 ‘-TCGATGTTG-
AAGAAATTGACG-3’) and an annealing temperature of
45°C.The reaction products were purified and size-fraction-
ated on a 1% agarose gel and the identity of the desired 875-
bp PCR product was confirmed by restriction analysis. A
562-bp BamHI/PvuII restriction fragment of this PCR prod-
uct was cloned into pBluescript KS (—).
DNA sequencing
Nucleotide sequence analysis was performed using the
Sanger dideoxynucleotide chain-termination method on de-
natured double-stranded plasmid templates. We used Seque-
nase (U.S. Biochemicals) with the usual nucleotides as well
as 7-deaza-dGTP and the PCR-basedfinol DNA Sequencing
System (Promega), with Taq polymerase and 7-deaza-
dGTP. Primers were synthetic oligonucleotides that were
either vector specific or derived from previously determined
sequence information. The eDNA sequence was confirmed
through the sequencing of both strands. Nucleic acid se-
quence data manipulations and amino acid sequence compar-
isons were performed using the DNA analysis suite Laser-
Gene (DNAstar, Madison, WI, U.S.A.).
Genomic mapping
Hybrid cells used in mapping the position of the 5-HT6
gene (HTR6 locus) were either isolated as previously de-
scribed (Haluska et al., 1988; Huebner Ct al., 1991) or were
obtained from the NIGMS Human Genetic Mutant Cell Re-
pository (Coriell Institute, Camden, NJ, U.S.A.) and are de-
scribed in detail in the Coriell catalogue. High-molecular-
weight DNA was obtained from cell lysates by phenol /chlo-
roform extraction and used for Southern blotting and hybrid-
ization as described (Huebner et al., 1991). The probe used
for Southern analysis was the eDNA clone HBI4c, radiola-
J. Neurochem., Vol. 66, No. I, 1996
32P by random priming. High-stringency washing
beled
of the with
filters was performed with 0.1 >< saline— sodium citrate
at 65°Cbefore autoradiography.
Northern blot analysis
A commercially available northern blot (Human Brain
Multiple Tissue Northern Blot III; Clontech) containing 2
~ig of poly (A) + RNA per lane was hybridized to a full-
length 5-HT 32P by random priming.
Hybridization6 and
eDNAwashes werewith
labeled done according to the manu-
facturer’s protocol. Hybridization signals were visualized us-
ing a phosphoimager (model GS-363; Bio-Rad). The blot
was exposed to an imaging plate at room temperature for 4
days and then scanned by a Molecular Imager scanning unit.
Scan data were analyzed with the Molecular Analyst soft-
ware package. To confirm that similar amounts of intact
RNA were loaded in each gel lane, the blot was also hybrid-
ized with a 32P-labeled ~-actin eDNA.
Expression studies
The human 5-HT
6 eDNA was subcloned into the XbaI/
Sad restriction sites of the eukaryotic expression vector
pSRa (Takebe et al., 1988). Preliminary studies were done
with 5-HT6 subcloned into the XbaIIXhoI sites of the vector
pSVK3 (Pharmacia). For transient expression of the 5-HT6
receptor, COS-7 cells were transfected using the DEAE-
dextran method described by Cullen (1987). For stable ex-
pression HeLa cells were cotransfected by electroporation
with 5-HT5-pSRa and pRC/CMVneo (Invitrogen). Stable
HeLa transfects were selected in the presence of G4 1 8 (ge-
neticin; GIBCO) at 600 ~sg/ml. We selected one transfected
HeLa cell line, expressing =3,400 fmol/mg of protein of 5-
HT6 receptor, for cyclic AMP (cAMP) analysis. All cells
were grown at 37°Cunder 5% CO2 in Dulbecco’s modified
Eagle’s medium with 10% fetal calf serum supplemented
with penicillin G (100 ~tg/ml) and streptomycin (100 UI
ml). At least 24 h before any functional assays medium
was replaced with medium containing dialyzed rather than
regular serum, because most commercial serum has high
(often >1 mM) concentrations of 5-HT.
Radioligand binding and cAMP assays
At 48—72 h after transient transfection COS-7 cells were
harvested by scraping into ice-cold Dulbecco’s phosphate-
buffered saline. The cells were then collected by centrifuga-
tion at 30,000 g and resuspended by Polytron homogen-
ization in ice-cold 2 x TME buffer [1 x TME = 50 mM
TrisHCl (pH 7.4 at 37°C), 10 mM MgSO4, and 0.5 mM
EDTA]. The crude membranes were again collected by cen-
trifugation and resuspended in ice-cold 2 x TME. For satura-
tion radioligand binding experiments, 0.25 ml of membrane
suspension was incubated in a final3H]5-HT
volume (New
of 0.5 England
ml with
increasing22.8
Nuclear; concentrations
Cilmmol; final
of either
concentration,
[ 1—60 nM) or
[3H]lysergic acid diethylamide ([3H]LSD; New England
Nuclear; 64.3 Ci/mmol; final concentration, 0.1—10 nM).
For competition binding experiments, 0.25 ml of membrane
suspension was incubated with 0.15 ml of competing drugs
in increasing concentration and 0.1 ml of 7.5 nM [3H]LSD
(final concentration, 1.5 nM). Drugs and radioligands had
all been prepared in 2 mM ascorbic acid to prevent oxidation.
Assays were done in triplicate, and mixtures were incubated
at 37°C for 30 mm in the case of [3H15-HT (saturation
binding experiments) and for 90 mm with [3H]LSD (satura-
tion and competition binding experiments). Binding assays
 CLONING OF A HUMAN 5-HT
were terminated by rapid filtration through Whatman GF/C
filters that had been pretreated with 0.5% polyethylenimine.
The filters were then rapidly washed with an additional 10
ml of ice-cold 50 mM Tris HC1 (pH 7.5), and the radioac-
tivity was quantified by liquid scintillation counting. Non-
specific binding was determined in the presence of 5 ~tM
methiothepin for saturation binding experiments (20—90%
of total binding over the range of concentrations used) or
by curve fitting for competition data (65—85% at the radioli-
gand KD). Protein content was measured by the method of
Bradford (1976), using bovine y-globulin as the standard.
The cAMP accumulation assays were performed as de-
scribed by Hamblin eta!. (1992a). Data were analyzed using
nonlinear regression analysis as in the analysis program
Prizm (GraphPad Software).
Materials
Compounds were purchased or obtained as gifts as fol-
lows: trifluoromethyiphenylpiperazine (TFMPP; Aldrich);
metergoline (Farmitalia); 5-carboxamidotryptamine and su-
matriptan (Glaxo); methiothepin (Hoffmann-La Roche);
ritanserin and ketanserin (Janssen Pharmaceutica); cypro-
heptadine and amitryptiline (Merck); quipazine (Miles);
5-methoxytryptamine (Regis); 8-hydroxy-2- ( di-n -propyl-
amino)tetralin (8-OH-DPAT), amoxapine, and mianserin
(Research Biochemicals); RU24969 (Roussel-Uclaf); bro-
mocryptine, clozapine, mesulergine, and methysergide (San-
doz); and 5-HT and N,N-dimethyltryptamine (Sigma). The
sources of other drugs used in this study were previously
specified (Roth et al., 1992, 1994).
RESULTS
Cloning of the human 5-HT6 receptor
In screening a human caudate eDNA library we
identified four positive clones with high sequence ho-
mology to the rat 5-HT6 receptor. Comparison with
the rat sequence suggested that if these encoded the
human homologue, alignment could be made as in Fig.
I. These were clone HB 16a, containing ‘—S 1 .4 kb of 5’
untranslated region (UTR) and the first 204 bp of the
presumed receptor open reading frame (ORF); clone
HC21a, a 1.3-kb fragment starting at bp 135 of the
ORF and containing part of an apparent unspliced in-
tron beginning at bp 715; and the identical clones
HB9a and HB 14c, extending from what later proved
to be bp 890 in the ORF through the 3’ end of the
coding region and containing ‘-.~ 1.4 kb of 3’ UTR.
Missing among these fragments was the piece ex-
tending from bp 715 to 889 and spanning at least one
intron. In search of the missing fragment we then
screened a human genomic library and obtained three
positive clones, among which MT IA I, containing the
entire 5-HT6 gene, was selected for further investiga-
tion. A 3,000-bp Sad fragment, l,100-bp SacIINotI
fragment, and an 800-hp BamHIINotl fragment of
MT IA I were subcloned into pBluescript KS (—) and
sequenced across the portion missing. Sequencing re-
vealed the presence of two putative introns of 1 .8 kb
and 191 bp located at bp 714 and 873, respectively,
separated by a 159-bp exon (Fig. 1). The donor and
acceptor sequences at the presumed splice junctions
6 RECEPTOR 49
(Table 1) conform to the consensus sequences de-
scribed (Mount, 1982). These exon/intron boundaries
correspond to those previously proposed for the rat
gene (Monsma et a!., 1993; Ruat et al., 1993). To
confirm that the 5-HT6 gene is spliced as presumed
and to obtain a eDNA fragment that could be used in
the assembly of a 5-HT~,expression construct, we used
PCR to amplify across the eDNA gap with the caudate
eDNA library as the template. A 562-bp BamHIIPvuII
restriction fragment of the resulting PCR product was
used in the assembly of our 5-HT6 eDNA. We first
joined together HB16a and HC2Ia at the l72-bp Avill
site and then connected this piece to two Sf1 fragments
originating from our PCR fragment (PCR-SJil/frag-
ment I from bp 694 to 793 and PCR-SfiI/fragment 2
from bp 793 to 1,042), which were joined downstream
to HB9a. The correct assembly of this product was
confirmed by sequence analysis. Comparison of the
genomic clones and our PCR product confirmed that
there were no PCR-introduced base changes (see
Fig. I).
The ORF of the human 5-HT6 eDNA is 1,320 bp
long, encoding a 440-amino-acid polypeptide with a
molecular size of 46.96 kDa. We partially sequenced
the first (1.8-kb) intron occurring after bp 714 and
completely sequenced the second 191 -bp long intron
following bp 873 (see Table 1). The length of the first
intron was determined by restriction analysis (data not
shown) and PCR. Comparison of eDNA and genomie
sequences tentatively identified a silent RsaI polymor-
phism at bp 267 (tyrosine in position 89; Figs. I and
2), which may be informative in linkage studies exam-
ining the role of this receptor in various human dis-
eases. Hydropathy analysis of the deduced amino acid
sequence indicates the presence of seven hydrophobic
regions (data not shown), predicted to represent puta-
tive transmembrane domains. The human 5-HT,~,also
contains one potential N-linked glycosylation site at
Asn ‘° in the presumed extracellular amino-terminus.
Several threonines and serines are present in the pre-
sumed third cytoplasmic loop and the intracellular ear-
boxyl-terminal tail, representing potential phosphory-
lation sites.
Comparison of human and rat 5-HT6 receptor
sequences
Comparing our human 5-HT6 eDNA sequence with
the published rat sequences (Monsma et al., 1993; Ruat
et al., 1993) we noticed an apparent frame shift be-
tween the rat and human receptor sequences at bp
1,034. We therefore resequenced the rat clone St-B 17
used by Monsma et a!. (1993), using a 19-bp oligonu-
cleotide primer corresponding to bp 902—920 of the
human and rat gene (5 ‘-TCTTCGATGTCCTCA-
CATG-3’), which are identical over this stretch.
We discovered a probable frame shift error in the
sequence from bp 1,030 to 1,040 described by both
Monsma et al. (1993) and Ruat et a!. (1993) as 5’-
CACCGGCCAG-3’. Our sequence for the correspond-
J. Neurochem., Vol. 66, No. 1, 1996
50 R. KOHEN ET AL.

FIG. 1. Comparison of the nucleic

acid sequences of human (top line)
and rat (bottom line) 5-HT
6 cDNA se-
quences. Only the ORF5 are shown.
The rat sequence is that of the clone
ST-B17 by Monsma et al. (1993)
with our corrections: (1) a probable
error leading to a frame shift at bp
1,034 (marked with an asterisk); (2)
a probable error in the sequence of
Monsma et al. (1993) conforming
with the sequence reported by Ruat
et al. (1993) (underlined with a bar);
and (3) a possible silent Smal poly-
morphism in the rat gene (circled). In
the human sequence, the silent Rsal
polymorphism 89, at isbphighlighted by
276, corre-
asponding
box. Thetoarrows
Tyr mark intron/exon
splice junctions in both the rat and
human 5-HT
6 genes. Human and rat
sequences have been deposited
with GenBank, having accession
nos. L41147 and [41146, respec-
tively.

ing stretch of the rat gene is 5 ‘-CACCGGGCCAG-3’.
The additional guanine base we found (marked with
an asterisk in Fig. 1) realigns the reading frames for
the rat and human receptors.
In a second location, at bp 1,110—1,111, the se-
quences published by the aforementioned authors dif-
fer: By resequeneing St-B 17, we confirmed the se-
quence of Ruat et a!. (1993) from bp 1,105 to bp
1,115, reading 5 ‘-GTGCTGCCTCT-3’ as opposed to
the sequence by Monsma et a!. (1993), reading 5’-
GTGCT~CTCT-3’ (underlined with a bar in Fig.
1). Both probable errors occurred in G/C-rich areas

TABLE 1. Exon—intron organization of the human 5-HT6 receptor gene ORF

Sequenee of exon —intron junction

Intron 5’ boundary Intron (total length in bp) 3’ boundary

gtacctgggg . . . (—1.800) . . . ctttccgcag GTG CCC AGG
A ACG CTG CAd 238 Va123°
B ATA GTC Gin
CAG gtaatgccac . . . (191) . . . ctccccccag GCC GTG TGC
GIn29’ Ala292

The exon—intron boundaries are shown for those junctions within the ORF. The exon between these two introns
is 159 bp long. Possible additional introns in the 5’ and 3’ UTRs have not been determined. These junctions are
in good agreement with the consensus sequences described by Mount (1982).

J. Neurochem., Vol. 66, No. 1, 1996

 CLONING OF A HUMAN 5-HTO RECEPTOR
FIG. 2. Deduced amino acid sequence and structure of the human 5-HT
the two intronssite
glycosyiation areismarked
marked.with arrows. with an asterisk is the site of a possible silent Rsal polymorphism (Tyr
Highlighted
of the eDNA marked by severe compressions and
strong stops in sequencing reactions using Sequenase
and native nucleotide triphosphates. These problems
could be only partially alleviated by using 7-deaza-
dGTP with Sequenase. However, using the thermosta-
ble Taq po!ymerase in conjunction with 7-deaza-dGTP
(see Experimental Procedures) markedly reduced
these problems, yielding unambiguous sequence data.
In a third location, at bp 1,209 (threonine in position
403), the sequences of St-B 17 and the rat 5-HT
6 eDNA
of Ruat et al. (1993) apparently diverge: These authors
reported the sequence from bp 1,205 to 1,215 as 5’-
CcACçCGGGAC-3’ with an Smal site at bp 1,210.
Monsma et a!. (1993) reported the corresponding St-
B17 sequence as 5 ‘-CCACACGGGAC-3’, and our re-
striction analysis of St-B17 (data not shown) con-
firmed the absence of an Smal site at bp 1,210. This
discrepancy between the two rat 5-HT6 cDNAs may
represent a silent Smal polymorphism in the rat gene
(circled in Fig. I).
The corrected rat 5-HT6 sequence has an ORF of
1,314 bp, encoding a 438-amino-acid protein with a
5’
89). The positions of
6 receptor. An asparagine residue representing a putative
molecular mass of 46.81 kDa. The nucleic acid se-
quence of the human 5-HT6 gene is 85% identical
to the corrected sequence of its rat homologue. The
predicted polypeptides of the human and rat receptors
are 89% identical and 95% similar with conservative
substitutions.
The pharmacological characteristics of human
and rat 5-HT6 are similar
Transient transfection of COS-7 cells with pSRct-5-
3H I 5-HT
HT6 resulted in the appearance of a single classand
of
high-affinity,
the saturable
serotonergic ligand binding
[3H]LSDsites for 3).
(Fig. [ No specific
binding was observed in nontransfected COS-7 cells
(Hamblin and Metcalf, 199!). Analysis of the [3HI5-
HT saturation binding data revealed a KD of 32.2 nM
(PKD 7.49 ±0.45, six experiments) and Bma. values
of 0.1—1.7 pmol/mg of protein. Analysis of the [3H]-
LSD binding data showed a KD of 3.16 nM (pK
0
= 8.50 ±0.02, three experiments) and Bma. values of
0.5—1.6 pmo!/mg of protein. Further characterization
of the human 5-HT6 pharmacology used various drugs
.1. Neurochem., Vol. 66, No. 1, 1996
52 R. KOHEN ET AL.
that exhibit specificity for various serotonergic recep-
tor subtypes and other binding sites as well as an array
of typical and atypical antipsychotic agents. The aver-
age K~values are listed in 3HILSD
Table 2, with representative
binding shown in
competition
Fig. 4. curves for [
The human 5-HT
6 receptor is positively coupled
to adenylyl cyclase
5-HT caused a 2.5- to eightfold dose-dependent in-
crease in cAMP levels in transfected HeLa cells, with
an average EC50 value of 3.2 nM (pEC50 = 8.49
± 0.55, three experiments). There was no detectable
response in nontransfeeted cells (data not shown). The
5-HT6 receptor-mediated stimulation of cAMP accu-
mulation could be surmountably antagonized by do-
zapine (Fig. 5).
The 5-HT6 receptor is expressed in several human
brain regions
We investigated the anatomical distribution of 5-
HT6 mRNA expression in human brain by northern
blot analysis (Fig. 6). The 5-HT6 eDNA probe hybrid-
ized to two mRNAs of -—4.0 and 3.2 kb in length,
predominantly in the caudate nucleus, with lower con-
centrations in hippocampus and amygdala. Very low
levels of expression could be detected in the thalamus,
subtha!amic nucleus, and substantia nigra.
The human 5-HT6 gene maps to chromosome
region lp35—p36
In Southern analysis. our 5-HT6 eDNA probe de-
tected a human genomic EcoRI fragment, containing
the HTR6 locus, of ‘-—6.5 kb and did not cross-hybridize
well with any rodent bands. Twenty rodent—human
hybrid DNAs, each carrying a small subset of human
chromosomes so that most chromosome regions were
represented in the panel, were tested for the presence
of this HTR6-specifie EcoRI fragment by filter hybrid-
ization to the radio!abeled eDNA probe. Hybrids car-
J. Neurochem., Vol. 66, No. 1, 1996
FIG. 3. Radioligand binding analysis of human 5-
HT
6 transiently expressed in COS-7 cells. Tran-
sient expression and radioligand binding assays
were performed as described in Experimental3H]5- Pro-
HT binding
cedures. A: to membranes
Saturation prepared
analysis from COS-7
of specific [
cells transiently transfected with construct pSRa-
5-HT
5. The experiment shown is representative of
six independent experiments, each conducted in
triplicate. Inset: Scatchard analysis of saturation
binding data. In this experiment, the KD and ~
values were 27.9 nM and 1.2 pmol/mg of protein,
3H]LSDbinding
respectively. to membranes
B: Saturation prepared
analysis from
of specific
COS-7
[ cells transiently transfected with construct
pSRa-5-HT
6. The experiment shown is represen-
tative of three experiments conducted in triplicate.
Inset: Scatchard analysis of saturation binding
data. In this experiment, the K0 and Bne, values
were 3.1 nM and 0.47 pmol/mg ofprotein, respec-
tively.
tying human chromosome region lp showed hybrid-
ization, whereas those without did not, as depicted in
Fig. 7A. A chromosome 1 mapping panel in which
each hybrid DNA carried a portion of chromosome I
was similarly tested for presence of the human HTR6
locus after EcoRI cleavage 35 —as1 p36
shown in Fig.of 7B.
portion Only
chromo-
hybrids1 contained
some retaining the 1p
the 5-HT(, gene.
DISCUSSION
We report the cloning and characterization of a hu-
man 5-HT
6 receptor with typical homology to the pre-
viously described 5-HT8, rat receptor (Monsma et al.,
1993; Ruat et al., 1993). The two receptors differ
mostly in their earboxyl-terminal region, with the hu-
man receptor being two amino acids longer than its rat
homologue (440 vs. 438 amino acids).
We also report a corrected sequence for the rat 5-
HT6 receptor. Noticing an apparent frame shift be-
tween the nucleotide sequences of the human 5-HT9
receptor and the rat sequences as previously described
(Monsma et al., 1993; Ruat et al., 1993) we rese-
quenced both our rat and human 5-HT6 clones in paral-
Id, using the same rat receptor, St-B 17, that was de-
scribed by Monsma et al. (1993). For this as for part
of our sequencing of the human 5-HT6 receptor, we
used a dideoxynucleotide chain termination method
with PCR and Taq polymerase. Before this, we had
used another dideoxynucleotide chain termination
method with Sequenase, which is the same method
that was originally used by Monsma et al. (1993) to
sequence St-B17. Comparing these two sequencing
methods we found the PCR-based method to be less
vulnerable to compressions and strong stops, thus
yielding better sequence data for the very G/C-rich 5-
HT6 clones.
The coding regions of both rat and human receptors
contain two introns in homologous positions. The first
 CLONING OF A HUMAN 5-HT
5 RECEPTOR 53

TABLE 2. Drug affinities for the human 5-HT5 receptor

Drug pK, K, (nM)

“Serotonergie” agents
Methiothepin 9.38 ±0.05 0.42
Bromocryptine 7.49 ± 0.09 33
5-Methoxytryptamine 7.37 ±0.05 43
Amoxapine 7.31 ±0.01 50
Ritanserin 7.27 ± 0.19 53
Mianserin 7.26 ±0.16 55
Amitryptiline 7.19 ±0.07 65
5-HT 7.19 ±0.10 65
N,N-Dimethyltryptamine 7.17 ±0.17 68
Cyproheptadine 6.82 ±0.12 iso
Methysergide 6.75 ±0.07 180
Metergoline 6.40 ± 0.08 400
TFMPP 6.37 ±0.06 430
RU24969 6.24 ± 0.04 570
5-Carboxamidotryptamine 6.14 ±0.04 720 3H]LSDbinding to COS-7
Sumatriptan 5.59 ± 0.16 2,600 cell
FIG. membranes
4. Pharmacological
expressinganalysis
5-HT of [
Ketanserin 5.55 ±0.03 2,800
Quipazine 5.44 ±0.05 3,600
5. Competition curves shown
Mesulergine 5.42 ±0.01 3,800 are representative of three independent experiments conducted
8-OH-DPAT <5.0 >10,000 in triplicate. Mean K, values are given in Table 2. 5-CT, 5-carbox-
“Antipsychotic” agents amidotryptamine.
“Atypical” drugs
(—)-Octoclothepin 8.75 ±0.04 1.8
Clozapine 8.02 ±0.15 9.5
Olanzapine 8.00 ±0.02 10 sponds to the location of introns in the 5-HT,A, 5-
1C1169369 7.96 ±0.03 11 HT~ (Matthes et al., 1993), dopamine D2 (Selbie et
Rilapine 7.77 ±0.13 17
Fluperlapine 7.54 ±0.08 29 a!., 1989; Monsma et al., 1989; Chio et al., 1990), and
Seroquel 7.48 ±0.17 33 D3 receptors (Sokoloff et al., 1990). We have excluded
Perlapine 6.82 150 the presence of other introns in the ORF.
Tenilapine 6.24 ±0.07 570 Using hybridization of a 5-HT6 probe to somatic
Tiospyrone 6.02 ±0.16 950
Amperozide 5.80 ±0.03 1,600 cell hybrid panels, we localized the HTR6 gene to
Risperidone 5.62 ±0.31 2,400 lp35—p36. The 5-HTID,. receptor gene has previously
1p34.3—
“Typical” drugs been(Libert
p36 mappedet to
a!.,human chromosome
1991) and region
the syntenic mouse chro-
(+)-Octoclothepin 8.59 ±0.16 2.6 mosome 4 (Wilkie et al., 1993). This predicts a likely
Chlorpromazine 7.72 ±0.18 19
Loxapine 7.57 ±0.02 27 location of the murine 5-HT
Fluphenazine 7.42 ±0.08 38 6 gene on chromosome 4
Trifluperazine 6.77 ±0.01 170
Thiothixine 6.50 ±0.01 320
Mesoridazine 6.42 380
Haloperidol 5.18 ±0.08 6,600
3H]LSD binding to washed cell
membranes from COS-7
Drug competitions forcells
1.5 transiently
nM [ transfected with the human
5-HT
6 receptor were performed as described in Experimental Proce-
dures. Data are mean ± SD pK, values determined by nonlinear
regression analysis and represent three experiments (two experiments
in the ease of perlapine and mesoridazine). Mean K, values were
determined from mean pK, values.

of these, described238
in rat by human
in the Ruat et receptor)
a!. (1993),and
occurs
lies
after bpthe
within 714 (G!n encoding the presumed third cyto-
sequence
plasmic loop, corresponding to a position 30 amino
acids downstream from the carboxyl-terminal end of FIG. 5. 5-HT-induced stimulation of cAMP accumulation in
the fifth presumed transmembrane region. The second HeLa cells stably expressing the human 5-HT
intron following bp 873 (GIn29’ in the human recep- 5 receptor and its
tor), proposed for the rat by Monsma et al. (1993), inhibition by clozapine. Shown is one representative experiment
of two, each performed in triplicate. The EC50 values of 5-HT
lies within the sequence encoding the putative third were 1.8 nM in the absence (.) and 53 nM in the presence (0)
extracellular loop. The position of the first intron corre- of 1 ,jM clozapine, leading to an apparent K, value of 35 nM for
the antagonist.

J. Neurochem., Vol. 66, No. 1, 1996

54 R. KOHEN ET AL.

FIG. 6. Northern blot analysis
of 5-HT
6 transcripts in human
brain. The northern blot was
probed and analyzed as de-
scribed in Experimental Pro-
cedures. The locations of
RNA size markers (kb) are in-
dicated. The blot was hybrid-
ized with a human ~3-actin
eDNA probe to check levels
of RNA between lanes (small
panel below the blot).
(Abbott et al., 1993) and indicates that the 5-HT6 and
the 5~HTi0uloci may be closely linked.
One difference was found between the sequences of
the 5-HT6 eDNA and exons of the human genomid
clone where a silent base exchange in position 267
produces an RsaI site in the eDNA, but not in the
genomic clone. The eDNA and genomic clones were
presumably derived from different individuals. This
restriction fragment length polymorphism may prove
to be useful in linkage studies, although we did not
obtain any information regarding its frequency.
The pattern of 5-HT9 mRNA expression in human
brain we observed by northern blot analysis parallels
the distribution previously found for the rat 5-HT6 re-
ceptor. We saw the highest level of expression in the
caudate nucleus, as has been described in rat by Mon-
sma et al. (1993) and Ruat et al. (1993). Ward et al.
(1995) also discovered a very high level of rat striatal
5-HT9 mRNA expression by in situ hybridization his-
tochemistry. They saw an equally high level of expres-
sion in the hippocampus, whereas we found a much
lower concentration in this area. Northern analysis and
in situ hybridization histochemistry by Ruat et al.
(1993) showed moderate expression in rat hippocam-
pus. We detected low levels of expression in the amyg-
dala, as described in the rat by Monsma et al. (1993)
and in the tha!amus, as found in rat by Ward et al.
(1995). We found two transcripts of ‘=4.0 and 3.2kb

FIG. 7. Localization of the 5-HT6 receptor gene (HTR6 locus) to chromosome lp35—p36. A: Presence of the HTR6 locus in a panel
of 20 rodent—human hybrids. Stippling indicates that a hybrid contains the chromosome indicated in the upper row. Partial stippling
indicates the presence ofa chromosome fragment derived from the short (upper left) or long (lower right) arms. The pattern of retention
of the HTR6 locus in the panel is shown to the right where the presence of the locus in a hybrid is indicated by a stippled box with a
plus sign and the absence of the locus is indicated by an open box enclosing a minus sign. The hybridization pattern for the human
5-HT6 probe is consistent with localization to the short arm of chromosome 1. B: Regional localization of the HTR6 locus on ip. The
portion of chromosome 1 present in specific hybrids is represented by the solid lines to the right of the chromosome 1 ideogram.
These hybrids were tested for the presence of the HTR6 gene by filter hybridization, and the presence or absence of the HTR6 gene
is indicated below the lines representing specific hybrids. The HTR6 gene is present only in hybrids that retain 1 p35—p36.

J. Neurochem., Vol. 66. No. I, 1996

 CLONING OF A HUMAN 5-HTO RECEPTOR
in length, which has similarly been described for the
rat by Ruat et al. (1993), who found two mRNAs of
4.! and 3.2 kb.
We confirmed that the human 5-HT
6 receptor is pos-
itively coupled to adenylyl cyclase in transfected mam-
malian cells, as was previously reported for the rat 5-
HT6 by Monsma eta!. (1993) and Ruat et al. (1993).
5-HT caused a 2.5- to eightfold dose-dependent in-
crease in cAMP levels that could be surmountably an-
tagonized by c!ozapine. The observed 5-HT EC50 is
lower than the K, as determined in binding experi-
ments, as has been observed for several other receptors
when expressed at high levels (Adham et a!. 1993).
In receptor binding studies, the human 5-HT6 shows
drug affinities similar to its rat homologue, with the
exception of two of the tested compounds, methio-
thepin (human 5-HT6 K~= 0.42 nM vs. rat K, = 1.84
nM) and metergo!ine (human 5-HT6 K = 400 nM vs.
rat K = 30 nM). The receptor displays a rank order
of potency of methiothepin > elozapine olanzapine
> ritanserin mianserin ~ risperidone ketanserin.
Among putative agonists, the rank order is 5-methoxy-
tryptamine 5-HT N,N-dimethy!tryptamine
> TFMPP RU24969 5-carboxamidotryptamine
> sumatriptan 2~8-OH-DPAT.
Like the rat receptor, human 5-HT6 shows high af-
finity for the atypical antipsychotic clozapine. About
40% of elozapine binding sites in rat brain pharmaco-
logically resemble the 5-HT6 receptor (G!att et al.,
1995). The high affinity of the human 5-HT6 receptor
for clozapine relative to D2 and 5-HT2A affinities im-
plies that 5-HT6 receptors would be occupied at clini-
cally relevant doses, as 20% of D2 and >80% of 5-
HT2A receptors are occupied under those conditions
(Nordstroem et al., 1993).
The mechanism(s) by which atypical antipsychotic
drugs exert their beneficial effects remain unknown. It
is clear, though, that different mechanisms may con-
tribute to atypicality. Drugs such as sulpiride and ami-
sulpiride may be atypical because of selectivity for a
subgroup of D2 receptors. A group of drugs of which
clozapine is the prototype may be atypical because of
their high affinity for 5-HT2A relative to their affinity
for D2 receptors (Meltzer et al., 1989). Many of these
compounds have been shown to produce low levels
of extrapyramidal symptoms in preelinical models or
human studies, and for some of those, e.g., olanzapine
and zotepine, comparable affinities for 5-HT2A and 5-
HT6 receptors have been demonstrated (Roth et al.,
1994; Bymaster et a!., 1995). This contrasts with an
atypical antipsychotic drug such as risperidone, which
is 1,000-fold weaker at the 5-HT6 than at the 5-HT2A
receptor and which does possess substantial ability to
induce extrapyramida! symptoms at high doses. It is
noteworthy that those atypical antipsychotic drugs that
in humans produce the fewest extrapyramidal symp-
toms (clozapine, olanzapine, and fiuperlapine) possess
high affinity for the human 5-HT6 receptor. Several
typical antipsychotic drugs, such as chlorpromazine,
55
loxapine, and fiuphenazine, had moderate affinities for
the human 5-HT6 receptor as well. These compounds,
however, have much higher D2 affinities (Roth et a!.,
1994). Occupancy of 5-HT6 receptors may thus very
well contribute to clinically significant differences be-
tween typical and atypical antipsychotie drugs and
within the group of atypical antipsychotics, especially
if further studies reveal interactions among D2-, 5-
HT2A-, and 5-HT6-mediated effects on neuronal excit-
ability.
Acknowledgment: This work was supported by a Geriat-
ric Psychiatry Fellowship (to R.K.) and the Merit Review
program (to M.W.H.) of the Department of Veterans Affairs
as well as grants CA51083 (to T.D. and K.H.), MH41649
and MH41684 (to H.Y.M. and B.L.R.), and GM52213 (to
N.K. and B.L.R.) from the National Institutes of Health. We
thank Doris Heidmann and James Leverenz for their help
with experiments and Frederick J. Monsma for helpful dis-
cussions.
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