Note: Descriptions are shown in the official language in which they were submitted.
0050/43234 213~0~
~ . .. . .
Serotonin receptor, ~reparation and use thereof
The invention relates to a novel serotonin receptor and to the
5 use thereof, and to methods for discove~ing functional ligands
for this receptor. .~
The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) plays
an important part in a large number of physiological functions,
10 in respect of cognitive abilities or else in the area of behav-
ior. Disturbances of serotonergic stimulus transmission are in- `~
volved in numerous pathological states such as depression,
migraine, high blood pressure or bulimia. Serotonin displays its `-~
physiological and pathophysiological effect via receptors with
15 differing affinity for serotonin binding. These receptors can be
divided into 4 classes: 5-HTl, 5-HT2, 5-HT3, S-HT4. This division ~
reflects both differences in receptor coupling and differences in -
receptor binding profiles for a number of 5-HT receptor ligands.
In rodents; at least 4 subtypes of the 5-HTl receptor subclass
20 have been~described (5-HTlA to 5-HTlD). A11 5-HT receptors have
high~affinity for serotonin ~Ki ~ 100 nm) and are coupled via G
prot~eins`~to~adenylate cyalase or phospholipase C.
The primary~structure of 5 serotonin~receptors, 5-HTlA, 5-HT~C, j~2
2~5 ~5-HTlD_}ike,~5-HT2~and 5-HT3,`has been elucidated to date. There
ha~s~been~merely pharmacological `tbinding data) or functional ~.
sign~a~ trans~duction second messenger) description of the other
subt~ype~
30 A~5-HT1B~receptor has to date been found only in rodents, and
S-HT1B~roceptors have~not to date been detected in humans by
elther~pharmacological~or molecuLar biological methods. -`
To d~àte thère~are no antagonists whi~ch are seleative/specific for
;3~5~the~5-HT~receptor subclass. All the known antagonists bind with
high affinity to at least one other class of neurotransmitter
receptors. In the~particular case of the 5-HTlB receptor, virt-
`- ually no slubtype-specifici/~s~bstances are knownfso that to date it
~- has also been possible to say little about the physiological im-
~ ;40 portance of this receptor.
=- ~ It i9 an object of the present invention to elucidate the molec-
ular~structure of a human 5-HTlB receptor and provide processes
for preparing it in high purity. Another object is to provide
45;methods for discovering specific functional ligands for this re-
ceptor.
: ,
~,
~ - ,
0050/43234
3 ~ 9 0 ;~ `
i.. . ................................................................ . .
We have found that this object is achieved by a novel human sero-
tonin receptor of class 1 (5-HTlB) (SEQ ID NO 3)r as well as DNA
sequences which code for such receptors. A cDNA which codes for a
protein according to the invention is depicted in SEQ ID NO 2.
Other suitable DNA sequences are those which, although they have
a nucleotide sequence different from that listed in SEQ ID NO 2,
code for the polypeptide chain listed in SEQ ID NO 3, or parts
thereof, as a consequence of the degeneracy of the genetic code.
10 Also suitable are those DNA sequences which code for 5-HTl~ recep-
tors and which hybridize under standard conditions with the
nucleotide sequence depicted in SEQ ID NO 2 or with a nucleotide
sequence which codes for the protein depicted in SEQ ID NO 3. The
experimental conditions for DNA hybridization are described in
15 textbooks of genetic engineering, for example in Maniatis et al.,
Molecular Cloning, Cold Spring Harbor Laboratory, 1989.
Standard conditions mean, for example, temperatures- of from 42 to
58 C in an aqueous buffer solution with a concentration of from
20 0.1 to l x SSC (1 x SSC: 0.15 M NaCl, 15 mM sodium citrate pH
7.2).
' ~ ";
We have also found genetic engineering processes for preparing
this receptor. We have additionally found that the DNA sequences
25 coding-for this receptor can be used to discover functional
~ ~ ligands for this receptor. The invention furthermore relates to
-~; methods for identifying functional ligands for 5-HTlB receptors,
which~comprise using a DNA sequence which codes for a 5-HTlB re-
ceptor to transfect cells, isolating the membranes of these
30 cells, and carrying out conventional receptor binding experiments
with these membranes. Another method according to the invention
for identifying functional ligands for 5-HTlB receptors comprises
~ using a DNA sequence which codes for a 5-HTlB receptor to trans-
-- fect cells, and detecting by a reporter system the change in the
35 second messenger level caused in these cells by binding of the
ligand to the receptor.
The novel polypeptide~s and DNAs can be prepared using known
methods of genetic engineering. Thus, mRNA can be isolated from
40 brain tissue and converted into double-stranded cDNA. This cDNA
- can be used as template for the polymerase chain reaction. It is
thus possible by using specific primers under suitable reaction
conditions to amplify the corresponding cDNA. It is possible by
using suitable primers to sequence the amplified cDNA without
45 previous cloning. The methods used for this are described, for
example, in Current Protocols in Molecular Biology (edited by
F.M. Ausubel et al.) 1989, ISBN 0-471 50338-x (Vol. 1 and 2 set),
0050/43~34 ~ ~ 3 ~
~.. .. . .
and for the polymerasP chain reaction in Saiki et al., Science
230 (1985) 1350-54 and Mullis and Faloona, Meth. Enzymol. 155
(1987) 335-350.
. .
5 The cDNA characterized in this way can easily be obtained using
restriction enzymes. The fragments produced in this way can, -~
where appropriate in conjunction with chemically synthesized
oligonucleotides, adaptors or gene fragments, be used to clone
the sequences coding for the protein. The incorporation of the
10 gene fragments or synthetic DNA sequences into cloning vectors,
eg. the commercial plasmid Ml3mpl8 or Bluescript, is carried out
in a conventional way. The genes or gene fragments can also be
provided with suitable control regions which have~been chemically
synthesized or isolated from bacteria, phages, eukaryotic cells
15 or their viruses and which make the expression of the proteins ~;
possib1e in various host systems.
The transformation or transfection of suitable host organisms ~`
with the hybrid plasmids obtained in this way has likewise been
20 described in detail (M. Wigler et al., Cell, 16 (1979), 777 ~
785; F.L. Graham and A.J. van der Eb, Virology 52 (1973), 456 -
467).
Vectors~which can be used for expression in mammalian cells are
2~S~those;~which~place~the gene to be expressed, in this case the cDNA
coding~for the 5-HTl3 receptor described herein, under the control
of the mous~ metallothionein or the viral SV40 promoter or under
the control of the cytomega}ovirus promoter (J. Page Martin,
Gene, 37 (19853, 139 to 144). It is necessary for expression that
30 the methionine start codon of the gene which codes for this 5-HTlB -
; receptor is present. Clones which have copies of these vectors as
episomes or integrated into the genome are then isolated. It is
,
particularly advantageous to integrate the foreign gene into a
vector which contains the cytomegalovirus promoter.
Alternatively, cells can be transfected with a suitable vector in
such a way that the transient expression of the DNA introduced in
this way i~ sufficient for pharmacological char!acterization of;
the expressed heterologous polypeptides. In this case too it is
40 particularly advantageous for expression to be controlled by the
cytomegalovirus promoter.
The use of shuttle vectors is very suitable in conjunction with
prokaryotic sequences which code for replication in bacterial
45 cells and re~istance to an antiobiotic. The construction and rep-
lication of the plasmid take place initially in bacterial cells;
there is subsequent transfer into eukaryotic cells, eg. into the
-
,~ .
OOS0/43234 ~ l 3
human embryonic kidney cell line HEK 293. Particularly suitable
shuttle vectors are the commercially available plasmids vcCMV,
pCDM8 and pCDNAI ( INVITROGEN, San Diego, USA).
5 It is also possible to use other cell systems, eg. yeast and
other fungi, insect cells as well as animal and human cells such
as CH0, CoS and L cells, in conjunction with suitable expression
vectors for expressing the cloned cDNA.
10 The eukaryotic expression systems have the advantage that they
are able to express their products efficiently and usually in
native form. They furthermore have the ability to carry out post-
translational modification of their products. ;~
15 Expressed receptor proteins can be solubilized by detergents suchas CHAPS ~3-~(3-cholamidopropyl)-dimethylammonio]-1-propanesulfo-
nate), and purified by affinity chromatography, for example using
receptor-specific antibodies, by known methods. The pure polypep- ;
tide can be used, after crystallization and X ray structural
20 analysïs or other suitable physical methods such as NMR or scan-
ning tunneling microscopy, for elucidating the spatial structure
of the ligand binding site. - ;
The expressed receptor proteins can also be used, after appropri-
25 ate purification, as antigens for generating polyclonal or mono-
clonal antibodies. These antibodies in turn can be used where
~- ~ appropriate for diagnostic purposes. Another possible use of such
antibodies is as aids to rational drug design. Thus, receptor-
- specific antibodies can be employed, for example, as antigen for
30 generating anti-idiotype antibodies. Such antibodies may repre-
sent an image of the receptor for defined regions and can be used
~; for screening for specific receptor ligands or for rational drug
~- design.
35 Receptor-expressing cell lines represent an important instrument
in the screening for specific receptor ligands. For this purpose,
the membranes of these cells can be used for receptor binding ~-
assays.~Equippediwithlappropriate reporter systems, eg. lucifer-
ase, which are coupled to a promoter system which is controlled
40 by compounds of signal transduction pathways such as Ca++, cAMP,
IP3 metabolites (second messenger), it is possible to obtain
direct information about the mode of action (agonism/antagonism)
of a receptor ligand (Science 252 (1991) 1424; Proc. Natl. Acad.
Sci. USA 88 (1991) 5061). It is also possible to detect these
45 compounds of the signal transduction pathway in the receptor-
0050/43234
~135~U~ `~
5expressing cells directly by suitable methods (RIA, ELISA, fluo-
rescent dyes) after ligand binding.
It is furthermore possible to measure the f~ow of current through
5 the cell membrane as a function of the ligand binding.
Because of the degeneracy of the genetic code it is possible to
use DNA sequences other than those described here, eg. chemically
synthesized genes with a different DNA sequence, for expressing
10 the human 5-HTlB receptor described.
,.
The invention makes it possible to identify and characterize sub- -
stances which bind to the receptor described herein and have an
15 agonistic or antagonistic effect there.
Further e~bodiments of the invention are described in detail in ~
the examples.
20 For genetic engineering methods, reference may be made, for exam-
ple, to the handbook of Maniatis et al., Molecular Cloning, Cold
Spring Harbor Laboratory, 1989, or DNA cloning, Vol. I to III,
- IRI Press 1985 to 1987, edited by D.M. Glover.
:
,
25 Example 1
, ~ ,,
Isolation of a cDNA which codes for the rat 5-HT1~ receptor
0.5 g of rat cerebrum was homogenized in an ULTRA-TURRAX in 6 M
30 guanidinium thiocyanate, 5 mM sodium citrate (pH 7.0), O.1 M
2-mercaptoethanol, 0.5 ~ sarcosyl. Large cell detritus was re-
~- moved ~by centrifugation at 3000 rpm. The RNA was removed by cen-
trifu~ation through a 5.7 M CsCl cushion at 45,000 rpm for
12 hours. Subsequently the polyA+-containing RNA fraction was re-
35 moved by affinity chromatography on oligo(dT)-cellulose.
Using the enzyme AMV reverse transcriptase and oligo(dT) 12-18 as
starter, the polyA~'RNA was trànscribed into single-stranded cDNA. ~`
The second strand was synthesized using E. coli DNA polymerase I.
40 An EcoRI adaptor with the following sequence: 5'AATT CCATGGATG-
CATGC 3' was attached to the double-stranded cDNA using the
enzyme T4 DNA ligase. The commercially obtainable phage vector ~ ~`
- gt 10 was linearized with the restriction enzyme EcoRI. Phage DNA
and cDNA were ligated together and packaged using a commercially
45 obtainable packaging extract to give infectious phages. The re-
combinant phages were plated out with E. coli C 600 Hfl on NZYDT
plates and incubated at 37 C overnight. The resulting cDNA library
:
0050/43234 ~
~, 1 3 5 ~
contained 2 x 106 independent clones. After amplification of the
cDNA library by conventional methods, 500,000 phages were plated
out with C 600 Hfl cells. The phages were transferred to nitro-
cellulose filters and lyzed with 0.5 N NaOH/1.5 M NaCl, and the
5 denaturated DNA was firmly bound to the filter by baking at 80C
for 2 hours. The filters were prehybridized in 6 x SET buffer
(1 x SET = 0.15 M NaCl, 15 mM Tris / HC1, pH 7.4, 1 mM EDTA),
0.1 % SDS and 5 x Denhardt's solution (100 x Denhardt = 1 g of
Ficoll, 1 g of polyvinylpyrrolidone, 1 g of BSA per S0 ml) at 6 a c
10 for 4 h.
A nick-translated cDNA probe (SEQ ID NO 1) which codes for the
rat 5-HT1~ receptor was used for hybridization.
15 The filters were incubated in a solution which contained 5 x SET,
0.1 % SDSI 30 % formamide, 5 x Denhardt's and 10 % dextran
sulfate at 42 C, shaking gently, overnight. They were then washed
several times in 2 x SET/0.1 % SDS at 42 C, partially dried and
exposed to an X-ray film. Clones which gave a radioactiv~
20 response in the screening were isolated and cultured further to
obtain the correspondlng phage DNA.
Phage DNA was prepared by incubating the purified phages with
proteinase K (ad 60 ~g/ml) at 55 C for 1 h and subsequent phenol~ ;
25 chloroform extraction. After addition of 3 volumes of ethanol
(-20 C), the phage DNA precipitated out and was transferred with a
sterile injection needle into 70 % strength ethanol, washed and
briefly sedimented. The pellet was briefly dried in air and then
suspended in TE buffer.
~ -
~ Example 2
Preparation of single-stranded DNA which codes for the rat 5-HTlB
receptor
;~ 35
The starting point was the phage DNA described in Example 1. It
was cut preparatively with the restriction enzyme Eco RI. The Eco
RI fragments whichl`contai~ed the cDNA inserts were eluted !from
the gel by electrophoreqis. 30 ng portions of these fragments
40 were ligated to 100 ng of the commercially available cloning vec-
tor M13mpl8 or M13mpl9, which had been cut with Eco RI, at 4 C for
12 h. The volume of the ligation mixture was 10 ~1. The ligation
- was stopped by heating at 80 C for 5 minutes.
45 1/10 of the volume of each ligation mixture was employed for ;
transforming 100 ~1 of competent JM 101 cells. After the trans-
formation was complete, 60 ~1 of 0.2 M IPTG solution and 120 ~1 of -~
0050/43234 ~35~0. ~:
.. , ~
XGal ~20 mg/ml) were added to the transformation mixture. This
mixture was plated out in NZYDT top agar on NZYDT agar plates
with 200 ~l of JM 101 cells (OD6no = 1). The NZYDT medium is
commercially available (GIBCO-BRL). It was possible to identify
5 clones which contained cDNA inserts from the lack of a blue
coloration of the plaques.
One clone contained a cDNA which codes for the 5-HTlB receptor.
The DNA sequence of this clone is depicted in sequence listing 1.
,
Example 3
Cloning of the human 5-HT1B receptor
15 The starting point for this was commercially available polyA+ RNA
from human brain (Clonetech; order No. 6516-2).
Using the enzyme reverse transcriptase ~AMV) and oligo~dT~12_18 as
starter, 5 ~g of this polyA~ RNA was transcribed into single-
20 stranded cDNA. The second strand was synthesize~ using E. coli -
~- ~ DNA polymerase. 5 ng of this cDNA were employed as template in a
polymerase chain reaction. The Perkin Elmer DNA amplification kit
GeneAmpTM~(order~No. 182 414) was used for this in accordance wi~h
~- the~manufacturer's instructions. The primer sequence was derived
25~from the sequence for the rat S-HT1B receptor which is indicated
b ~ n~sequence~ listing 1. The primers had the following sequence:
(Shtlb start)
A: 5'~CGC/TCC/GCA/GCC/AGG/ACGtAGG!GCT/ATG 3
; (5htlb Stop)
B: 5' CTT/AGG/CGA/CCC/CCA/TGC/CAT/TGA/CAA/GTC/A 3'
The annealing temperature was 60 C, which was maintained for
35~3~min.~After this,~the~primers were extended at 72 C for 2 min.
~` Denaturation was carried out at 94 C for 1 min. This temperature
cycle was repeated 40 times. 20 pmol of each of primer A and B
~were empl~yed in~one poly~erase chain;reaction. 10 ~ of thislmix-
ture was, after the reaction was complete, transferred to 1 %
40 agarose gel in order to analyze the reaction products. The domi-
nant band comigrated with a DNA band of about 1200 base pairs. It
was eluted from the geI by electrophoresis, taken up in One-Phor-
all buffer (Pharmacia) and incubated with the Klenow fragment of
E. coli DNA polymerase I. The concentration of deoxynucleotide
45 triphosphates for this was S0 ~M (for each of dATP, dTTP, dCTP and
dGTP).
~: :
0050/4323~ ;
3 ~ 9 l~ a t~
Single-stranded DNA was prepared in a similar way tG Example 2
but with one difference in that the PCR fragment was cloned into
the SmaI cleavage site of mpl8. The DNA sequence of the human
5-HTlB receptor gene is described in sequence listing 2 (SEQ ID
5 No 2). The homology to the rat 5-HT1B receptor gene is about 90 %.
The amino-acid sequence derived from SEQ ID No 2 is depicted in
sequence listing 3.
A clone with the cDNA sequence depicted in SEQ ID N0 2 has been
10 deposited at the Deutsche Sammlung von Mikroorganismen und Zell-
kulturen GmbH, Braunschweig, under number DSM 6860.
Example 4
15 Transient expression of the cloned human receptor gene in human
embryonic kidney (HEK) 293 cells
Unless described otherwise, the methods of Lindl and Bauer ~Zell-
und Gewebekultur, Gustav Fischer Verlag) were used for the cell
20 culture.
Double-stranded mp 18 DNA which contained the receptor cDNA as
insert was cleaved with the enzymes EcoRI and HindIII. The frag-
ment codi~g~for the receptor, which resulted from this with pro-
25 truding ends,~ was treated with the aid of the enzymes T4 DNApolymer-ase and the Rlenow fragment of E. coli DNA polymerase
under standard conditions ~cf. Current Protocols in Molecular
Biology see above) in order to generate blunt ends. The commer-
cially~ available linkers (Invitrogen) with the sequence
30~5'-CTTAGAGCAC-3~' and 3'-GAATCTC-S' were then ligated to this
fragment.~ The DNA fragment provided with these linkers was
ligated under standard conditions into the commercially available
~ ~ vectors pCDM8 and rcCMV (Invitrogen) which had been cut with `
-~ ~stXI. The recombinant plasmids resulting from this were replic-
35 ated in a conventional way. HEK 293 cells were cultivated under
st~ndard conditions in a 10 cm cell culture dish until the cell
- count was 7-8 x 106. After trypsini2ation, the cells were diluted1:3 in MEM medium (Gibco)~which contained 2.2 g/l NaHC03, and were
again inoculated in 10 cm Petri dishes. The cells were then
40 cultivated at 37 C for 40-48 h.
The DNA to be transfected was prepared as follows: 20 ~g of the
DNA solution (1 mg/ml), purified on a CsCl gradient, were mixed
with 437 ~l of H20 and then 62.5 ~l of 2 M CaCl2 were added and
45 finally 500 ~l of P3S. Ca++ precipitates formed within 10 min at
room temperature.
0050/43234 i
~ ~ 3 5 ~
The solution was placed on a 10 cm cell culture dish with the 293
cells cultivated by the above methodO After careful mixing, the
cells were cultivated in a 3 ~ co~ incubator at 37 c for 15-20 h.
Then 5 ml of serum-free medium was cautiously added. Removal of
5 all the medium and repetition of the washing step with 5 ml of
serum-free medium were followed by addition of 10 ml of complete
medium to the cells. After incubation in a 5 ~ C02 incubator for
48 h it was possible to use the cells for pharmacological and
electrophysiological investigations.
Alternatively, liposome-mediated insertion of the DNA into the
cells was carried out using lipofectin from GIBCo-~RL in accor-
dance with the manufacturer's instructions.
15 Example 5
Receptor binding assay
2.5 ml of cold P~S were added to the cells transfected and culti-
20 vated in Example 4. After incubation at room temperature for5 min, a further 5 ml of PBS were added, and the cells were care-
fully removed from the surface of the culture dish. The cell sus-
pension was~transferred into a centrifuge tube and centrifuged at
about 20~ g for 10 min. After careful removal of the superna~ant,
25 the cells were used for membrane preparation. `
Thé~cell~pellet was resuspended in 1.5 ml of 10 mM Tris-HCl pH
7.2 and then the cells were homogenized usinq an ULTRA-TURRAX.
The homogenate was centrifuged in a Sorvall SS 34 rotor at
30 50,000 q and 4 C for 20 min. The membrane pellet was taken up in
1.5 ml of lO ~M Tris-HCl pH 7.2 and again homoqenized using an
ULT~A-TUR~AX. After centrifugation as above, the pellet was taken
` up in~3.5 ml of BB (10 mM Tris-HCl pH 7.2, 100 mM Nacl). After
brief homogenization with a tissue homogenizer it was possibIe to
35 use the membranes in the actual binding assay.
800 ~l of membranes prepared in this way were incubated with
[3H]-serotonin (fiha~ con!ceintration 2 nM) and ~arious concentra-
tions of the substance to be tested at 4'C for 2 h. The membranes
~ ~ 40 were then ~iltered on glass fiber filters (Schleicher & Schuell
-~ No. 34) in order to separate the unbound radiolabeled serotonin.
The amount of bound ~3H~-serotonin was determined in a liquid
scintlllation counter.
,
0050/43234
l 3 5 ~
.. ~
The following substances (obtainable from RBI, Natick, MA, USA
under the stated catalog no.) were employed as test substances
for the human 5-HTl~ receptor:
5 5-Carboxyamidotryptamine (5-CT) ~No. C117)
(-)-Propranolol (No. P110)
Methysergide (No. M137
Rauwolscine (No. R104)
MDL 7222 (No. T102)
10 8-OH-DPAT (No. S002)
The following Ki values (nM) were determined:
5-CT 5 :
15 (-)-Propranolol 5
Methysergide 500
Rauwolscine 4500
MDL 7222 > 10,000
8-OH-DPAT > 10,000
:
~ 25~ :
~' ` ".
. 30
:~ 35 ~:
~: 45
0050/43234
1 ~3~9U;~
11 :
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT:
(A) NAME: BASF Aktiengesellschaft
(B) STREET: Carl-~osch-Strasse 38
(C) CITY: Ludwi~shafen
(E) COUNTRY: Federal Republic of Germany
(F) POSTAL CODE: D-6700
(G) TELEPHONE: 0621/6048526
(H) TELEFAX: 0621/6043123 :
(I) TELEX: 1762175170
(ii) TITLE OF APPLICATION: Serotonin receptor: Preparation and use
thereof - `
(iii) NUM3ER OF SEQUENCES: 3
~: (iv) COMPUTER READABLE FORM:
A):MEDIUM $YPE::Floppy disk ~:
(B)~ COMPUTER: IBM PC compatible ~`
(C) OPERATING SYSTEMs PC-DOS/MS-DOS '~-
(D) SOFI~ARE: PatentIn Relea~e #I.0, Version #1.25 (EPA)
(2)~ INFORMATION FOR SEQ ID NO~
i) SEQUENCE CHARACTERISTICS:
- (A)~:LENGTH: 1414 base pairs
(B) TYPE: Nucleic acid :
(Cj STRANDEDNESS: single
D);TOP0LOGY: l1near
ii) MOLECULE TYPE: cDNA to mRNA :~`
:'
~iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
CCTGTGACCT~CTCCTTTCGG CTGAGA~CAC AGGCGGAGGA GTTTACTGAG GAACCCACGG 60
A~CTGGCTAG CCAAAGGAGA CAAGCCTATA GTCTCCATGA TCCTCCCGTC CTCTGTTCTT l20
:
0050/43234
~ ~35~Ua
12
TCCCTGGTCC GCTCCATGTT CAAGAGCTGC GCTCCGCAGC CAGGACGAGG AGAGCTATGG 180
AGGAGCAGGG TATTCAGTGC GCTCCGCCGC CTCCCGCCAC CTCCCAGACA GGGGTACCTC 240
TCGCCAACCT CTCCCACAAC TGCAGCGCCG ACGACTACAT TTACCAGGAC TCCATCGCCC 300
TGCCCTGGAA AGTCCTGCTG GTTGCTTTGT TAGCGCTCAT CACCTTGGCC ACCACGC~CT 360
CCAACGCCTT TGTAATCGCT ACGGTGTATC GGACCCGGAA GCTGCACACC CCGGCTAACT 420
ACCTGATCGC CTCGCTGGCA GTCACTGACC TGCTCGTGTC CATCCTGGTG ATGCCCATCA 480
GCACCATGTA CACGGTCACT GGACGCTGGA CACTAGGCCA GGTGGTCTGC GACTTCTGGC 540
TGTCGTCGGA TATCACCTGT TGCACTGCTT CCATCATGCA TCTCTGTGTC ATCGCCCTGG 600
ACCGCTACTG GGCCATCACT GATGCGGTGG ACTATTCTGC TA~AAGAACT CCCAAAAGGG 660
CGGCCATCAT GATCGTGCTG GTGTGGGTCT TCTCCATCTC GATTTCGCTG CCACCCTTCT 720
TCTGGCGTCA AGCCAAAGCG GAGGAGGAGG TGCTGGACT~- CTTTGTGAAC ACCGACCACG 780
TCCTCTACAC GGTCTACTCC ACGGTGGGGG CTTTCTATTT ACCCACCCTG CTCCTCATCG 840
CCCTCTATGG CCGTATCTAT GTGGAAGCCC GCTCTCGGAT TTTGAAACAG ACACCCAACA 900
AGACCGGCA~ GCGCTTGACC CGAGCCCAGT TGATAACAGA CTCTCCAGGA TCCACGTCCT 960
CGGTCACCTC CATTAACTCC CGGGTTCCGG AGGTGCCCAG TGAGTCCGGG TCTCCTGTGT 1020
ACGTGAACCA AGTCAAAGTG CG M TCTCAG ACGCCCTGCT GGAAAAGAAG AAACTCATGG 1080
CCGCTAGGGA GCGCAAAGCC ACCAAGACCC TAGGGATCAT TTTAGGAGCA TTTATTGTGT 1140
GCTGGCTCCC CTTCTTCATC ATCTCCCTGG TGATGCCTAT CTGCAAGGA~T GCCTGCTGGT 1200
TTCACATGGC CATTTTTGAC TTTTTCAA~T GGCTAGGCTA TCTTAACTCT CTCATCAACC 1260
CCATCATCTA CACCATGTCC AATGAGGACT TCAAACAPGC ATTCCATAAA CTGATACGCT 1320
TTAAGTGCAC AGGTTGACCT GTCAATGGCA TTGGGGTCGC CTAAGGCCAT TTGGGGACCA 1380
AGTTGGGTCT TTTCACAGGA AAGACAGGAA CAAA 1414
2)~ INFORMATION FOR SEQ ID NO: 2:
ti):~5EQUENE:CHARACTERISTICS:
(A)~ LENGTH: 1228 base pairs
~B) TYPE: Nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
MoLECULE TYPE: cDNA to mRNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(ix) FEATURES:
: ~A) NAME/KEY: CDS
(8) LOCATION: 28..1197
(D) OTHER INFORMATION: /product= Human 5-HT 1~ Recep-
tor
. ~
0050/43:234 ~t
1 3 ~
13
( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
CGCTCCGCAG CCAGGACGAG GAGAGCT ATG GAG GAA CCG GGT GCT CAG TGC 51
Met Glu Glu Pro Gly Ala G}n Cys
S
GCT CCA CCG CCG CCC GCG GGC TCC GAG ACC TGG GTT CCT CAA GCC AAC 9 9
Ala Pro Pro Pro Pro Ala Gly Ser Glu Thr Trp Val Pro Gln Ala Asn
10 15 20 ;:
TTA TCC TCT GCT CCC TCC CGA AAC TGC AGC GCC AAG GAC TAC ATT TAC 14 7
Leu Ser Ser Ala Pro Ser Arg Asn Cys Ser Ala Lys Asp Tyr Ile Tyr
25 30 35 40
CAG GAC TCT ATC TCC CTA CCC TGG AAA GTA CTG CTG GTT ATG CTA TTG 19 5 ' :~
Gln Asp Ser Ile Ser Leu Pro Trp Lys Val Leu Leu Val ~et Leu Leu -,
45 50 55 ~''`
GCG CTC ATC ACC TTG GCC ACC ACG CTC TCC AAT GCC TTT GTG ATT GCC 2 4 3 ,`Ala Leu Ile Thr Leu Ala Thr Thr Leu Ser Asn Ala Phe Val Ile Ala ~:
60 65 70 ;,-
ACA GTG TAC CGG ACC CGG AAA :CTG CAC ACC CCG GCT AAC TAC CTG ATC 291
Thr Val Tyr Arg Thr Arg Lys Leu His Thr Pro Ala Asn Tyr Leu Ile
75~ 80 ' 85
GCC TCT ~CTG GCG GTC :ACC~ GAC~ CTG CTT GTG TCC ATC CTG: GTG ATG CCC 339
Ala~ ~S:er:~Leu~Ala~Val~ Thr: Asp Lèu Leu Val Ser~ Ile:Leu Val Met Pro
n ~ : 9 5 10 0
ATC~AGC,~ACC~ATG~AC ACT G~C ~GCC :GGC CGC TGG ACA CTG GGC CAG GTG 387 :~
I le Ser' ~Thr ~Met Tyr'~ Thr Val Ala Gly Arg Trp Thr Leu Gly Gln Val -',.
1 0 5' ~ 1 1 0~ 1 1 5 ; 1 2 0
GTC T.GT GAC TTC TGG CTG TCG TCG GAC ATC ACT TGT TGC ACT GCC TCC 4 3 5 '
Val Cys P.sp Phe Trp Leu Ser Ser Asp Ile Thr Cy9 Cys Thr Ala Ser :~
1 2 5 1 3 0 1 3 5
ATC~ ~CTG~CAC~ ~CTC TGT GTG ATC GCC CTG GAC CGC TAC TGG GCC ATC ACG 483
lei~Leu His~' Leu Cyo Val Ile ~Ala ~Leu Asp Arg Tyr Trp Ala Ile Thr
140 : ~ 145 ~ 150
GAC~:~GAC ~GTG~GXG~ TAC TCA GCT AAP~ AGG ACT CCC AAG AGG GCG GCG GTC 531
As:p- Asp Val Glu Tyr Ser Ala Lys Arg Thr Pro Lys Arg Ala Ala Val
155 I60 165
ATG ATC GCG CTG GTG TGG GTC TTC TCC ACC TCT GCC TCG CTG CCG CCC 5 7 9
Met Ile Ala Leu Val ~Trp~Val Phe;:~Ser, Thr Ser Ala Ser Leu Pro Pro
170 175 180
TTC TTC TGG CGT CAG GCT AAG GCC GAA GAG GAG GTG TCG GAA TGC GTG 6 2 7
Phe Phe'Trp Arg Gln Ala Lys Ala GlU Glu Glu Val Ser Glu Cys Val
185 ~ ~ 190 195 200
GTG AAC ACC GAC CAC ATC CTC TAC ACG GTC TAC TCC ACG GTG GGT GCT 6 7 5
Val Asn:Thr~Asp~His I}e heu Tyr Thr-Val Tyr Ser Thr Val Gly Ala
205 210 215
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TTC TAC TTC CCC ACC CTG CTC CTC ATC GCC CTC TAT GGC CGC ATC TAC 723
Phe Tyr Phe Pro Thr Leu Leu Leu Ile Ala Leu Tyr Gly Arg Ile Tyr
220 225 230
GTA GAA GCC CGC TCC CGG ATT TTG AAA CAG ACG CCC AAC AGG ACC GGC~ 771
Val Glu Ala Arg Ser Arg Ile Leu Lys Gln Thr Pro Asn Arg Thr Gly .
235 240 245
AAG CGC TTG ACC CGA GCC CAG CTG ATA ACC GAC TCC CCC GGG TCC ACG 819
Lys Arg Leu Thr Arg Ala Gln Leu Xle Thr Asp Ser Pro Gly Ser Thr
250 255 260 :~
TCC TCG GTC ACC TCT ATT AAC TCG CGG GTT CCC GAC GTG CCC AGC GAA 867
Ser Ser Val Thr Ser Ile Asn Ser Arg Val Pro Asp Val Pro Ser Glu
265 270 275 2ao
TCC GGA TCT CCT GTG TAT GTC AAC CAA GTC AAA GTG CGA GTC TCC GAC 915
Ser Gly Ser Pro Val Tyr Val Asn Gln Val Lys Val Arg Val Ser Asp ;:
285 290 295 .:~
GCC CTG CTG GAA AAG AAG AAA CTC ATG GCC GCT AGG GAG CGC AAA GCC 963 :~
Ala Leu Leu Glu Lys Lys Lys Leu Met Ala Ala Arg Glu Arg Lys Ala .
300 ~ 305 31~0 :~
ACC AAG ACC CTA GGG ATC ATT TTG GGA GCC TTT ATT GTG TGT TGG CTA 1011
Thr:Lys:Thr Leu Gly Ile Ile Leu Gly Ala Phe Ile~Val Cys Trp Leu
315 : 320 : 325 : .
CCC TTC:~TTC ATC ATC TCC CTA GTG ATG CCT ATC TGC~AAA GAT GCC TGC 1059
~ Pro Phe ~he Ile Ile Ser Leu Val Met Pro Ile Cys Lys Asp Ala Cys `~
- 330: ~ : ~335 : 340 -~.
: TGG TTC CAC CTA GCC ATC TTT GAC TTC TTC ACA TGG CTG GGC TAT CTC 1107
Trp~Phe Hls Leu Alà~ Ile Phe Asp Phe Phe Thr Trp Leu Gly Tyr Leu
345~ 350 355 360
:AAC:TCC CTC ATC AAC CCC ATA ATC TAT ACC CTG TCC A~T GAG GAC TTT 1155
~ :Asn~Ser Leu I~le Asn Pro Ile Ile Tyr Thr Leu Ser Asn Glu:Asp Phe
: 365 370 375
~: AAA~CAA GCA TTC CAT AAA CTG ATA CGT TTT AAG TGC ACA AGT . 1197
:- Lys~Gln Ala~Phe His Lys Leu Ile Arg Phe Lys Cys Thr Ser
380 385 390
-: TGACTTGTC~ ATGGCATTGG GGTCGCCTAA G 1228
(2) INFORMATION FOR SEQ ID NO: 3: ~-
~i) SEQ OE NCE CHARACTERISTICS:
:(A) LENGTH: 390 am1no acids
tB) TYPEs amino acid
D) TOPOLOGY: linear
. (ii) MOLECULE TYPE: Protein
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~,
0~0/43234
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( xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Met Glu Glu Pro Gly Ala Gln Cys Ala Pro Pro Pro Pro Ala Gly Ser
5 10 15 ~ ~'
Glu Thr Trp Val Pro Gln Ala Asn Leu Ser Ser Ala Pro Ser Arg Asn
20 25 30 .
Cys Ser Ala Lys Asp Tyr Ile Tyr Gln Asp Ser Ile Ser Leu Pro Trp
35 ` 40 45 -
Lys Val Leu Leu Val Met Leu Leu Ala Leu Ile Thr Leu Ala Thr Thr
50 55 60 . ;~
Leu Ser Asn Ala Phe Val Ile Ala Thr Val Tyr Arg Thr Arg Lys Leu ~.
His Thr Pro Ala Asn Tyr Leu Ile Ala Ser Leu Ala Val Thr Asp Leu `
. 90 95
Leu Val Ser Ile Leu Val Met Pro Ile Ser Thr Met Tyr Thr Val Al.a
100 105 110 '`~
Gly Arg Trp Thr Leu Gly Gln Val Val Cys Asp Phe Trp Leu Ser Ser
11.5 120 125
Asp Ile Thr Cys Cys Thr Ala Ser Ile Leu His Leu Cys Val Ile Ala
130 135 140
Leu Asp Arg Tyr Trp Ala Ile Thr Asp ~3p Val Glu Tyr Ser Ala Lys
145 : ~ 50 ~ 155 160
Arg~ Thr~:- Pro :Lys ~Arg Ala ALa Val Met Ile Ala Leu Val Trp Val Phe
165 170 ~ 175
Ser~:~Thr~Ser ~Ala Ser Leu Pro Pro Phe Phe Trp Arg Gln Ala Lys Ala
180 185 : 190
Glu Glu Glu ~Val Ser Glu Cys Val Val Asn Thr Asp His Ile Leu Tyr
195 200 205
Thr Val ~Tyr Ser Thr V~al Gly Ala Phe Tyr Phe Pro Thr Leu Leu Leu
210 ~ 215 ~ 220
e;:~Ala Léu~ Tyr Gly Arg Ile Tyr Val Glu Ala Arg Ser Arg Ile Leu
225~ ; 230. : ~ 235 240
Lys: Gln Thr Pro: Asn Arg Thr Gly Lys Arg Leu Thr Arg Ala Gln Leu
::: 2:45: 250 255
Ile Thr A~p Ser: Pro Gly Ser Thr Ser Ser Val Thr Ser Ile Asn Ser
260 265 270
Arg Val Pro A~p.Val PFo1 Selr Glju Ser Gly Ser Pro Val Tyr Val Asn
275 280 285
Gln~Val Lys Val Arg Val Ser Asp Ala Leu Leu Glu Lys Lys Lys Leu
290 ~ 295 300
Met Ala AIa Arg Glu A g Lys Ala Thr Lys Thr Leu Gly Ile Ile Leu
305 310 ~ 315 320
Gly Ala Phe Ile Val cy5 Trp Leu Pro Phe Phe Ile Ile Ser Leu Val
325 330 335
~-~;'
:~:
00~0/43234
~35~0a
16 l:
-;~
Met Pro Ile Cys Lys Asp Ala Cys Trp Phe His Leu Ala Ile Phe Asp ;
3~0 345 350
Phe Phe Thr Trp Leu Gly Tyr Leu Asn Ser Leu Ile Asn Pro Ile Ile
355 360 365 ~
Tyr Thr Leu Ser Asn Glu Asp Phe Lys Gln Ala Phe 8is Lys Leu Ile
370 375 380
Arg Phe Lys Cys Thr Ser
385 390
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