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Molecular cloning, structural characterization and functional expression of the human substance P receptor
Vol.
179,
No.
September
3, 1991
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
1232-1240
MOLECULAR CLONING, STRUCTURAL CHARACTERIZATION AND FUNCTIONAL EXPRESSION OF THE HUMAN SUBSTANCE P RECEPTOR
Y. Takeda,
K.B.
Chou,
Department Washington
Received
August
5,
J.
Takeda,
B.S.
Sachais
and J.E.
Krause1
of Anatomy and Neurobiology University School of Medicine 660 South Euclid Avenue St. Louis, MO 63110
1991
A cDNA encoding the human substance P receptor (SPR) was isolated and the primary structure of the protein was deduced by nucleotide sequence analysis. This SPR consists of 407 residues and is a member of the G-protein coupled receptor superfamily. Comparison of rat and human SPR sequences demonstrated a 94.5% identity. The receptor was expressed in a COS-7 cell line and displayed a Kd for Tyr-l-SP binding of 0.24 nM. Ligand displacement by naturally occurring tachykinin peptides was SP >> neurokinin A > neurokinin B. SP stimulation of transfected cells resulted in a rapid and transient inositol 1,4,5-trisphosphate response. RNA blot hybridization and solution hybridization demonstrated that SPR mRNA was about 4.5 Kb in size, and was expressed in IM-9 lymphoblast and U373-MG astrocytoma cells, as well as in spinal cord and lung but not in liver. Q 1991 RcademlcPress, Inc.
Substance detected
in
P (SP)
is
1931 based
a peptide
on its
SP was isolated
based
on its
was established
(2).
SP is
since
been
excitatory neurons.
shown
to regulate
agent
released
In addition,
secretions,
aids
sites,
immunological
disorders
are
mediated
conserved type
largely tachykinin
receptor.
specificity.
'To
Amino Activation
whom correspondence
Abbreviations: chain reaction;
muscle
sialagogic
activity,
a member of the diverse
from
and neuromodulator
contractile tachykinin
biological
central,
endocrine
regulation
of blood
pressure
and has been
suggested
a receptor
carboxyl
terminal
terminal
interacts domain,
sequences
of the
should
SP, substance PBS, plasmid
that
of the
SPR stimulates
family
(3,4,5).
and has It
and exocrine by acting
to be involved
states.
In 1971
structure is
gland
both in certain
The biological specif
G-protein
actions
tally
and has been tachykinins
at central
with
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1232
P receptor;
of SP
the
called
an NK-1
dictate
receptor
dependent
second
be addressed. P; SPR, substance BLUESCRIPT.
an
and gastrointestinal
certain
via
peptide
first
(1).
primary
activities
peripheral
and inflammatory
activity
and its
SP regulates
in the
and peripheral
neurotransmitter
smooth
PCR, polymerase
Vol.
BIOCHEMICALANDBIOPHYSICALRESEARCH
179, No. 3, 1991
messenger Yokota
systems et al.
functionally G-protein characterized patterns
(6)
that
the specific
and Hershey
expressed coupled
mediate the
receptor
and Krause
rat
(7)
In this
expressed
responses.
molecularly
SPR, and established
superfamily.
and functionally
biological
COMMUNICATIONS
it work,
Recently,
characterized
and
to be a member
of the
we have
molecularly
the human SPR and determined
some
of mRNA expression.
MATERIALS
AND METHODS
Materials. Most reagents used have been described previously (7-9). The plasmid pMZ was obtained from Dr. Irving Boime, Washington University School of Medicine (10). Oligonucleotides were obtained from the Washington University Protein Chemistry Facility. IM-9 cells were obtained from Dr. Norman Boyd and Susan Leeman, University of Massachusetts Medical Center, and U373-MG cells were obtained from the ATCC. Tyr-l -Substance P (Tyr-Arg-ProLys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) was synthesized and purified to homogeneity by HPLC using the general procedures described (11). Radioiodination was performed using the chloramine T oxidative iodination procedure and monoiodo Tyr-' -SP was purified by HPLC (SA-2175 Ci/mmole). RNA isolation. cDNA and zenomic cloninv PCR methods and nucleotide seauence analvsis. Methods for RNA isolation, poly(A)+ RNA selection, cDNA synthesis and polymerase chain reaction (PCR) have been described (7-9). The coding region of the human SPR cDNA for expression studies was generated by PCR after sequence analysis of a partial cDNA and gene exons 1 and 5. A partial SPR cDNA was generated from IM-9 cDNA by PCR using oligonucleotide primers corresponding to G-protein coupled receptor membrane spanning domains II and VII (7). This 671 bp cDNA was cloned into plasma BLUESCRIPT (PBS) and sequenced; it contained an open reading frame with 90.5% identity to the corresponding rat SPR cDNA (6,7) and gene (9) sequence (nucleotides 237-908 in The 5' and 3' extents of the human SPR cDNA coding region were Fig. 1). determined by isolation and characterization of human SPR genomic exons 1 and 5, respectively, using the PBS-hSPRII-VII insert and rat genomic exons (9) as probes. An amplified human x Dash II genomic library (Stratagene) was screened and 10 positive phage were isolated and characterized. Hybridizing sequences corresponding to exons l-5 were identified, and exons 1 and 5 were isolated as 1.2 kb EcoRI and 1.4 kb EcoRI fragments, subcloned and sequenced. The initiator methionine and termination codons were identified by comparison to the rat SPR gene sequence (9), and the predicted coding region of the human SPR was generated by PCR with IM-9 cDNA with oligonucleotides corresponding to the coding region 5' [S'CCACCATGGATAACGTCCTCCCGGTG 3'; with the 5' end modified to an optimal Kozak sequence (12)] and 3'(antisense, S'CTAGGAGAGCACATTGGAGGAGAA 3') ends as primers. The cDNA generated was isolated and was blunt-end ligated into SmaI-digested PBS. Electroporation of E. coli XL-l Blue cells with ligated DNA yielded a cDNA corresponding to bases -5 to +1227 shown in Fig. 1. The cDNA was restricted with Hind111 and BamHI (in PBS polylinker) and was made blunt-ended with Klenow fragment. The pM2 was similarly prepared after BamHI digestion. After ligation and bacterial transformation, plasmid DNA was isolated and orientation of inserts was determined by restriction analysis. Two plasmids, called pM2-hSPR and pM2hSPR antisense, were identified and used for expression studies. Transfection of COS-7 1.4.5 trisnhosohate assay. transfected as described (7). were incubated with 1251-Tyr-1 determined with a filtration performed with 100,000 cells
cells. 1iKand binding exneriments and inositol COS-7 cells plated at 50 to 90% confluence were Cells harvested 48-72 hours after transfections -SP (for 2 hours at 4") and binding was assay (11). Typical binding experiments were per assay tube. The Binding data was analyzed
1233
by
Vol.
179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
the LIGAND program (13). Inositol 1,4,5-trisphosphate with a radioreceptor assay (14) with rat cerebellar extraction and assay conditions as described (16).
RESEARCH COMMUNICATIONS levels membranes
were (15)
determined using
RNA blot and solution hvbridizations. The general methods have been described previously (8,9,17,18). A random-primer labeled cDNA was prepared with Klenow fragment of DNA polymerase I for the PBS-hSPRII-VII cDNA insert, and an antisense RNA was prepared by transcription using T7 RNA polymerase and EcoRI-linearized PBS-hSPRII-VII. RNA gels (1.0%) were blotted onto Nytran membranes, and protected species from solution hybridizations were electrophoresed on 6% polyacrylamide gels containing 7M urea. Autoradiography was performed at -70" with an intensifying screen.
RESULTS AND DISCUSSION A human Fig.
SPR cDNA fragment
1 was generated
by PCR from
as described
(7).
The 5'
and sequence
analysis
corresponding
to nucleotides
cDNA prepared
end of the
coding
from region
of the human SPR gene exon
IM-9
+237 lymphoblast
was determined 1, and the
3'
to +908 cell
in RNA
by isolation end of the
cDNA
(-2lO)MTTCAGAGCCACCGCGGGCAGGCGGGCAGTGCATCCAG~GCG~ATA~CTGAGCGCCAGTTCAGCT~G~GAGTGCTGCCCAT~
-115
GGCTTCCACCCTCCTGTCTGCTTTAGAAGGACCCTGACCCTGAGCCCCAGG~GCCAGCCACAGGACTCTG~TGCAGAGGGGGGTTGTGTA~AGATAGTAGGCTTTACGCGTAGCTTCG~ ATGGATMCGTCCTCCCGGTGGACTCAGACCTCTCCC~CATCTCCACT~CACCTCGG~CCC~TCAGTTCGTGCMCCAGCCTGGC~~GTCC~GGG~GCTGGC ~e~spAs"ValLeuProValA~pSerAspLcuSerProAsnIleSer~rAs"~rSerGluProAs"Gl"~eValGl"ProAl~T~ln~leV~l~u~ a 1 21 MI TAWLCGGTCATTGTGGTGACCTCTGTGGTGGGC~CGTGGTAGTGATGTGGATCATCTTAGCC~C~G~TGAGGACAGTGACGMCTATT~CTGGTGMCGTGGCG~C T~rThrV~lIl~V~lV~l~hrS~rV8lV~lGl~A~"V~lV~lV~lH~tTrpIl~Il~~~l~Hi~Ly~ArSle~S~rV~l~rA~"~~e~~V~~~"~~l~~~ 41 61 MI1 GCGGAGGCCTCCATGGCTGWTCMTACAGTGGTGMCTTCACCTATGCTGTCCAC~CGMTGGTACTACGGCCTGTTCTAGTGC~G~CCAC~CTTC~TGCCATGGCC AlaGluAlaSerHetAl~la~~sn~rValValAanPhe~r~AlaValHisAsnGluT~~~Gly~~h~~Cy~Ly~~eH~~Asn~~~~pr"~~~l~ 81 101 MI11 GCTGTCTTCGCCAGTATCTACTCCATGACGGCTGTGGCCT~GATAGGTACATGGCCATCATACATCCCCTCGAGCCCCGGGTGTCAGG~CAGCCACC~GTGGTCATCTGT AlaV~lPheAlaSerIle~SerWetThrAlaValAl~PheAspArS~rUe~l~IleIleHi~Pro~uGlnProArSLe~erAl~~rAl~~rLy~~ 121 141 HIV GTCATCTGGGTCCTGGCTCTCCTGCTGGCCTTCCCCCAGGGCTACTACTC~CCACAGAGACGATGCCGAGCAGAGTCGTGTGCATGATCG~TGGCCAGffi~TCCGM~G ValIleT~ValLeuAlaLeuleuLeuAlaKeProClnGl~TvrSer~r~rGlu~r~etProS~rArSValValGysHetIleGluTrpPr~l~i~Pro~nLya 161 181 ATTTATGAGAAAGTGTACCACATCTGTGTGTGACTGTGCTGATCTACTTCCTCGCGCTGCTGGTGA~GGCTATGCATACACC~AGT~G~TCA~CTATGGGCCAGTGAGATC IleTyrGluLysV~l~HiSIlcCrsVolThrValLsuIleTvrPheLeuProLe"~uValIl~Gl~~Al~~~~ V~lV.s1GlyIleTl1rLeuTr@laSerGluIlc m 201 221 CCCGGGGACTCCTCTGACCGCTACCACGAGCMGTCTCTGCC~GCGCMGGTGGTC~TGATGA~GTCGTGGTGTGCAGCTTCGCCATCTGCTGGCTGCGC~G~~TC ProGlyAspSarSerAs~S~Hi~Gl~l"V~lS~rAlaLys~SLysValValLyS~et~etIleValV~lValC~s~rPheAlaIleCvsT~~uPro~eHlsIl~ 241 HVI 261 TTCTTCCTCCTGCCCTACATCMCCCAGATCTCTACCTG~GMG~TATCCAGCAGGTCTACCT~CCATCATGTGGCTGGC~~GAGCTC~CCATGTAC~CCC~TCATC phePheLeuLcuPro~I1~snProAsp~u~~~ysLys~~IleGlnGlnV~l~~~l~IleNetT~~~l~etSe~er~r~et~A m MVII 3:1 281 TACTGCTGCCTCMTGACACGTTCCGTCTGGGCTTC~G~TGC~CCGGTGCTGCCCCTTGAT~GCGCGGGCGACTATGAGGGGCTGG~TG~TCCACCCGGTATGTC TyrGvsC~sLeuAsnAr~S~~S~uGly~eLysHi~Al~Ph~rSCysCysRo~eIleSerAlaGlyA~p~Gl~ly~~l~~tLy~S~r~r~S~~u 321 341 CAGACCCAGGGCAGTGTGTAC~GTCAGCCGCCTGGAGAC~C~TCTCCACAGTGGTGGG~GCCACGAGGAGGAGCGAGAGGAG~CCC~GGCCACAGCCTCGTCCCTG GlnThrGlnGl~arV~l~Ly~V~lS~rArS~uGlu~r~rIl~S~r~rV~lV~lGlyAl~H~~GluGluGluPr~l~spGlyProLysAl~~rProSarSer~u 361 GACCTGACCTCCAACTGGTCTTCACGMGTGACTC~GAC~TGACAGAGAGCTTGAGC~CTGCTCG~TGTGCTCTCCTAGGC~~GGGCC~~~~TGCAGCCCCC AspLauThrSarA~nCy~SsrSseArgSsrAspSerLya~~et~rGluSer~eSer~~S~rSerAsnV~l~~erEnd 381 401 407 ACTGCCmGACCTGCCTCCCTTCATGCATGGAAATTCCCTTCCCTTCATC~GMCCATCAG~~CCCT~GAGTGGGAC~G~ffiGT~GTATG~~A~~GA~ CCATCCTTGAGTGAAAMA TCTCAATTCTTGCGTATCmGCCACCCCT~TGCTGAC
1.
+1556
Nucleotide
numbering initiator methionine. Transmembrane domains number is M74290.
a +22S +342 +456 +570 dab
+79S +912 +1026 +1140 +1254 +136S +14S2
C~CGTGMOLGGCCMTGCA~GGA~CT~MGTGAG~GGGTG~TGCGAGTGCT~~CAGGATG
Figure Nucleotide
-I +114
and deduced amino acid sequence of the human SPR. shown on the right starts with +l beginning with A of the Amino acids are numbered below the displayed sequence. labeled MI-MVII are underlined. The GenBank accession
1234
Vol.
A
179,
40000
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
n0ng‘g-d
30000
-
pM2 hSPR antisense
pM2 hSpR
nnnn
pM2 rSPR
Expression of human SPR in COS-7 cells. A. Comparison of '*'Ito nontransfected and transfected cells. B. Competition of -SP binding by tachykinin peptides. Transfection conditions and ligand binding were performed with 0.1 nM '*'I-Tyr-'-SP as described in Methods. Each datum represents the Xf SEH of four duplicate determinations performed with different preparations of transfected cells.
was determined
by isolation
5, as described coding
region,
coding
region.
which
the
and a PCR using This
IM-9
COS-7 cells
were
transfected
that
contain
plasmids antisense
the
were
assay.
25,000
cpm ligand
cells,
or cells
that
binding.
Consequently,
performed
with
is
that
tachykinins
or
the
potent
were
IC5D values
displacer values
peptides
y, neuropeptide
much less
experiments
related
for
with
to neurokinin nM, 0.63
(11,19)
or pM*rSPR 2A).
were
1251-Tyr-1-SP
concentrations
P free
-SP binding.
PM, and 1.12
binding B,
eledoisin
-SP binding.
site.
of other
A, neurokinin acid,
to
no specific
specific
higher
a
15,000
the binding
A and neurokinin
1235
bound
analyses
and senktide,
Additional
SP, NKA and NKB at various
+ 0.06
using
Nontransfected
showed
neurokinin
l*51-Tyr-l
and
characterize
l*51-Tyr-l
displacing
displacing
f 0.09
including K, substance
performed
compared of 0.72
in
lo-fold
in
From 48 to 72 hours
and saturation
at 10 nM SP or physalaemin,
(10)
the human SPR cDNA inserted
by 1 PM SP (Fig.
to pharmacologically
a complete vector
antisense
of 1251-Tyr-1-SP pM*hSPR
SPR
expression.
SPR cDNA (7).
displacement
by 85 to 95%, whereas
were
with
to generate
pM*hSPR
pM 2hSPR antisense,
with
was reduced neuropeptide
rat
exon
of the
pM* expression
controls
pM*hSPR,
binding
displaced
ligand
pM*hSPR
2B shows
for
transfected
transfected
the
LTR (10)
with
SPR gene
5' and 3' portions
cDNA was used into
and the
examined Cells
the
cell
virus
of the human
the human SPR cDNA,
orientation,
cells
analysis
provided
cDNA was subcloned sarcoma
filtration
Fig.
This
murine
the
later
in Methods.
Harvey
pM*rSPR, in
and sequence
doses
to determine
SP was the most B (Fig. f 0.21
3A),
with
potent
IC50
PM, respectively,
Vol.
179,
No.
3, 1991
-11
-12
-10
BIOCHEMICAL
-9
-8
-7
-6
AND
-5
BIOPHYSICAL
RESEARCH
1
-4
Hill
nM, with
coefficients
of 0.94
LIGAND program
determine experiments 15,
20,
with
performed
resting
a return
Some patterns isolated single
from
RNA isolated with the
cell
hybridizing both
of human
IM-9
region
hybridization-nuclease base) that
anneals this
preparations
completely
not
probe
region
or liver
of about and exons
partially
spliced
liver.
RNA. 150 bases 1 plus
cord
In IM-9
cell
RNA
but
they
2 protected
RNAs as previously
blot
in
species
that
were
observed
in
observed to exon
in which
the
two
to exon (9).
Fig.
1 of
but
(712 4 shows
and U373 cell
These
rat
probe
species.
IM-9
appear
a
in poly(A)+
in a solution
preparations,
noted.
RNA
analysis,
RNA preparations,
may correspond
using
corresponding
a 671 base
were
1236
examined
used
to
stimulation
shown).
also
(7,9,18)
and lung
and 350 bases
not
of 2.5
after
signals
was also
of 671 bases
of 5, 10,
kb was identified
Similar
experiment
Two
response
By northern 4.5
and a DNA probe
a species
characterized,
species
4).
RNA probe
in spinal
were
3B).
stimulation
(data
&
1pM SP to
(14,15).
A transient
SPR mRNA and protects
protected
and also
HepGP cell species
to human
after
of
analysis of 0.24
(Fig. with
at 10 to 15 seconds
(Fig.
protection
probe
points
SPR RNA expression
but
cell
responses
of approximately
cells
The coding
per
from
analysis
and its
a Kd value
stimulated
analyzed.
or tissues
a RNA coding
SPR gene.
were
data
with
sites
by 20 to 30 seconds
species
from
time
was observed
level
lines
fit
were
binding
of the
trisphosphate
in which
levels
to basal
f 8,000
COS-7 cells
60 and 120 seconds
above
Saturation plot
a 1 site
of 151,000
inositol-1,4,5
were 30,
3-fold
provides
transfected
cellular
- 0.96.
and a Scatchard
(13)
an average
Transiently
of
b
1251-Tyr-l -SP was performed 0.01
(nM)
of ligand binding characteristics. A. Displacement substance P, neurokinin A and neurokinin B. B. of P 251-Tyr-1-SP binding. Cells were transfected and performed as described in Methods. The data shown are performed in duplicate on separate transfected cell
binding
Saturation analysis ligand binding was four determinations preparations.
the
3
Concentration
Analysis
173!$$?-&
with
2 Ligand
Log[peptide(M)]
each with
COMMUNICATIONS
RNA
not
additional
have 1 alone
not
been
protected
to represent
with
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ewon 1 Coding region probe
probe
hSPR cDNA
5’--
I
-
I
”
”
“I
“I, 100 - bp
axon 1 Probe Coding
t--3’
Region Probe
Figure 4. Analysis of SPR mRNA expression patterns by RNA blot and solution hybridization methods. The upper left shows the RNA blot results, the upper right shows the solution hybridization results, and the lower portion illustrates the probes. For RNA blots, 2 pg poly(A)+ RNA was denatured, electrophoresed on 1% gels and transferred to Nytran. For solution hybridization, 25 pg total RNA was annealed with the coding region probe, and non-hybridized probe was digested with nuclease Sl (see Methods). An autoradiogram of 6% polyacrylamide gel is shown. Standards for the RNA blot were 0.24 to 9.5 Kb RNA ladder (BRL), and for the nuclease protection gel ware radiolabeled
MspI-digested
Fig.
5 compares
deduced
from
residues
are
distributed
different
with
hydrophobicity
domain,
residues
carboxyl
potential
serine
cytoplasmic
the
and are
generally
of human and rat
analysis.
sequences;
a-helical
and a potential to the MVII terminal
these
transmembrane
SPR has 2 consensus
and threonine
Twenty
differences domains
to other N-linked
domain.
1237
sites and most
exist are
encode
coupled
glycosylation
transmembrane
domain,
are
G-protein site
as
407
Both sequences based on
palmitoylation
phosphorylation
SPR protein,
two of the
conservative.
and by comparisons
terminal
and carboxyl
structures
between
7 putative
The human
terminal
primary
and sequence
plotting
receptors. amino
the
cDNA cloning throughout
receptors
pBR322.
sites
(~~~-323)
in
the
15
Multiple in the 3rd
conserved
between
the
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1 MDNVLPVDSDLSP~IST~TSEPNQFVQPAWQIVLWAAAYTVIVVTSVVGNVVVMWIILAH I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I MDNVLPHDSDLFPNISTNTSESNQFVQPTWQIVLWAAAYTVIVVTSVVGNVVVIWIILAH . 1 MI1 100 KRMR~V~NYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPIAAVFAS~ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I KRMRTVTNYFLVNLAFAEACMAAFNTVVNFTYAVHNVWYYGLFYCKFHNFFPIAALFAS~ . . HIV . . YSHTAVAFDRYMAIIHPLQPRLSATA~KVVICVIWVLALLLAFPQGYYSTTETMPSRVVC I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I YSMTAVAFDRYMAIIHPLOPRLSATATKVVIFVIWVLALLLAFPDGYYSTTETNPSRVVC . . . 200 MIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGI~LWA~EIPGD~~DRYHEQV I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I MIEWPEHPNRTYEKAYHICVTVLIYFLPLLVIGYAYTVVG~TLWASEIPGDSSDRYHEOV
COMMUNICATIONS
MI I I I I I I I I I I I I I
I I I I I I
HI11 I I I I I I I I I I I I I I I
I I I I
I I I I I I I I I I I I I I I I I I I I
I I I I I I I I I I I I I I I I I I I I
.
.
‘M;II
MVI ~AKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKFIQQVYLAIHWLAHSSTNY I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I SAKRKVVKHHIVVVCTFAICWLPFHVFFLLPYINPDLYLKKFIQQVYLASMWLAMSSTNY .
I I I I I I I I I I
NPIIYCCLNDRFRLGFKHAFRC~PFI~AGDYEGLEMK~~RYLQ~QG~VYKV~RLE~~I~~ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I NPIIYCCLNDRFRLGFKHAFRC~PFISAGDYEGLEMKSTRYLQTQSSVYKVSRLETTIST . . . . . . . . . 407 . . . . . . . 400. VVGAHEEEPEDGFKATPSSLDL;:NC::RSDtKTMTESFSF;;NVL; human I I I I I I I I I I I I I I I I I I I I I II I I I I I IIIIIII II II I VVGAHEEEPEEGPKATPSSLDLTSNGSSRSNSKTMTESSSFYSNMLA rat . . . . . . . . . . . . . . .
Figure 5. Comparison of the amino acid sequences of human and rat SPRs. Identical residues are indicated by the vertical line. Putative membrane spanning domains MI-MVII are overlined. The closed triangles indicate consensus N-linked glycosylation sites, the filled circles indicate potential intracellular serine and threonine phosphorylation sites, and the arrow depicts a potential palmitoylation site.
two sequences. acidic
The cytoplasmic
region
about
rich
regions.
ser/thr
Previous
studies
preparations potency
indicate profile,
SPRs display agonist. sequences
within
with It
(25). agonist
high
as well
high
the
cloned
terminal
high
domain
sequence,
which
and human
(23,24)
affinity rat
for
(6,7)
of responsiveness
the primary
of sequence
structure
essential
to binding
of a recently
be of interest
to elucidate
ligand
rat the
will
studies
SP binding
as on IM-9
lymphoblast
work)
agonist SPRs.
presence
it
is
likely
that
coupling,
responses,
and human have
been
SPR nonpeptide
high are
reported determining
and the differences be useful in this analysis.
reported
between
sites (23)
human tissues were
expressed
and U373-MG
1238
and cell in the
astrocytoma
lines
brain (24)
to
antagonist
residues
with
Both of
receptor
performed
affinity
(this
G-protein
specificity,
structures
tissue
and a similar
identity
described
two
and/or
continuing
for
by an
the
SPR cell
of receptor
between
separated
separates
in the
and desensitization
is
and human
degree
differences
and antagonist Previous
that
a similar
binding,
regard will
two primary
(20-22)
the
Important
the
on rat
losses
upon
agonist
conserved.
way into
as with similar
Based
affinity exist
half
carboxyl
the
indicated and periphery, cells.
The
I I . . SPR SPR
Vol.
179, No. 3, 1991
experiments
BIOCHEMICAL
performed
in this
report
demonstrate
and expressed
in
IM-9
cells
is
prepared
U373-MG
cells
and from
Like
from
with
variety
the of
These
cell
high
affinity
correspond in
posttranslational affinity
the
rat
compared
sites
are
similar
are
SPRs and consequently
data
important
that in
receptor
the
not
Also,
liver.
expressed
in a
attributed
to SP. mechanisms
of SPR mRNA is
even
though
the
about
number
significant
experiments
precursor
but
SPR is
from
in RNAs
transcriptional
level
(23-24).
suggest
type
responses
protection
SPR nuclear
present
and lung
the
to lJ373 cells
in nuclease
These
mechanisms
diverse
in determining
spliced (9).
NK-1
SPR mRNA cloned
also
cord
The steady-state
observed
to partially
the
many of the
be useful
the
and is
human spinal that
RESEARCH COMMUNICATIONS
that
kb in size
appears
mediate
cells
SP binding were
it
-4.5
expression.
in IM-9
of mRNA species
high
that should
SPR gene higher
observed
SPR (7),
tissues lines
regulating IO-fold
rat
AND BIOPHYSICAL
that
of
amounts may
RNA as previously
posttranscriptional regulation
of the
and number
of
responsiveness.
ACKNOWLEDGMENTS We thank Dr. Irving Boime technical assistance. Supported Monsanto grant.
for the pM2 vector and Phil Dykema for by NIH grant NS21937 and by a Washington-
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
von Euler, U.S. and J.H. Gaddum (1931) J. Physiol. 72, 74-87. Chang, M.M., S.E. Leeman and H.D. Niall (1971) Nature New Biol. 232, 86-87. Pernow, B. (1983) Pharmacol. Rev. 35, 85-141. Maggio, J.E. (1988) Ann. Rev. Neurosci. 11, 13-28. Helke, C.J., J.E. Krause, P.W. Mathyh, R. Couture and M.J. Bannon (1990) FASEB J. 4, 1606-1615. Yokota, Y., Y. Sasai, K. Tanaka, J. Fujiwara, K. Tsuchida, R. Shigemoto, A. Kakizuka, H. Ohkubo and S. Nakanishi (1989) J. Biol. Chem. 264, 1764917652. Hershey, A.D. and J.E. Krause (1990) Science 247, 958-962. Carter, M.S., J.D. Cremins and J.E. Krause (1990) J. Neurosci. 10, 2203-2214. Hershey, A.D., P.E. Dykema and J.E. Krause (1991) J. Biol. Chem. 266, 4366-4374 Matzuk, M.M., M. Krieger, C.L. Corless and I. Boime (1987) Proc. Natl. Acad. Sci. USA 84, 6354-6358. Takeda, Y. and J.E. Krause (1989) Proc. Natl. Acad. Sci. USA 86, 392-396. Kozak, M. (1987) Nut. Acids Res. 15, 8125-8148. Munson, P.J. (1983) Methods Enzymol. 92, 543-576. Challis, R.A.J., 1-H. Battey and S.R. Nahorski (1988) Biochem. Biophys. Res. Comm. 157, 684-691. Biochem. Biophys. Res. Bredt, D.S., R.J. Mourey and S.H. Snyder (1989) comm. 159, 976-982. Hershey, A.D. (1991) Ph.D. Thesis. Washington University, St. Louis, MO. Krause, J.E., J.M. Chirgwin, M.S. Carter, Z.S. Xu and A.D. Hershey (1987) Proc. Natl. Acad. Sci. USA 84, 881-885. Krause, J.E., J.D. Cremins, M.S. Carter, E.R. Brown and M.R. MacDonald (1989) Methods Enzymol. 168, 634-652. Lavielle, S., G. Chassaing, S. Julien, J. Besseyre, A. Marquet, J.C. Beaujouan, Y. Torrens and J. Glowinski (1986) Neuropeptides 7, 191-200. 1239
Vol. 179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
20. Lee, C.-M., Iversen, L.L., M.R. Hanley and B.E.B. Schmiedeberg's Arch. Pharmacol. 318, 281-287. 21. Cascieri, M.A. and T. Liang (1983) J. Biol. Chem. C.F. White, R. Cerpa, E.T., 22. Boyd, N.D., Kaiser and Biochem 30, 336-342. 23. Payan, D.G., J.P. McGillis and M.L. Organist (1986) 14321-14329. 24. Lee, C.M., W. Kum, C.S. Cockran, R. Teoh and J.D. 488, 328-331. 25. Snider, R.M., J.W. Contantine, J.A. Lowe III, K.P. Woody, S.E. Drozda, M.C. Desai, F.J. Vinick, R.W. (1991) Science 251, 435-437.
1240
Sandberg
(1982)
Naunyn
258, 5158-5164. S.E. Leeman (1991) J.
Biol.
Young Longo, Spencer
Chem. 261,
(1989)
Brain
Res.
W.S. Lebel, H.A. and H.J. Hess
179,
No.
September
3, 1991
30,
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Pages
1991
1232-1240
MOLECULAR CLONING, STRUCTURAL CHARACTERIZATION AND FUNCTIONAL EXPRESSION OF THE HUMAN SUBSTANCE P RECEPTOR
Y. Takeda,
K.B.
Chou,
Department Washington
Received
August
5,
J.
Takeda,
B.S.
Sachais
and J.E.
Krause1
of Anatomy and Neurobiology University School of Medicine 660 South Euclid Avenue St. Louis, MO 63110
1991
A cDNA encoding the human substance P receptor (SPR) was isolated and the primary structure of the protein was deduced by nucleotide sequence analysis. This SPR consists of 407 residues and is a member of the G-protein coupled receptor superfamily. Comparison of rat and human SPR sequences demonstrated a 94.5% identity. The receptor was expressed in a COS-7 cell line and displayed a Kd for Tyr-l-SP binding of 0.24 nM. Ligand displacement by naturally occurring tachykinin peptides was SP >> neurokinin A > neurokinin B. SP stimulation of transfected cells resulted in a rapid and transient inositol 1,4,5-trisphosphate response. RNA blot hybridization and solution hybridization demonstrated that SPR mRNA was about 4.5 Kb in size, and was expressed in IM-9 lymphoblast and U373-MG astrocytoma cells, as well as in spinal cord and lung but not in liver. Q 1991 RcademlcPress, Inc.
Substance detected
in
P (SP)
is
1931 based
a peptide
on its
SP was isolated
based
on its
was established
(2).
SP is
since
been
excitatory neurons.
shown
to regulate
agent
released
In addition,
secretions,
aids
sites,
immunological
disorders
are
mediated
conserved type
largely tachykinin
receptor.
specificity.
'To
Amino Activation
whom correspondence
Abbreviations: chain reaction;
muscle
sialagogic
activity,
a member of the diverse
from
and neuromodulator
contractile tachykinin
biological
central,
endocrine
regulation
of blood
pressure
and has been
suggested
a receptor
carboxyl
terminal
terminal
interacts domain,
sequences
of the
should
SP, substance PBS, plasmid
that
of the
SPR stimulates
family
(3,4,5).
and has It
and exocrine by acting
to be involved
states.
In 1971
structure is
gland
both in certain
The biological specif
G-protein
actions
tally
and has been tachykinins
at central
with
0006-291X/91 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
1232
P receptor;
of SP
the
called
an NK-1
dictate
receptor
dependent
second
be addressed. P; SPR, substance BLUESCRIPT.
an
and gastrointestinal
certain
via
peptide
first
(1).
primary
activities
peripheral
and inflammatory
activity
and its
SP regulates
in the
and peripheral
neurotransmitter
smooth
PCR, polymerase
Vol.
BIOCHEMICALANDBIOPHYSICALRESEARCH
179, No. 3, 1991
messenger Yokota
systems et al.
functionally G-protein characterized patterns
(6)
that
the specific
and Hershey
expressed coupled
mediate the
receptor
and Krause
rat
(7)
In this
expressed
responses.
molecularly
SPR, and established
superfamily.
and functionally
biological
COMMUNICATIONS
it work,
Recently,
characterized
and
to be a member
of the
we have
molecularly
the human SPR and determined
some
of mRNA expression.
MATERIALS
AND METHODS
Materials. Most reagents used have been described previously (7-9). The plasmid pMZ was obtained from Dr. Irving Boime, Washington University School of Medicine (10). Oligonucleotides were obtained from the Washington University Protein Chemistry Facility. IM-9 cells were obtained from Dr. Norman Boyd and Susan Leeman, University of Massachusetts Medical Center, and U373-MG cells were obtained from the ATCC. Tyr-l -Substance P (Tyr-Arg-ProLys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) was synthesized and purified to homogeneity by HPLC using the general procedures described (11). Radioiodination was performed using the chloramine T oxidative iodination procedure and monoiodo Tyr-' -SP was purified by HPLC (SA-2175 Ci/mmole). RNA isolation. cDNA and zenomic cloninv PCR methods and nucleotide seauence analvsis. Methods for RNA isolation, poly(A)+ RNA selection, cDNA synthesis and polymerase chain reaction (PCR) have been described (7-9). The coding region of the human SPR cDNA for expression studies was generated by PCR after sequence analysis of a partial cDNA and gene exons 1 and 5. A partial SPR cDNA was generated from IM-9 cDNA by PCR using oligonucleotide primers corresponding to G-protein coupled receptor membrane spanning domains II and VII (7). This 671 bp cDNA was cloned into plasma BLUESCRIPT (PBS) and sequenced; it contained an open reading frame with 90.5% identity to the corresponding rat SPR cDNA (6,7) and gene (9) sequence (nucleotides 237-908 in The 5' and 3' extents of the human SPR cDNA coding region were Fig. 1). determined by isolation and characterization of human SPR genomic exons 1 and 5, respectively, using the PBS-hSPRII-VII insert and rat genomic exons (9) as probes. An amplified human x Dash II genomic library (Stratagene) was screened and 10 positive phage were isolated and characterized. Hybridizing sequences corresponding to exons l-5 were identified, and exons 1 and 5 were isolated as 1.2 kb EcoRI and 1.4 kb EcoRI fragments, subcloned and sequenced. The initiator methionine and termination codons were identified by comparison to the rat SPR gene sequence (9), and the predicted coding region of the human SPR was generated by PCR with IM-9 cDNA with oligonucleotides corresponding to the coding region 5' [S'CCACCATGGATAACGTCCTCCCGGTG 3'; with the 5' end modified to an optimal Kozak sequence (12)] and 3'(antisense, S'CTAGGAGAGCACATTGGAGGAGAA 3') ends as primers. The cDNA generated was isolated and was blunt-end ligated into SmaI-digested PBS. Electroporation of E. coli XL-l Blue cells with ligated DNA yielded a cDNA corresponding to bases -5 to +1227 shown in Fig. 1. The cDNA was restricted with Hind111 and BamHI (in PBS polylinker) and was made blunt-ended with Klenow fragment. The pM2 was similarly prepared after BamHI digestion. After ligation and bacterial transformation, plasmid DNA was isolated and orientation of inserts was determined by restriction analysis. Two plasmids, called pM2-hSPR and pM2hSPR antisense, were identified and used for expression studies. Transfection of COS-7 1.4.5 trisnhosohate assay. transfected as described (7). were incubated with 1251-Tyr-1 determined with a filtration performed with 100,000 cells
cells. 1iKand binding exneriments and inositol COS-7 cells plated at 50 to 90% confluence were Cells harvested 48-72 hours after transfections -SP (for 2 hours at 4") and binding was assay (11). Typical binding experiments were per assay tube. The Binding data was analyzed
1233
by
Vol.
179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
the LIGAND program (13). Inositol 1,4,5-trisphosphate with a radioreceptor assay (14) with rat cerebellar extraction and assay conditions as described (16).
RESEARCH COMMUNICATIONS levels membranes
were (15)
determined using
RNA blot and solution hvbridizations. The general methods have been described previously (8,9,17,18). A random-primer labeled cDNA was prepared with Klenow fragment of DNA polymerase I for the PBS-hSPRII-VII cDNA insert, and an antisense RNA was prepared by transcription using T7 RNA polymerase and EcoRI-linearized PBS-hSPRII-VII. RNA gels (1.0%) were blotted onto Nytran membranes, and protected species from solution hybridizations were electrophoresed on 6% polyacrylamide gels containing 7M urea. Autoradiography was performed at -70" with an intensifying screen.
RESULTS AND DISCUSSION A human Fig.
SPR cDNA fragment
1 was generated
by PCR from
as described
(7).
The 5'
and sequence
analysis
corresponding
to nucleotides
cDNA prepared
end of the
coding
from region
of the human SPR gene exon
IM-9
+237 lymphoblast
was determined 1, and the
3'
to +908 cell
in RNA
by isolation end of the
cDNA
(-2lO)MTTCAGAGCCACCGCGGGCAGGCGGGCAGTGCATCCAG~GCG~ATA~CTGAGCGCCAGTTCAGCT~G~GAGTGCTGCCCAT~
-115
GGCTTCCACCCTCCTGTCTGCTTTAGAAGGACCCTGACCCTGAGCCCCAGG~GCCAGCCACAGGACTCTG~TGCAGAGGGGGGTTGTGTA~AGATAGTAGGCTTTACGCGTAGCTTCG~ ATGGATMCGTCCTCCCGGTGGACTCAGACCTCTCCC~CATCTCCACT~CACCTCGG~CCC~TCAGTTCGTGCMCCAGCCTGGC~~GTCC~GGG~GCTGGC ~e~spAs"ValLeuProValA~pSerAspLcuSerProAsnIleSer~rAs"~rSerGluProAs"Gl"~eValGl"ProAl~T~ln~leV~l~u~ a 1 21 MI TAWLCGGTCATTGTGGTGACCTCTGTGGTGGGC~CGTGGTAGTGATGTGGATCATCTTAGCC~C~G~TGAGGACAGTGACGMCTATT~CTGGTGMCGTGGCG~C T~rThrV~lIl~V~lV~l~hrS~rV8lV~lGl~A~"V~lV~lV~lH~tTrpIl~Il~~~l~Hi~Ly~ArSle~S~rV~l~rA~"~~e~~V~~~"~~l~~~ 41 61 MI1 GCGGAGGCCTCCATGGCTGWTCMTACAGTGGTGMCTTCACCTATGCTGTCCAC~CGMTGGTACTACGGCCTGTTCTAGTGC~G~CCAC~CTTC~TGCCATGGCC AlaGluAlaSerHetAl~la~~sn~rValValAanPhe~r~AlaValHisAsnGluT~~~Gly~~h~~Cy~Ly~~eH~~Asn~~~~pr"~~~l~ 81 101 MI11 GCTGTCTTCGCCAGTATCTACTCCATGACGGCTGTGGCCT~GATAGGTACATGGCCATCATACATCCCCTCGAGCCCCGGGTGTCAGG~CAGCCACC~GTGGTCATCTGT AlaV~lPheAlaSerIle~SerWetThrAlaValAl~PheAspArS~rUe~l~IleIleHi~Pro~uGlnProArSLe~erAl~~rAl~~rLy~~ 121 141 HIV GTCATCTGGGTCCTGGCTCTCCTGCTGGCCTTCCCCCAGGGCTACTACTC~CCACAGAGACGATGCCGAGCAGAGTCGTGTGCATGATCG~TGGCCAGffi~TCCGM~G ValIleT~ValLeuAlaLeuleuLeuAlaKeProClnGl~TvrSer~r~rGlu~r~etProS~rArSValValGysHetIleGluTrpPr~l~i~Pro~nLya 161 181 ATTTATGAGAAAGTGTACCACATCTGTGTGTGACTGTGCTGATCTACTTCCTCGCGCTGCTGGTGA~GGCTATGCATACACC~AGT~G~TCA~CTATGGGCCAGTGAGATC IleTyrGluLysV~l~HiSIlcCrsVolThrValLsuIleTvrPheLeuProLe"~uValIl~Gl~~Al~~~~ V~lV.s1GlyIleTl1rLeuTr@laSerGluIlc m 201 221 CCCGGGGACTCCTCTGACCGCTACCACGAGCMGTCTCTGCC~GCGCMGGTGGTC~TGATGA~GTCGTGGTGTGCAGCTTCGCCATCTGCTGGCTGCGC~G~~TC ProGlyAspSarSerAs~S~Hi~Gl~l"V~lS~rAlaLys~SLysValValLyS~et~etIleValV~lValC~s~rPheAlaIleCvsT~~uPro~eHlsIl~ 241 HVI 261 TTCTTCCTCCTGCCCTACATCMCCCAGATCTCTACCTG~GMG~TATCCAGCAGGTCTACCT~CCATCATGTGGCTGGC~~GAGCTC~CCATGTAC~CCC~TCATC phePheLeuLcuPro~I1~snProAsp~u~~~ysLys~~IleGlnGlnV~l~~~l~IleNetT~~~l~etSe~er~r~et~A m MVII 3:1 281 TACTGCTGCCTCMTGACACGTTCCGTCTGGGCTTC~G~TGC~CCGGTGCTGCCCCTTGAT~GCGCGGGCGACTATGAGGGGCTGG~TG~TCCACCCGGTATGTC TyrGvsC~sLeuAsnAr~S~~S~uGly~eLysHi~Al~Ph~rSCysCysRo~eIleSerAlaGlyA~p~Gl~ly~~l~~tLy~S~r~r~S~~u 321 341 CAGACCCAGGGCAGTGTGTAC~GTCAGCCGCCTGGAGAC~C~TCTCCACAGTGGTGGG~GCCACGAGGAGGAGCGAGAGGAG~CCC~GGCCACAGCCTCGTCCCTG GlnThrGlnGl~arV~l~Ly~V~lS~rArS~uGlu~r~rIl~S~r~rV~lV~lGlyAl~H~~GluGluGluPr~l~spGlyProLysAl~~rProSarSer~u 361 GACCTGACCTCCAACTGGTCTTCACGMGTGACTC~GAC~TGACAGAGAGCTTGAGC~CTGCTCG~TGTGCTCTCCTAGGC~~GGGCC~~~~TGCAGCCCCC AspLauThrSarA~nCy~SsrSseArgSsrAspSerLya~~et~rGluSer~eSer~~S~rSerAsnV~l~~erEnd 381 401 407 ACTGCCmGACCTGCCTCCCTTCATGCATGGAAATTCCCTTCCCTTCATC~GMCCATCAG~~CCCT~GAGTGGGAC~G~ffiGT~GTATG~~A~~GA~ CCATCCTTGAGTGAAAMA TCTCAATTCTTGCGTATCmGCCACCCCT~TGCTGAC
1.
+1556
Nucleotide
numbering initiator methionine. Transmembrane domains number is M74290.
a +22S +342 +456 +570 dab
+79S +912 +1026 +1140 +1254 +136S +14S2
C~CGTGMOLGGCCMTGCA~GGA~CT~MGTGAG~GGGTG~TGCGAGTGCT~~CAGGATG
Figure Nucleotide
-I +114
and deduced amino acid sequence of the human SPR. shown on the right starts with +l beginning with A of the Amino acids are numbered below the displayed sequence. labeled MI-MVII are underlined. The GenBank accession
1234
Vol.
A
179,
40000
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
n0ng‘g-d
30000
-
pM2 hSPR antisense
pM2 hSpR
nnnn
pM2 rSPR
Expression of human SPR in COS-7 cells. A. Comparison of '*'Ito nontransfected and transfected cells. B. Competition of -SP binding by tachykinin peptides. Transfection conditions and ligand binding were performed with 0.1 nM '*'I-Tyr-'-SP as described in Methods. Each datum represents the Xf SEH of four duplicate determinations performed with different preparations of transfected cells.
was determined
by isolation
5, as described coding
region,
coding
region.
which
the
and a PCR using This
IM-9
COS-7 cells
were
transfected
that
contain
plasmids antisense
the
were
assay.
25,000
cpm ligand
cells,
or cells
that
binding.
Consequently,
performed
with
is
that
tachykinins
or
the
potent
were
IC5D values
displacer values
peptides
y, neuropeptide
much less
experiments
related
for
with
to neurokinin nM, 0.63
(11,19)
or pM*rSPR 2A).
were
1251-Tyr-1-SP
concentrations
P free
-SP binding.
PM, and 1.12
binding B,
eledoisin
-SP binding.
site.
of other
A, neurokinin acid,
to
no specific
specific
higher
a
15,000
the binding
A and neurokinin
1235
bound
analyses
and senktide,
Additional
SP, NKA and NKB at various
+ 0.06
using
Nontransfected
showed
neurokinin
l*51-Tyr-l
and
characterize
l*51-Tyr-l
displacing
displacing
f 0.09
including K, substance
performed
compared of 0.72
in
lo-fold
in
From 48 to 72 hours
and saturation
at 10 nM SP or physalaemin,
(10)
the human SPR cDNA inserted
by 1 PM SP (Fig.
to pharmacologically
a complete vector
antisense
of 1251-Tyr-1-SP pM*hSPR
SPR
expression.
SPR cDNA (7).
displacement
by 85 to 95%, whereas
were
with
to generate
pM*hSPR
pM 2hSPR antisense,
with
was reduced neuropeptide
rat
exon
of the
pM* expression
controls
pM*hSPR,
binding
displaced
ligand
pM*hSPR
2B shows
for
transfected
transfected
the
LTR (10)
with
SPR gene
5' and 3' portions
cDNA was used into
and the
examined Cells
the
cell
virus
of the human
the human SPR cDNA,
orientation,
cells
analysis
provided
cDNA was subcloned sarcoma
filtration
Fig.
This
murine
the
later
in Methods.
Harvey
pM*rSPR, in
and sequence
doses
to determine
SP was the most B (Fig. f 0.21
3A),
with
potent
IC50
PM, respectively,
Vol.
179,
No.
3, 1991
-11
-12
-10
BIOCHEMICAL
-9
-8
-7
-6
AND
-5
BIOPHYSICAL
RESEARCH
1
-4
Hill
nM, with
coefficients
of 0.94
LIGAND program
determine experiments 15,
20,
with
performed
resting
a return
Some patterns isolated single
from
RNA isolated with the
cell
hybridizing both
of human
IM-9
region
hybridization-nuclease base) that
anneals this
preparations
completely
not
probe
region
or liver
of about and exons
partially
spliced
liver.
RNA. 150 bases 1 plus
cord
In IM-9
cell
RNA
but
they
2 protected
RNAs as previously
blot
in
species
that
were
observed
in
observed to exon
in which
the
two
to exon (9).
Fig.
1 of
but
(712 4 shows
and U373 cell
These
rat
probe
species.
IM-9
appear
a
in poly(A)+
in a solution
preparations,
noted.
RNA
analysis,
RNA preparations,
may correspond
using
corresponding
a 671 base
were
1236
examined
used
to
stimulation
shown).
also
(7,9,18)
and lung
and 350 bases
not
of 2.5
after
signals
was also
of 671 bases
of 5, 10,
kb was identified
Similar
experiment
Two
response
By northern 4.5
and a DNA probe
a species
characterized,
species
4).
RNA probe
in spinal
were
3B).
stimulation
(data
&
1pM SP to
(14,15).
A transient
SPR mRNA and protects
protected
and also
HepGP cell species
to human
after
of
analysis of 0.24
(Fig. with
at 10 to 15 seconds
(Fig.
protection
probe
points
SPR RNA expression
but
cell
responses
of approximately
cells
The coding
per
from
analysis
and its
a Kd value
stimulated
analyzed.
or tissues
a RNA coding
SPR gene.
were
data
with
sites
by 20 to 30 seconds
species
from
time
was observed
level
lines
fit
were
binding
of the
trisphosphate
in which
levels
to basal
f 8,000
COS-7 cells
60 and 120 seconds
above
Saturation plot
a 1 site
of 151,000
inositol-1,4,5
were 30,
3-fold
provides
transfected
cellular
- 0.96.
and a Scatchard
(13)
an average
Transiently
of
b
1251-Tyr-l -SP was performed 0.01
(nM)
of ligand binding characteristics. A. Displacement substance P, neurokinin A and neurokinin B. B. of P 251-Tyr-1-SP binding. Cells were transfected and performed as described in Methods. The data shown are performed in duplicate on separate transfected cell
binding
Saturation analysis ligand binding was four determinations preparations.
the
3
Concentration
Analysis
173!$$?-&
with
2 Ligand
Log[peptide(M)]
each with
COMMUNICATIONS
RNA
not
additional
have 1 alone
not
been
protected
to represent
with
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
Ewon 1 Coding region probe
probe
hSPR cDNA
5’--
I
-
I
”
”
“I
“I, 100 - bp
axon 1 Probe Coding
t--3’
Region Probe
Figure 4. Analysis of SPR mRNA expression patterns by RNA blot and solution hybridization methods. The upper left shows the RNA blot results, the upper right shows the solution hybridization results, and the lower portion illustrates the probes. For RNA blots, 2 pg poly(A)+ RNA was denatured, electrophoresed on 1% gels and transferred to Nytran. For solution hybridization, 25 pg total RNA was annealed with the coding region probe, and non-hybridized probe was digested with nuclease Sl (see Methods). An autoradiogram of 6% polyacrylamide gel is shown. Standards for the RNA blot were 0.24 to 9.5 Kb RNA ladder (BRL), and for the nuclease protection gel ware radiolabeled
MspI-digested
Fig.
5 compares
deduced
from
residues
are
distributed
different
with
hydrophobicity
domain,
residues
carboxyl
potential
serine
cytoplasmic
the
and are
generally
of human and rat
analysis.
sequences;
a-helical
and a potential to the MVII terminal
these
transmembrane
SPR has 2 consensus
and threonine
Twenty
differences domains
to other N-linked
domain.
1237
sites and most
exist are
encode
coupled
glycosylation
transmembrane
domain,
are
G-protein site
as
407
Both sequences based on
palmitoylation
phosphorylation
SPR protein,
two of the
conservative.
and by comparisons
terminal
and carboxyl
structures
between
7 putative
The human
terminal
primary
and sequence
plotting
receptors. amino
the
cDNA cloning throughout
receptors
pBR322.
sites
(~~~-323)
in
the
15
Multiple in the 3rd
conserved
between
the
Vol.
179,
No.
3, 1991
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
1 MDNVLPVDSDLSP~IST~TSEPNQFVQPAWQIVLWAAAYTVIVVTSVVGNVVVMWIILAH I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I MDNVLPHDSDLFPNISTNTSESNQFVQPTWQIVLWAAAYTVIVVTSVVGNVVVIWIILAH . 1 MI1 100 KRMR~V~NYFLVNLAFAEASMAAFNTVVNFTYAVHNEWYYGLFYCKFHNFFPIAAVFAS~ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I KRMRTVTNYFLVNLAFAEACMAAFNTVVNFTYAVHNVWYYGLFYCKFHNFFPIAALFAS~ . . HIV . . YSHTAVAFDRYMAIIHPLQPRLSATA~KVVICVIWVLALLLAFPQGYYSTTETMPSRVVC I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I YSMTAVAFDRYMAIIHPLOPRLSATATKVVIFVIWVLALLLAFPDGYYSTTETNPSRVVC . . . 200 MIEWPEHPNKIYEKVYHICVTVLIYFLPLLVIGYAYTVVGI~LWA~EIPGD~~DRYHEQV I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I MIEWPEHPNRTYEKAYHICVTVLIYFLPLLVIGYAYTVVG~TLWASEIPGDSSDRYHEOV
COMMUNICATIONS
MI I I I I I I I I I I I I I
I I I I I I
HI11 I I I I I I I I I I I I I I I
I I I I
I I I I I I I I I I I I I I I I I I I I
I I I I I I I I I I I I I I I I I I I I
.
.
‘M;II
MVI ~AKRKVVKMMIVVVCTFAICWLPFHIFFLLPYINPDLYLKKFIQQVYLAIHWLAHSSTNY I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I SAKRKVVKHHIVVVCTFAICWLPFHVFFLLPYINPDLYLKKFIQQVYLASMWLAMSSTNY .
I I I I I I I I I I
NPIIYCCLNDRFRLGFKHAFRC~PFI~AGDYEGLEMK~~RYLQ~QG~VYKV~RLE~~I~~ I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I NPIIYCCLNDRFRLGFKHAFRC~PFISAGDYEGLEMKSTRYLQTQSSVYKVSRLETTIST . . . . . . . . . 407 . . . . . . . 400. VVGAHEEEPEDGFKATPSSLDL;:NC::RSDtKTMTESFSF;;NVL; human I I I I I I I I I I I I I I I I I I I I I II I I I I I IIIIIII II II I VVGAHEEEPEEGPKATPSSLDLTSNGSSRSNSKTMTESSSFYSNMLA rat . . . . . . . . . . . . . . .
Figure 5. Comparison of the amino acid sequences of human and rat SPRs. Identical residues are indicated by the vertical line. Putative membrane spanning domains MI-MVII are overlined. The closed triangles indicate consensus N-linked glycosylation sites, the filled circles indicate potential intracellular serine and threonine phosphorylation sites, and the arrow depicts a potential palmitoylation site.
two sequences. acidic
The cytoplasmic
region
about
rich
regions.
ser/thr
Previous
studies
preparations potency
indicate profile,
SPRs display agonist. sequences
within
with It
(25). agonist
high
as well
high
the
cloned
terminal
high
domain
sequence,
which
and human
(23,24)
affinity rat
for
(6,7)
of responsiveness
the primary
of sequence
structure
essential
to binding
of a recently
be of interest
to elucidate
ligand
rat the
will
studies
SP binding
as on IM-9
lymphoblast
work)
agonist SPRs.
presence
it
is
likely
that
coupling,
responses,
and human have
been
SPR nonpeptide
high are
reported determining
and the differences be useful in this analysis.
reported
between
sites (23)
human tissues were
expressed
and U373-MG
1238
and cell in the
astrocytoma
lines
brain (24)
to
antagonist
residues
with
Both of
receptor
performed
affinity
(this
G-protein
specificity,
structures
tissue
and a similar
identity
described
two
and/or
continuing
for
by an
the
SPR cell
of receptor
between
separated
separates
in the
and desensitization
is
and human
degree
differences
and antagonist Previous
that
a similar
binding,
regard will
two primary
(20-22)
the
Important
the
on rat
losses
upon
agonist
conserved.
way into
as with similar
Based
affinity exist
half
carboxyl
the
indicated and periphery, cells.
The
I I . . SPR SPR
Vol.
179, No. 3, 1991
experiments
BIOCHEMICAL
performed
in this
report
demonstrate
and expressed
in
IM-9
cells
is
prepared
U373-MG
cells
and from
Like
from
with
variety
the of
These
cell
high
affinity
correspond in
posttranslational affinity
the
rat
compared
sites
are
similar
are
SPRs and consequently
data
important
that in
receptor
the
not
Also,
liver.
expressed
in a
attributed
to SP. mechanisms
of SPR mRNA is
even
though
the
about
number
significant
experiments
precursor
but
SPR is
from
in RNAs
transcriptional
level
(23-24).
suggest
type
responses
protection
SPR nuclear
present
and lung
the
to lJ373 cells
in nuclease
These
mechanisms
diverse
in determining
spliced (9).
NK-1
SPR mRNA cloned
also
cord
The steady-state
observed
to partially
the
many of the
be useful
the
and is
human spinal that
RESEARCH COMMUNICATIONS
that
kb in size
appears
mediate
cells
SP binding were
it
-4.5
expression.
in IM-9
of mRNA species
high
that should
SPR gene higher
observed
SPR (7),
tissues lines
regulating IO-fold
rat
AND BIOPHYSICAL
that
of
amounts may
RNA as previously
posttranscriptional regulation
of the
and number
of
responsiveness.
ACKNOWLEDGMENTS We thank Dr. Irving Boime technical assistance. Supported Monsanto grant.
for the pM2 vector and Phil Dykema for by NIH grant NS21937 and by a Washington-
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
von Euler, U.S. and J.H. Gaddum (1931) J. Physiol. 72, 74-87. Chang, M.M., S.E. Leeman and H.D. Niall (1971) Nature New Biol. 232, 86-87. Pernow, B. (1983) Pharmacol. Rev. 35, 85-141. Maggio, J.E. (1988) Ann. Rev. Neurosci. 11, 13-28. Helke, C.J., J.E. Krause, P.W. Mathyh, R. Couture and M.J. Bannon (1990) FASEB J. 4, 1606-1615. Yokota, Y., Y. Sasai, K. Tanaka, J. Fujiwara, K. Tsuchida, R. Shigemoto, A. Kakizuka, H. Ohkubo and S. Nakanishi (1989) J. Biol. Chem. 264, 1764917652. Hershey, A.D. and J.E. Krause (1990) Science 247, 958-962. Carter, M.S., J.D. Cremins and J.E. Krause (1990) J. Neurosci. 10, 2203-2214. Hershey, A.D., P.E. Dykema and J.E. Krause (1991) J. Biol. Chem. 266, 4366-4374 Matzuk, M.M., M. Krieger, C.L. Corless and I. Boime (1987) Proc. Natl. Acad. Sci. USA 84, 6354-6358. Takeda, Y. and J.E. Krause (1989) Proc. Natl. Acad. Sci. USA 86, 392-396. Kozak, M. (1987) Nut. Acids Res. 15, 8125-8148. Munson, P.J. (1983) Methods Enzymol. 92, 543-576. Challis, R.A.J., 1-H. Battey and S.R. Nahorski (1988) Biochem. Biophys. Res. Comm. 157, 684-691. Biochem. Biophys. Res. Bredt, D.S., R.J. Mourey and S.H. Snyder (1989) comm. 159, 976-982. Hershey, A.D. (1991) Ph.D. Thesis. Washington University, St. Louis, MO. Krause, J.E., J.M. Chirgwin, M.S. Carter, Z.S. Xu and A.D. Hershey (1987) Proc. Natl. Acad. Sci. USA 84, 881-885. Krause, J.E., J.D. Cremins, M.S. Carter, E.R. Brown and M.R. MacDonald (1989) Methods Enzymol. 168, 634-652. Lavielle, S., G. Chassaing, S. Julien, J. Besseyre, A. Marquet, J.C. Beaujouan, Y. Torrens and J. Glowinski (1986) Neuropeptides 7, 191-200. 1239
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