Vol.
187,
No.
September
3, 1992
30,
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
Eggerickx’,
Gilbert
and 2Service
de GCnCtique
3DCpartement
Raspel,
Daniel
and Marc
Mtdicale,
de Lennik,
313
Servet,
Bertrand3,
Parmentierl
Universite B-1070
de Physiologie,
1 rue Michel
27,
Eric
Vassart1,2
808 route
July
1306-l
FUNCTIONAL EXPRESSION AND P HARMACOLOGICAL OF A HUMAN BRADYKININ B2 RECEPTOR GENE
Dominique
‘IRIBHN
COMMUNICATIONS Pages
MOLECULAR CLONING, CHARACTERIZATION
Received
AND
1992
libre
de Bruxelles,
Bruxelles,
Centre
Medical
120.5, Genke,
Campus
Erasme,
Belgium Universitaire,
Switzerland
1992
The gene encoding a putative G protein-coupled receptor (HGlO) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor (1). The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HGIO encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca2+phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype (2). This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable. ‘d 1992 Acadrmrc Press, 1°C.
Kinins serine
are a group
proteases,
BK)
and met-lys-BK.
venous
smooth
Kinins
(4).
intestine
and uterus
specific
These
neuronal
bradykinin vascular potent (smooth Phe7]-BK
the
and kallidin smooth
biological
receptors
injections
membrane muscle
of actions
agonists
for which been
muscle)
subtypes,
on the basis
of their
(7-8).
Bradykinin
stimulates
a variety
inc. reserved.
the kininase
cells.
Bl
via
pharmacological events,
the
by the action
receptors
(BK),
pain libers,
(5). The
hypertensive,
have
des-Argg-BK
response including
of (lys-
contract
relaxing
factor tree,
existence analgesic
of and
neurotransmitter
(6).
including
and
been
central
divided
of B2 receptors
was characterized
presynaptic
kallidin
bronchopulmonary
cell types
occupancy
receptor
into
of
release
with
in many
I metabolites
sensory
role as a possible
Kinin
mediated
of intracellular
1306
together a central present
subdivided
respective
endothelium-derived
contraction
(3). The
further
activate
and
and cytokine
and endothelial
(kininogens) bradykinin
and
motility
suggest
are
precursors
including
receptors
of kinins
potent
have
0006-29 I X/92 $4.00 Copwight 0 1992 hi Academic Press, All rights of reproduction in nay form
processes,
bradykinin,
smooth
preparations
B2
injury,
immunoreactive
by specific
large
are the peptides
of prostacyclin
sperm
are the most
muscle
to tissue
synthesis
bradykinin
majority
from
cell proliferation,
containing
of intracerebral
fibroblasts,
but
spasmogens.
other
derived
to this group
in response the
muscle,
are mediated
neurons,
B2 types,
stimulate
influence smooth
peptides
(3). Belonging
are released
and
systems
effects
actions
peripheral and
Kinins
active
kallikreins
muscle
(EDRF)
hyperthermic
of biologically
termed
into for
mainly
on rabbit
and des-ArgtO-Kallidi (neuronal)
and
to the BK analog accumulation
Bl
which
are
postsynaptic [Th&s,D-
of CAMP
and
Vol.
187,
No.
3.
cGMP,
activation
cloning
of a rat smooth
functional
BIOCHEMICAL
1992
of phospholipase
expression
muscle
AND
C and phospholipase B2 receptor
was recently
and pharmacological
BIOPHYSICAL
A2, reported
characterization
RESEARCH
and opening
of ion channels
(1). In the present
of a human
COMMUNICATIONS
bradykmin
study,
(5).
The
we report
molecular the cloning,
B2 receptor.
Material and Methods Material. The tritiated bradykinin analog [2,3,-prolyl-3,4,-3H(N)]-BK (102 Ci/mmole) and myo-[2-3H(N)]-inositol (16.5 Ci/mmole) were purchased from NEN. BK, Lys-BK, des-Arg9-BK Des-Arg9,[Leu*]-BK, [Hyp3]-BK, [DPhe7]-BK, [Th&*,D-Phe7]-BK, D-Arg,[Hyp3,D-Phe7]-BK, D-Arg,[Hyp3,Thi5.s,D-Phe7]-BK, Quin2-AM and bacitracin were purchased from Sigma. N-Adamantaneacetyl-D-Arg,[Hyp3,Thi5s8,D-Phe7]-BK and DArg,[Hyp3,Thij,D-Tic7,0ica]-BK (Hoel40) were obtained from Bachem. Library screening. A human genomic DNA library constructed in Acharon 4A was screened at high stringency (9) with the HGlO probe, a 600 bp PCR fragment amplified from genomic DNA by low stringency PCR (10-11) Sequencing was performed on both strands after subcloning in Ml3mp derivatives, using fluorescent primers and an automated DNA sequencer (Applied Biosystem 370A). Expression in Xenopus oocytes. The coding region of clone HGlO was amplified by PCR and cloned between the SalI and BumHI sites of the pFlip vector in which transcription is promoted by the SV40 early promoter. Purified plasmid DNA was resuspended in injection buffer (15 mM Hepes pH 7.0, 88 mM NaCl, 1mM KCl) and oocytes nuclei were microinjected with 10 nl of a 0.5 rig/ml DNA solution as descrihL,tl ( 12). The Ca2+-activated chloride currents were measured with a conventional two electrode voltage clamp. Expression in cell lines. The WI-BumHI PCR fragment of clone HGlO was cloned in the pSVL vector (Pharmacia). The resulting construct was co-transfected with pSV2Nco in CHQ-Kl cells as described (13), with the exception that no carrier DNA was added. Two days later, selection for transfectants was initiated by the addition of 400 pg/ml G418 (Gibco), and resistant clones were isolated at day 10. CHO-Kl cells were cultured in Ham’s F12 medium supplemented with 1 mM sodium pyruvate, 100 W/ml penicillin, 100 rig/ml streptomycin, 2.5 pg/ml amphotericin B and 1% foetal calf serum. Binding assays. Membranes were prepared from transfected CHO-Kl cells, either before or after clonal selection. Cells were rinsed once with Ca2+ and Mg 2t-free phosphate-buffered saline (PBS). After low speed centrifugation, the cell pellet was resuspended in buffer A (15 mM Tris-HCl pH 7.5,2 mM MgC12,0.3 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 PM leupeptin) and homogenized in a glass homogenizer. The crude membrane fraction was collected by centrifugation at 40,000 g for 30 min, washed once, resuspended in buffer B (7.5 mM Tris-HCl pH 7.5, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose) and flash frozen in liquid nitrogen. Protein content was assayed by the Lowry method (14). Binding assays were performed in 1.5 ml polypropylene tubes containing 0.25 ml binding buffer (25 mM potassium phosphate (pH 6.5), 0.2% BSA (fraction V, Sigma), 0.1 mM bacitracin, 1 mM dithiothreitol, 0.1 PM captopril), transfected CHO-Kl membranes (50 vg protein/tube), [3H]BK and the competing agents. After incubation for 45 mm at 4°C membranes were collected at 11,000 g, dissolved in 1 ml soluene, and counted in a liquid scintillation counter. IPJ measurements. Transfected CHO-Kl cells were seeded in glass tubes, incubated for 16 hours with 40 nCi [3H]-inositol, and further incubated as described (15) in 200 ~1 of fresh buffer containing 10 mM LiCl and BK analogs. [3H]-labelled inositol phosphates were fractionnated through an AGl-X8 column using a stepwise elution procedure (16). Intracellular free- calcium concentration ( [Ca2+]i) measurements. Intracellular free calcium measurements were carried out in CHO cells, as described previously (15) with the exception that captopril (100 FM) and BSA (1 mg/ml) were added to the incubation medium. Briefly, the cells were loaded with the Ca2+-sensitive indicator Quin-2AM and excited at 335 and 365 nm in a dual wavenlength excitation Spex CM-1 fluorimeter. The fluorescence of the indicator was recorded at 490 nm. The calibration of the fluorescence signals was performed using digitonin (30 FM) to obtain the maximal fluorescence of the Ca 2t-saturated dye, and MnClz (1 mM) to determine the autofluorescence background of the cells. The correct hydrolysis of quin-2 AM to the Ca2+-sensitive quin-2 was checked beforehand by recording the shift in the emission spectrum of the indicator. ATP (10 ,uM) was used as positive control.
Results and Discussion Cloning
and
receptors PCR clones,
structural
allowed
clones
amplified
corresponding
analysis.
the cloning from
The
sequence
by low stringency human
to a single
genomic
locus,
were
homology PCR
DNA isolated.
characterizing
of 15 new members (HGlO)
was used
A 3000 bp XL&
1307
genes
encoding
G protein-coupled
of this gene family to screen
fragment
a human was subcloned
(10,ll).
genomic
One library
in pBluescript
of the and 6 SK+
Vol.
187,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
(Stratagene) and the sequence revealed a single open reading frame of 364 codons (fig. 1) encoding a protein of 41,500 Da. The sequence surrounding the proposed initiation codon is in good agreement with the consensus as described by Kozak (17). The hydropathy profile (not shown) of the deduced amino acid sequence is consistent with the existence of 7 transmembrane segments. The amino acid sequence of clone HGlO was found to be 81% identical to that of the rat bradykinin B2 receptor described recently (1). Three potential sites for N-linked glycosylation (fig. 1) are found at identical positions in the human and rat sequences. Potential sites for phosphorylation by PKC and PKA are present in the third intracellular loop, and the C-terminal domain (fig. 1). These sites could be involved in the regulation of receptor function (18). By analogy with the p-adrenergic receptor, a conserved cysteine located in the C-terminal segment could be palmitoylated (19), and conserved serine and threonine residues could represent phosphorylation sites by PARK or PARK-related kinases (18). After completion of this work, cloning of the cDNA encoding a human B2 receptor was reported (20). Their cDNA and
-147 CTGCAG~CAGCCTGi\GCTCCACCT~GGCTTCTCC~TGCCCTGGC~GGTTGTCCT ~'A~CCCCTGT~TCCTTCTGG~\CCAGTTTTT~TCC~CCCT~GTGACCCTG~GGGGT~CA~CCTCTTTTC~ACTTTCT~~AG~GCCGAC ATGCTCnnTbTCACCTTGCAGGCCCACiCTTAACGGG~CCTTTGCCC~GAGC-TG~CCCC~GTG~AGTGGCTGG~CTG~CTC~~ MLNVTLQGPTLNCTFAQSKCPQVEWLGWLN
-91 -1 90 30
A~CA~CC~G~~CC~CTTCC'CT~GG~GC~~TTCG~GCTG~CCACCCTAG~G~CATCTT~GTCCTCAGC~TCTTCTGCC~GC~C~GA~~ F L F LBTLENIFVLSVFCL
1;:
AGCTTGGTVGiTCTGWGGGGT~TACGCTLGC~~CTGAGCTCA~CCATGCTGG~GT~CGGAC~ATG~GGAG~ACAGCGATG~GGGCCACM~ LVFRTMKEYSDEGHB rvG G~CA~CG~T;PIQTG~CATCA~CTACCCATC~CTCATCTGG~MGTGTTCA~CMCATGCT~CTGMTGTC~TGGGCTTCC~GCTGCCCCT~ SYPSLIWEVFTNMLLNVVGFLLPL
540 180
631 211
AGTG~CATC6CCT~CTGCA~GATGCAGAT~ATGCAGGT~TGCGG~C~CGAGATGCA~~GTTC~G~~GA~CC~GA~ 3 CTMQIMQVLRNNEMQKF
;:8
;:;
G~CA~GG~G~~AG~CCTGGITGTGCTGCT~CTATTCATC~~CT~CT~GC~T~CCA~A~CAGCACC~TCCTGGATA~GCTGCATCG~ LVVLLLFI STFLDTLHR
-90 : ;;
"1:; :;:
2;;
+ 1;;
l
i+:
C~CG~CA~C~~CT~CA~CT~~~TG~M~CA~~GA~CG~~TCCTTCA~~~CTACAGC~AGCTGCC~~
37; VIT
;;:
AnCCCACTG~TGTACGTGAiCGTGGGC~~CGCTTCCGA NPLVYVIVGKRFRKKSWEVYQG
990 330
3;;
TCAGAACCCATTCAGATGGiGAACTCCATGGGCACACTG~GGACCTCCA~GTGG~CGCCAGATT~AC-CTGC~GGACTGGGC~ SEPIQMENSMGTLRTS SVERQIHKLQDWA
1080 360
l!'X:
GGGAGCAGA~AGTPA~E~GCCAGCAGi;GCTGCTGTG~TTTGTGT~GGATTGAGG~ACAGTTGCT~TTCAGCATG~GCCCAGG~~ G S R Q
1170
1171 1261 1351 1441 1531 1621 1711 1801 1891 1981 2071
GCCAAGGAG~CATCTATGCiCGACCTTGG~~TGAGTT~ATGTCTCCG~T~CACC~GAGACT~T~CCTGNCCTG~CC~TTTTG~ AGGGAGCATi;GCTGTGAGGiTGGGGTG~~TCACGCACA~CC~GGACT~C~TCAC~CAGCATTA~TGTTCTTAT~TGCTGCCAC~ CCTGAGCCA~CCTGCTCCT~CCCAGGAGT~GAGGAGGCC~GGGGGCAGG~AGAGGAGTG~CTGAGCTTC~CTCCCGTGT~TTCTCCGTC~ CTGCCCCAG~MGACMC~AGATCTCCA~GAGMCTGC~ATCCAGCT TTTCTTTAAi'CTATTCAGC~AGMCTTTG~GGACAA
1260 1350 1440 1530 1620 1710 1600 1890 1980 2070
GACCAGGAT~‘TTATGGCTC~CCT~ACTGA~GGACMGGG~GGTCTGTGC~-GMGM~~~MTMGC~~ATATTGAG~A~TTG~TGT~
TATGCAGTAi'TGAGCACTG~AGGCAAGAG~GMG~GA~MGGAGCCA~CTCCATCTT~~GGMCTC~GACTC~~TGGG~CGA~ TGGCACTGC~ACCACCAGAGCTGTTCGliCGAGACGGT~GAGCAGGGT~CTGTGGGTG~TATGGACAG~AG~GGGGG~GACC~GGT~ CCAGCTCM~CMTMCTA~T~ACMCC~CCTGTCC~T~CCTCAGTTC~~T~TT~TGT~~ATGMGT~GTTGTGAGG~TT-GGCA~ TAACAGGTAi'ARnGTACTTAGARRRGCAAAGGGTGCTAC~TACATGTGA~GCATCATTA~GCAGACGT~CTGGGATAT~TTTACTAT~ GG-GACiCTGAGGTCTiGA 2092
F&&
Nucleotide
and deduced
amino acid sequences of the human B2 receptor gene. Numbering is relative to the codon. Putative transmembrane segments are indicated by the roman numbers I through VII. Potential N-linked glycosylation sites are indicated in bold characters and underlined. Potential phosphorylation sites by PKA and PKC are indicated respectively by * and +. A dot tags every tenth residue. The introa-cxon border located upstream of the open reading frame is indicated by an arrow.
putative initiation
1308
Vol.
187,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
our genomic DNA clones appear identical all along the coding and 3’ non coding sequences, but diier abruptly from base 16 ahead of the initiation codon. This interruption of homology in a context consistent with an intron acceptor site (fig. 1) strongly suggest the presence of an intron in the 5’ untranslated region of the mRNA. The 5 sequences displayed in fig. 1 represent therefore intronic material rather than 5’ untranslated sequence and upstream regulatory regions. Expression in Xenopus oocytes and CHO-Kl cells. Clone HGlO was expressed in different eukaryotic systems in order to confirm that it was encoding a BK receptor, and to characterize the functional and pharmacological
properties of the receptor. Xenopus oocytes were injected with the pFlip-HGlO construct and assayed for BKinduced chloride currents. Uninjected oocytes did not respond to any of the tested BK analogs, whereas injected oocytes displayed a 250 nA current in response to BK (not shown). The Bl agonist [des-Arg9]-BK (1 yM) and [Thi5,8,D-Phe7]-BK (1 mM) had no effect. The pSVL-HGIO construct was co-transfected with the pSV2Neo plasmid in CHO-Kl cells, and stably transfected lines were generated. Individual clones and control cells were tested for their response to BK in a [Ca2+], assay using the Quin-2 indicator. Out of the 24 clones tested, 8 responded significantly to BK whereas control cells were negative. The strongest response was obtained for clone #14 and this clone was used for all subsequent assaysand membrane preparations. Saturation binding curves with [3H]-BK as labelled ligand were consistent with a single binding site characterized by an apparent Kd of 1.50 pM (fig. 2). From the Bmax value, it was estimated that clone #14 displays approximately 8,000 binding sites per cell. Displacement curves were obtained for a variety of BK analogs reported to be either agonist or antagonist and to differentiate the BK receptor subtypes (fig. 3). Amongst the agonists, [Hyp3]-BK, which is considered as the most potent agonist for BK B2 receptors, had the lowest ICso value (60 PM), followed by BK and Lys-BK, The I& for the Bl agonist Des-Arg9-BK was over 0.1 mM, demonstrating unambiguously that clone HGlO encodes a B2 receptor. For antagonists, the highest affinity was obtained with Hoel40, a recently developped analog reported to be antagonist to all B2 and B3 subtypes (21). The order of potency of antagonists in this binding assay was Hoe140 (IC50:420 PM) > D-Arg,[Hyp3,ThiS%*,D-Phe’]-BK > D-Arg,[Hyp3,D-Phe’]-BK > [D-Phe’]-BK > [Th$*,DPhe’]-BK = N-Adamantaneacetyl-D-Arg,[Hyp3,This,8,D-Phe7]-BK.
4000
-
I
I
3H-Bradykinin
(nM)
Fig. Saturatable binding of [‘HI-BK to the human 82 receptor. Saturation biding experiment performed on a stable. CHO cell line expressing the human recombinant B2 receptor. Total(O), specific(O) and non specific(v) binding are represented. Curve fitting using a non linear regression algorithm and a one site model yielded an
apparent Kd of 150 pM.
1309
Vol.
187,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-10 -13 Log[analog]
-12
-9
-8
140
lys-EK
0
D
[Thi5’B.D-Phe7]-BK
V
v
[NA-D-Ar~“.Hy~3.Thia’n,D-Ph~‘]-DK
F~JQ.
Pharmacological
to elicit
were
measured
a response.
The
concentration
somewhat
the presence
of extracellular
the basal
level
sustained
rise
whereas
within
a clear
phosphates
continued
inactive BK
except
Phe7]-BK
(10
and
were
5E,F),
blocking
toward
the
that
higher
concentrations
Arg,[HypqTh@,D-Phe7]-BK
level
were
(fig.
PM)
4A).
The
role
at which full
D-Arg,[Hyp3,D-Phe7]-BK
addition
of any ago&tic the
when (10 PM),
BK
response
added
after
at a concentration BK.
was also effective (10 PM,
Hoe140
fig. 5G,H)
cells
assay
(fig. agonist
(not
were
partial
shown).
BK was
(InM),
[Hyp3]-
D-Arg,[Hyp3,ThiS,8,DThe
B2
(10 nh4)
and the Bl
was the most
efficient
antagonist
and reducing
antagonists
as antagonist
(not
shown).
1310
Despite (10 PM,
N-
antagonist
Des-
of BK action the elevated
N-Adamantaneacetyl-D-Arg,[Hyp3,This~8,D-Phe7]-BK,
and D-Arg,[Hyp3,D-Phe’]-BK
for
of LiCl.
Des-Arg9
and Hoe140
of 10 nM,
BK
Inositol
was higher
Lys-BK
(1 PM), agonists.
agonist.
effect
was
shown).
in presence
5), the 81 agonist
to by a
of [Ca”+]i (not
of the
for incubations
[Thi 5q8,D-Phe7]-BK
(10 FM)
activity.
addition
down
was preserved,
rise
CHO
a
to BK in
was followed
of BK, the accumulation as expected
it was a weak
,uM)
response
able
30 pM,
went
of the response
in transfected after
around
that gradually
initial
phase
effect
PM) the response
of IP3 in the BK dependent
as 15 seconds
agonists. (10
S,8,D-Phe7]-BK
devoid
this
accumulation
after
(~30
of (Ca2+]i,
concentration
in the [Ca’+]i
were
increase
Ca 2+, the transient
as early
tested
over 1 VM (10
completely
basal
higher
up to 15 minutes
Adamantaneacetyl-D-Arg,[Hyp3,Thi Arg9,[Leu8]-BK
At
than for IP2 and IP3 (2 fold increase),
[D-Phe7]-BK
PM)
4D).
of extracellular
as low as 1 pM were
and the half maximal
At low concentrations
by a rapid
concentrations
to accumulate level)
at 300 pM,
Kd value.
of inositol-phosphate
in IP3
at concentrations and
(fig.
was abolished
the BK analogs
(1 nM),
was obtained
In the absence phase
to BK (fig. 4). BK concentrations
was characterized
2 to 3 minutes
increase
in response
effect
measurement
IPl (10 fold the basal Amongst
#14
than the apparent Ca2+
of [Caz+]i.
by the direct
promoted
maximal lower
the sustained
verified
in clone
0
of the human BZ receptor. Displacement curves were obtained for a (A) or antagonists (B) of B2 receptors. The ordinate is expressed as
characterization
variety of analogs reported as agonists percentage of the total specific binding.
[Ca2+]i
-5
.
[D-Phe’]-BK
0
-6
.
D-Arg.[Hyp3.D-Phe’]-BK
0
Des-Arg’-BK
-7 (Id)
D-Arg.[Hyp3.ThiS”.D-Ph~‘]-EK
.
BK
-10
Log(analog] HOE
[Hyp*]-BK
-1,
(M)
(fig.
[Ca2+]i at much
their
agonistic
not shown)
activity, were
D-
able to
Vol.
187,
No.
3,
A
BIOCHEMICAL
1992
EGTA
Bk.
I
900 BOO 700
AND
BIOPHYSICAL
RESEARCH
C
ATP 900
I rl
I
2, ,(
400 300 200 100
12
.” 500 400 300 “2 zoo i
1000 900 800 700 600 . 500 400 ^ 300 200 100 O-
02
lo:t 0
6
12 Time
100
,000
(PM)
0
+-
JP
10
[Elradykinine]
1000 900 800 700 600
s E
1
0
18 ATP 10 UM
Bk. 333 DM
t3
mean peak [CE?+]~
500
2
6
r
.
E
700 600
5z 9 -t
0
COMMUNICATIONS
0
10
1
2
3
4
5
6
7
Time (min.)
(min.)
F~J& Functional coupling of the human BZ receptor in CHO ceils. A: A CHO clonal cell Line stably transfected with pSVL-HGIO (done #14) was assayed for [Ca’+]i in response to BK and ATP, in presence (plain line) or absence (dashed line) of extracellular Ca*+. B: The JP02 line, transfected with pSV2neo alone was used as control. Vertical bars indicate the addition of agents. C: dose response curve of [Ca*+], values averaged at the peak of the response to BK (about 1 min after the agonist addition). Error bars represent the standarddeviation on the mean. The data from 3 independent experiments were pooled. D: [Ca*+]i response elicited by the addition of BK (3,30, 100, and 300 PM) in transfected CHO cells (clone #14). Each trace in panels A, B and D is representative of at least 3 independent experiments.
antagonize
partially
Phe7]-BK
were
Subtyping
HGlO
of the analog
“muscular”
guinea
receptor
pig
(7,8).
trachea,
as
Ms,Ww3,Thi
S,8D-Phe7]-BK ,
subsequently
demonstrated
(22),
suggesting
Moreover, other
in many
from In our
early
retained
an
that
tissues agents,
the biological transfected
used
Bl antagonist
receptor.
B2 receptors
[Th@,D-Phe7]-BK Similarly,
have been which
Farmer
as well as [D-Phe7]-BK
is agonist
to
and
[Hyp3,D-Phe7]-BK,
D-Arg,
other
the
preparations
observation
taken
in agonistic
or antagonistic
as bioassays,
the biological
and receptors response
for these
and [Th&*,D-
agonistic
Adamantaneacetyl-D-Arg,[Hyp3,Thi5,*,D-Phe7]-BK
properties.
guinea properties
response
BK receptor,
actually
the acted
two
classical
recently
as antagonists
1311
this
species
involving
in
as
D-
It
was
profile differences.
the release
interaction
of
is therefore
(23). B2 agonists agonists. were
developped with
such
preparation.
reflect
on
subtype
pharmacological
indirectly,
act as full or partial
and D-Arg,[Hyp3,D-Phe7]-BK Only
on
pig have a similar
reduced
and antagonist
antagonists,
The kinin-receptor
of the bioassay
the human
82
as agonists
is evoked
mediators.
on the basis
of a B3 receptor
classical
could
essentially
subtype
the existence
act
and [Th?,*,D-Phe7]-BK,
into subtypes,
on the “neuron&
that
from
secondary
and the precision
cell line expressing [D-Phe’]-BK
subdivided
et al. (2) proposed
explanation
D-Arg,[Hyp3,Th&*,D-Phe7]-BK moderate
des-Arg9,[Leu8]-BK
as antagonists.
differences
B2 antagonists,
B2 antagonists but
that
endogenous
remote
of BK. The
ineffective
of the human
of the properties the
the effect
totally
were
the most
The
second
able to inhibit antagonists
no residual
agonistic
active.
The
generation
a BK response, Hoe140 property.
and
N-
Vol.
187,
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No.
1
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2
3
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DP-Ek 10 ,LM Bk 100 $4
Fig, Agonistic and antagonistic properties of BK analogs. A-D: The ago&tic properties of des-Arg9-BK (dABK), Lys-BK, [D-Phe’J-BK (DP-BK), and [ThiS%s,D-Phe7]-BK (TDP-BK) were assayed on CHO cells expressing the human recombinant BK receptor (clone #14). E-H: The antagonistic properties of Hoe140 and DArg,[Hyp3,This~E,D-Phe7]-BK (DAHTDP) were assayed upon addition either before (E,G) or after (F,H) the BK addition. The presence of the agents in the incubation medium is represented by straight lines over the trace. The control response to BK (100 PM) is indicated as a dashed trace. Each trace is representative of duplicate coverslips from at least two independent experiments. The results obtained for [Hyp3]-BK (1 nM), N-Adamantaneacety-DArg [Hyp3,Thi5,*,D-Phe’]-BK (10 PM) and D-Arg,(Hyp3,D-Phe’]-BK (10 PM) were identical to those displayed reqkxtively for Lys-BK (B), Hoe140 (E,F) and D-Arg,[Hyp3,Thi5,8,D-Phe7j-BK (G,H).
Altogether,
binding
and functional
assays
on CHO
cells would
class our human
receptor
as a B2 receptor
of
with some properties of a B3 receptor the neuronal subtype (agonistic properties of [Tlu ‘5s8,D-Phe7]-BK), (agonistic properties of D-Arg,[Hyp3,Thi5)8,D-Phe7]-BK and D-Arg,[Hyp3,D-Phe7]-BK). However, when the same response. receptor was expressed in xenopus oocytes, [Thl‘5,8,D-Phe7]-BK was unable to generate a biological Similarly, the rat ortholog of our human receptor has been reported as a B2 receptor of the muscular type, based on the antagonistic properties of the same analog [Thiss8,D-Phe7]-BK (7-8). We therefore feel that the respective properties of the so-called neuronal, muscular and B3 subtypes could rather be the reflect of species differences, and/or of the characteristics of G protein-coupling in different cell types. Future work dealing with the expression of different levels of the cloned human receptor in various cell types could be of great help in the clarification of the BK receptor classification.
Acknowledgments We are grateful to Dr. J.E. Dumont for continual support, and to Dr S. Swillens for computer work. This work was supported by the Belgian programme on Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. It was also supported by the For&s de la Recherche Scientijique Mkdicale of Belgium, Boehringer Ingelheim and the Association Recherrhe Biomkiicale et Diagnostic. 1312
Vol.
187,
No.
3,
1992
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BIOPHYSICAL
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The scientific responsibility is assumed by the authors. D.E. and E.R. are respectively fellows of the IRSfA and Televie. M.P. is Chercheur Qua@! of the Fonds National de la Recherche Scientifique.
References 1. McEachern, A.E., Shelton, E.R., Bhakta, S., Obernolte, R., Bach, C., Zuppan, P., Fujisaki, J., Aldrich, R.W. and Jarnagin, K. (1991). Proc. Natl. Acad. Sci. USA 88: 7724-7728. 2. Farmer, S.G., Burch, R.M., Meeker, S.A. and Wilkins, D.E. (1989). Mol. Pharmacol. 36: 1-8. 3. Regoli, D., and Barabe, J. (1980). Pharmacol. Rev. 32: l-46. 4. Plevin, R. and Owen, P.J. (1988) Trends Pharmacol. Sci. 91: 387-389. 5. Bhoola, K.D., Figueiroa, C.D. and Worthy, K. (1992). Pharmacol. Rev. 44: l-80. 6. Steranka, L.R., Marming, D.C., De Haas, C.J., Ferkany, J.W., Borosky, SA., Connor, J.R., Vavrek, R.J., Stewart, J.N. and Snyder, S.H. (1988) Proc. Natl. Acad. Sci. USA 85: 3245-3249. 7. Braas, K.M., Manning, D.C., Perry, D.C. and Snyder, S.H. (1988) Br. J. Pharmacol. 94: 3-5. 8. Llona, I., Vavrek, R., Stewart, J. and Huidobro-Toro, J.P. (1987) J. Pharmacol. Exp. Ther. 241: 608-614. 9. Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. 10. Libert, F., Parmentier, M., Lefort, A., Dinsart, C., Van Sande, J., Maenhaut, C., Simons, M.J., Dumont, J.E. and Vassart, G. (1989) Science 2441 569-572. 11. Parmentier, M., Libert, F., Maenhaut, C., Lefort, A., Gerard, C., Perret, J., Van Sande, J., Dumont, J.E. and Vassart, G. (1989). Science 246: 1620-1622. 12. Voelhny, R., Ahmed, A., Schiller, P., Bromley, P., and Rungger, D. (1985) Proc. Natl. Acad. Sci. USA 82: 49494953. 13. Velu, T.J., Beguinot, L., Vass, W.C., Zhang, K., Pastan, I. and Lowry, D.R. (1989) J. Cell. Biochem. 39: 153166. 14. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193: 265-275. 15. Van Sande, J., Raspt, E., Perret, J., Lejeune, C., Maenhaut, C., Vassart, G. and Dumont, J.E. (1990) Mol. and Cell. Endo. 74: Rl-R6. 16. Berridge, M.J. (1983) Biochem. J. 212: 849-858. 17. Kozak, M. (1989) J. Cell. Biol. 108: 229-241. 18. Dohhnann, H.G., Thorner, J., Caron, M.G. and Lefkowitz R.J. (1991) Anuu. Rev. Biochem. 60: 653-688. 19. O’Dowd, B.F., Hnatowich, M., Caron, M.G., Lefkowitz, R.J. and Bouvier M. (1989). J. Biol. Chem. 264: 75647569. 20. Hess, J.F., Borkowski, JA., Gretcher, S.Y., Strader, C.D. and Ransom, R.W. (1992) Biochem. Biophys. Res. Commun. 184: 260-268. 21. Wirth, K., Hock, F.J., Albus, U., Linz, W., Alpermann, H.G., Anagnostopoulos, H., Her&e, St., Breipohl, G., Konig, W., Knolle, J. and Scholkens, B.A. (1991) Br. J. Pharmacol. 102: 774-777. 22. Field, J.L., Hall, J.M. and Morton, K.M. (1992). Br. J. Pharmacol. 105: 293-296. 23. Regoli, D., Rhaleb, N.E., Dion, S. and Drapeau, G. (1991). Trends Pharmacol. Sci. 11: 155-161.
1313
187,
No.
September
3, 1992
30,
BIOCHEMICAL
BIOPHYSICAL
RESEARCH
Eggerickx’,
Gilbert
and 2Service
de GCnCtique
3DCpartement
Raspel,
Daniel
and Marc
Mtdicale,
de Lennik,
313
Servet,
Bertrand3,
Parmentierl
Universite B-1070
de Physiologie,
1 rue Michel
27,
Eric
Vassart1,2
808 route
July
1306-l
FUNCTIONAL EXPRESSION AND P HARMACOLOGICAL OF A HUMAN BRADYKININ B2 RECEPTOR GENE
Dominique
‘IRIBHN
COMMUNICATIONS Pages
MOLECULAR CLONING, CHARACTERIZATION
Received
AND
1992
libre
de Bruxelles,
Bruxelles,
Centre
Medical
120.5, Genke,
Campus
Erasme,
Belgium Universitaire,
Switzerland
1992
The gene encoding a putative G protein-coupled receptor (HGlO) was cloned from human genomic DNA by low stringency PCR and found to be homologous to the recently described rat bradykinin B2 receptor (1). The receptor was expressed in xenopus oocytes and stably transfected CHO cell lines. Binding studies demonstrated that HGIO encodes a high affinity BK receptor with an apparent Kd of 150 pM. Displacement by BK agonists and antagonists allowed the characterization of the receptor as a B2 subtype. Functional coupling to the Ca2+phosphatidylinositol cascade was demonstrated in transfected CHO cells where inositol phosphates accumulation and intracellular calcium concentration were elevated in response to BK stimulation. The agonistic and antagonistic properties of BK analogs do not match strictly the pharmacological profile described for the rat or guinea pig B2 receptor subtypes or the putative B3 subtype (2). This discrepancy is attributed either to species variability or to differences in the coupling efficiency of receptors to the transduction cascade in different cell types. From our results, the existence of B3 receptors and of B2 subtypes appears questionable. ‘d 1992 Acadrmrc Press, 1°C.
Kinins serine
are a group
proteases,
BK)
and met-lys-BK.
venous
smooth
Kinins
(4).
intestine
and uterus
specific
These
neuronal
bradykinin vascular potent (smooth Phe7]-BK
the
and kallidin smooth
biological
receptors
injections
membrane muscle
of actions
agonists
for which been
muscle)
subtypes,
on the basis
of their
(7-8).
Bradykinin
stimulates
a variety
inc. reserved.
the kininase
cells.
Bl
via
pharmacological events,
the
by the action
receptors
(BK),
pain libers,
(5). The
hypertensive,
have
des-Argg-BK
response including
of (lys-
contract
relaxing
factor tree,
existence analgesic
of and
neurotransmitter
(6).
including
and
been
central
divided
of B2 receptors
was characterized
presynaptic
kallidin
bronchopulmonary
cell types
occupancy
receptor
into
of
release
with
in many
I metabolites
sensory
role as a possible
Kinin
mediated
of intracellular
1306
together a central present
subdivided
respective
endothelium-derived
contraction
(3). The
further
activate
and
and cytokine
and endothelial
(kininogens) bradykinin
and
motility
suggest
are
precursors
including
receptors
of kinins
potent
have
0006-29 I X/92 $4.00 Copwight 0 1992 hi Academic Press, All rights of reproduction in nay form
processes,
bradykinin,
smooth
preparations
B2
injury,
immunoreactive
by specific
large
are the peptides
of prostacyclin
sperm
are the most
muscle
to tissue
synthesis
bradykinin
majority
from
cell proliferation,
containing
of intracerebral
fibroblasts,
but
spasmogens.
other
derived
to this group
in response the
muscle,
are mediated
neurons,
B2 types,
stimulate
influence smooth
peptides
(3). Belonging
are released
and
systems
effects
actions
peripheral and
Kinins
active
kallikreins
muscle
(EDRF)
hyperthermic
of biologically
termed
into for
mainly
on rabbit
and des-ArgtO-Kallidi (neuronal)
and
to the BK analog accumulation
Bl
which
are
postsynaptic [Th&s,D-
of CAMP
and
Vol.
187,
No.
3.
cGMP,
activation
cloning
of a rat smooth
functional
BIOCHEMICAL
1992
of phospholipase
expression
muscle
AND
C and phospholipase B2 receptor
was recently
and pharmacological
BIOPHYSICAL
A2, reported
characterization
RESEARCH
and opening
of ion channels
(1). In the present
of a human
COMMUNICATIONS
bradykmin
study,
(5).
The
we report
molecular the cloning,
B2 receptor.
Material and Methods Material. The tritiated bradykinin analog [2,3,-prolyl-3,4,-3H(N)]-BK (102 Ci/mmole) and myo-[2-3H(N)]-inositol (16.5 Ci/mmole) were purchased from NEN. BK, Lys-BK, des-Arg9-BK Des-Arg9,[Leu*]-BK, [Hyp3]-BK, [DPhe7]-BK, [Th&*,D-Phe7]-BK, D-Arg,[Hyp3,D-Phe7]-BK, D-Arg,[Hyp3,Thi5.s,D-Phe7]-BK, Quin2-AM and bacitracin were purchased from Sigma. N-Adamantaneacetyl-D-Arg,[Hyp3,Thi5s8,D-Phe7]-BK and DArg,[Hyp3,Thij,D-Tic7,0ica]-BK (Hoel40) were obtained from Bachem. Library screening. A human genomic DNA library constructed in Acharon 4A was screened at high stringency (9) with the HGlO probe, a 600 bp PCR fragment amplified from genomic DNA by low stringency PCR (10-11) Sequencing was performed on both strands after subcloning in Ml3mp derivatives, using fluorescent primers and an automated DNA sequencer (Applied Biosystem 370A). Expression in Xenopus oocytes. The coding region of clone HGlO was amplified by PCR and cloned between the SalI and BumHI sites of the pFlip vector in which transcription is promoted by the SV40 early promoter. Purified plasmid DNA was resuspended in injection buffer (15 mM Hepes pH 7.0, 88 mM NaCl, 1mM KCl) and oocytes nuclei were microinjected with 10 nl of a 0.5 rig/ml DNA solution as descrihL,tl ( 12). The Ca2+-activated chloride currents were measured with a conventional two electrode voltage clamp. Expression in cell lines. The WI-BumHI PCR fragment of clone HGlO was cloned in the pSVL vector (Pharmacia). The resulting construct was co-transfected with pSV2Nco in CHQ-Kl cells as described (13), with the exception that no carrier DNA was added. Two days later, selection for transfectants was initiated by the addition of 400 pg/ml G418 (Gibco), and resistant clones were isolated at day 10. CHO-Kl cells were cultured in Ham’s F12 medium supplemented with 1 mM sodium pyruvate, 100 W/ml penicillin, 100 rig/ml streptomycin, 2.5 pg/ml amphotericin B and 1% foetal calf serum. Binding assays. Membranes were prepared from transfected CHO-Kl cells, either before or after clonal selection. Cells were rinsed once with Ca2+ and Mg 2t-free phosphate-buffered saline (PBS). After low speed centrifugation, the cell pellet was resuspended in buffer A (15 mM Tris-HCl pH 7.5,2 mM MgC12,0.3 mM EDTA, 1 mM EGTA, 1 mM PMSF, 1 PM leupeptin) and homogenized in a glass homogenizer. The crude membrane fraction was collected by centrifugation at 40,000 g for 30 min, washed once, resuspended in buffer B (7.5 mM Tris-HCl pH 7.5, 12.5 mM MgCl2, 0.3 mM EDTA, 1 mM EGTA, 250 mM sucrose) and flash frozen in liquid nitrogen. Protein content was assayed by the Lowry method (14). Binding assays were performed in 1.5 ml polypropylene tubes containing 0.25 ml binding buffer (25 mM potassium phosphate (pH 6.5), 0.2% BSA (fraction V, Sigma), 0.1 mM bacitracin, 1 mM dithiothreitol, 0.1 PM captopril), transfected CHO-Kl membranes (50 vg protein/tube), [3H]BK and the competing agents. After incubation for 45 mm at 4°C membranes were collected at 11,000 g, dissolved in 1 ml soluene, and counted in a liquid scintillation counter. IPJ measurements. Transfected CHO-Kl cells were seeded in glass tubes, incubated for 16 hours with 40 nCi [3H]-inositol, and further incubated as described (15) in 200 ~1 of fresh buffer containing 10 mM LiCl and BK analogs. [3H]-labelled inositol phosphates were fractionnated through an AGl-X8 column using a stepwise elution procedure (16). Intracellular free- calcium concentration ( [Ca2+]i) measurements. Intracellular free calcium measurements were carried out in CHO cells, as described previously (15) with the exception that captopril (100 FM) and BSA (1 mg/ml) were added to the incubation medium. Briefly, the cells were loaded with the Ca2+-sensitive indicator Quin-2AM and excited at 335 and 365 nm in a dual wavenlength excitation Spex CM-1 fluorimeter. The fluorescence of the indicator was recorded at 490 nm. The calibration of the fluorescence signals was performed using digitonin (30 FM) to obtain the maximal fluorescence of the Ca 2t-saturated dye, and MnClz (1 mM) to determine the autofluorescence background of the cells. The correct hydrolysis of quin-2 AM to the Ca2+-sensitive quin-2 was checked beforehand by recording the shift in the emission spectrum of the indicator. ATP (10 ,uM) was used as positive control.
Results and Discussion Cloning
and
receptors PCR clones,
structural
allowed
clones
amplified
corresponding
analysis.
the cloning from
The
sequence
by low stringency human
to a single
genomic
locus,
were
homology PCR
DNA isolated.
characterizing
of 15 new members (HGlO)
was used
A 3000 bp XL&
1307
genes
encoding
G protein-coupled
of this gene family to screen
fragment
a human was subcloned
(10,ll).
genomic
One library
in pBluescript
of the and 6 SK+
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187,
No.
3,
1992
BIOCHEMICAL
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RESEARCH
COMMUNICATIONS
(Stratagene) and the sequence revealed a single open reading frame of 364 codons (fig. 1) encoding a protein of 41,500 Da. The sequence surrounding the proposed initiation codon is in good agreement with the consensus as described by Kozak (17). The hydropathy profile (not shown) of the deduced amino acid sequence is consistent with the existence of 7 transmembrane segments. The amino acid sequence of clone HGlO was found to be 81% identical to that of the rat bradykinin B2 receptor described recently (1). Three potential sites for N-linked glycosylation (fig. 1) are found at identical positions in the human and rat sequences. Potential sites for phosphorylation by PKC and PKA are present in the third intracellular loop, and the C-terminal domain (fig. 1). These sites could be involved in the regulation of receptor function (18). By analogy with the p-adrenergic receptor, a conserved cysteine located in the C-terminal segment could be palmitoylated (19), and conserved serine and threonine residues could represent phosphorylation sites by PARK or PARK-related kinases (18). After completion of this work, cloning of the cDNA encoding a human B2 receptor was reported (20). Their cDNA and
-147 CTGCAG~CAGCCTGi\GCTCCACCT~GGCTTCTCC~TGCCCTGGC~GGTTGTCCT ~'A~CCCCTGT~TCCTTCTGG~\CCAGTTTTT~TCC~CCCT~GTGACCCTG~GGGGT~CA~CCTCTTTTC~ACTTTCT~~AG~GCCGAC ATGCTCnnTbTCACCTTGCAGGCCCACiCTTAACGGG~CCTTTGCCC~GAGC-TG~CCCC~GTG~AGTGGCTGG~CTG~CTC~~ MLNVTLQGPTLNCTFAQSKCPQVEWLGWLN
-91 -1 90 30
A~CA~CC~G~~CC~CTTCC'CT~GG~GC~~TTCG~GCTG~CCACCCTAG~G~CATCTT~GTCCTCAGC~TCTTCTGCC~GC~C~GA~~ F L F LBTLENIFVLSVFCL
1;:
AGCTTGGTVGiTCTGWGGGGT~TACGCTLGC~~CTGAGCTCA~CCATGCTGG~GT~CGGAC~ATG~GGAG~ACAGCGATG~GGGCCACM~ LVFRTMKEYSDEGHB rvG G~CA~CG~T;PIQTG~CATCA~CTACCCATC~CTCATCTGG~MGTGTTCA~CMCATGCT~CTGMTGTC~TGGGCTTCC~GCTGCCCCT~ SYPSLIWEVFTNMLLNVVGFLLPL
540 180
631 211
AGTG~CATC6CCT~CTGCA~GATGCAGAT~ATGCAGGT~TGCGG~C~CGAGATGCA~~GTTC~G~~GA~CC~GA~ 3 CTMQIMQVLRNNEMQKF
;:8
;:;
G~CA~GG~G~~AG~CCTGGITGTGCTGCT~CTATTCATC~~CT~CT~GC~T~CCA~A~CAGCACC~TCCTGGATA~GCTGCATCG~ LVVLLLFI STFLDTLHR
-90 : ;;
"1:; :;:
2;;
+ 1;;
l
i+:
C~CG~CA~C~~CT~CA~CT~~~TG~M~CA~~GA~CG~~TCCTTCA~~~CTACAGC~AGCTGCC~~
37; VIT
;;:
AnCCCACTG~TGTACGTGAiCGTGGGC~~CGCTTCCGA NPLVYVIVGKRFRKKSWEVYQG
990 330
3;;
TCAGAACCCATTCAGATGGiGAACTCCATGGGCACACTG~GGACCTCCA~GTGG~CGCCAGATT~AC-CTGC~GGACTGGGC~ SEPIQMENSMGTLRTS SVERQIHKLQDWA
1080 360
l!'X:
GGGAGCAGA~AGTPA~E~GCCAGCAGi;GCTGCTGTG~TTTGTGT~GGATTGAGG~ACAGTTGCT~TTCAGCATG~GCCCAGG~~ G S R Q
1170
1171 1261 1351 1441 1531 1621 1711 1801 1891 1981 2071
GCCAAGGAG~CATCTATGCiCGACCTTGG~~TGAGTT~ATGTCTCCG~T~CACC~GAGACT~T~CCTGNCCTG~CC~TTTTG~ AGGGAGCATi;GCTGTGAGGiTGGGGTG~~TCACGCACA~CC~GGACT~C~TCAC~CAGCATTA~TGTTCTTAT~TGCTGCCAC~ CCTGAGCCA~CCTGCTCCT~CCCAGGAGT~GAGGAGGCC~GGGGGCAGG~AGAGGAGTG~CTGAGCTTC~CTCCCGTGT~TTCTCCGTC~ CTGCCCCAG~MGACMC~AGATCTCCA~GAGMCTGC~ATCCAGCT TTTCTTTAAi'CTATTCAGC~AGMCTTTG~GGACAA
1260 1350 1440 1530 1620 1710 1600 1890 1980 2070
GACCAGGAT~‘TTATGGCTC~CCT~ACTGA~GGACMGGG~GGTCTGTGC~-GMGM~~~MTMGC~~ATATTGAG~A~TTG~TGT~
TATGCAGTAi'TGAGCACTG~AGGCAAGAG~GMG~GA~MGGAGCCA~CTCCATCTT~~GGMCTC~GACTC~~TGGG~CGA~ TGGCACTGC~ACCACCAGAGCTGTTCGliCGAGACGGT~GAGCAGGGT~CTGTGGGTG~TATGGACAG~AG~GGGGG~GACC~GGT~ CCAGCTCM~CMTMCTA~T~ACMCC~CCTGTCC~T~CCTCAGTTC~~T~TT~TGT~~ATGMGT~GTTGTGAGG~TT-GGCA~ TAACAGGTAi'ARnGTACTTAGARRRGCAAAGGGTGCTAC~TACATGTGA~GCATCATTA~GCAGACGT~CTGGGATAT~TTTACTAT~ GG-GACiCTGAGGTCTiGA 2092
F&&
Nucleotide
and deduced
amino acid sequences of the human B2 receptor gene. Numbering is relative to the codon. Putative transmembrane segments are indicated by the roman numbers I through VII. Potential N-linked glycosylation sites are indicated in bold characters and underlined. Potential phosphorylation sites by PKA and PKC are indicated respectively by * and +. A dot tags every tenth residue. The introa-cxon border located upstream of the open reading frame is indicated by an arrow.
putative initiation
1308
Vol.
187,
No.
3,
1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
our genomic DNA clones appear identical all along the coding and 3’ non coding sequences, but diier abruptly from base 16 ahead of the initiation codon. This interruption of homology in a context consistent with an intron acceptor site (fig. 1) strongly suggest the presence of an intron in the 5’ untranslated region of the mRNA. The 5 sequences displayed in fig. 1 represent therefore intronic material rather than 5’ untranslated sequence and upstream regulatory regions. Expression in Xenopus oocytes and CHO-Kl cells. Clone HGlO was expressed in different eukaryotic systems in order to confirm that it was encoding a BK receptor, and to characterize the functional and pharmacological
properties of the receptor. Xenopus oocytes were injected with the pFlip-HGlO construct and assayed for BKinduced chloride currents. Uninjected oocytes did not respond to any of the tested BK analogs, whereas injected oocytes displayed a 250 nA current in response to BK (not shown). The Bl agonist [des-Arg9]-BK (1 yM) and [Thi5,8,D-Phe7]-BK (1 mM) had no effect. The pSVL-HGIO construct was co-transfected with the pSV2Neo plasmid in CHO-Kl cells, and stably transfected lines were generated. Individual clones and control cells were tested for their response to BK in a [Ca2+], assay using the Quin-2 indicator. Out of the 24 clones tested, 8 responded significantly to BK whereas control cells were negative. The strongest response was obtained for clone #14 and this clone was used for all subsequent assaysand membrane preparations. Saturation binding curves with [3H]-BK as labelled ligand were consistent with a single binding site characterized by an apparent Kd of 1.50 pM (fig. 2). From the Bmax value, it was estimated that clone #14 displays approximately 8,000 binding sites per cell. Displacement curves were obtained for a variety of BK analogs reported to be either agonist or antagonist and to differentiate the BK receptor subtypes (fig. 3). Amongst the agonists, [Hyp3]-BK, which is considered as the most potent agonist for BK B2 receptors, had the lowest ICso value (60 PM), followed by BK and Lys-BK, The I& for the Bl agonist Des-Arg9-BK was over 0.1 mM, demonstrating unambiguously that clone HGlO encodes a B2 receptor. For antagonists, the highest affinity was obtained with Hoel40, a recently developped analog reported to be antagonist to all B2 and B3 subtypes (21). The order of potency of antagonists in this binding assay was Hoe140 (IC50:420 PM) > D-Arg,[Hyp3,ThiS%*,D-Phe’]-BK > D-Arg,[Hyp3,D-Phe’]-BK > [D-Phe’]-BK > [Th$*,DPhe’]-BK = N-Adamantaneacetyl-D-Arg,[Hyp3,This,8,D-Phe7]-BK.
4000
-
I
I
3H-Bradykinin
(nM)
Fig. Saturatable binding of [‘HI-BK to the human 82 receptor. Saturation biding experiment performed on a stable. CHO cell line expressing the human recombinant B2 receptor. Total(O), specific(O) and non specific(v) binding are represented. Curve fitting using a non linear regression algorithm and a one site model yielded an
apparent Kd of 150 pM.
1309
Vol.
187,
No.
3,
BIOCHEMICAL
1992
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
-10 -13 Log[analog]
-12
-9
-8
140
lys-EK
0
D
[Thi5’B.D-Phe7]-BK
V
v
[NA-D-Ar~“.Hy~3.Thia’n,D-Ph~‘]-DK
F~JQ.
Pharmacological
to elicit
were
measured
a response.
The
concentration
somewhat
the presence
of extracellular
the basal
level
sustained
rise
whereas
within
a clear
phosphates
continued
inactive BK
except
Phe7]-BK
(10
and
were
5E,F),
blocking
toward
the
that
higher
concentrations
Arg,[HypqTh@,D-Phe7]-BK
level
were
(fig.
PM)
4A).
The
role
at which full
D-Arg,[Hyp3,D-Phe7]-BK
addition
of any ago&tic the
when (10 PM),
BK
response
added
after
at a concentration BK.
was also effective (10 PM,
Hoe140
fig. 5G,H)
cells
assay
(fig. agonist
(not
were
partial
shown).
BK was
(InM),
[Hyp3]-
D-Arg,[Hyp3,ThiS,8,DThe
B2
(10 nh4)
and the Bl
was the most
efficient
antagonist
and reducing
antagonists
as antagonist
(not
shown).
1310
Despite (10 PM,
N-
antagonist
Des-
of BK action the elevated
N-Adamantaneacetyl-D-Arg,[Hyp3,This~8,D-Phe7]-BK,
and D-Arg,[Hyp3,D-Phe’]-BK
for
of LiCl.
Des-Arg9
and Hoe140
of 10 nM,
BK
Inositol
was higher
Lys-BK
(1 PM), agonists.
agonist.
effect
was
shown).
in presence
5), the 81 agonist
to by a
of [Ca”+]i (not
of the
for incubations
[Thi 5q8,D-Phe7]-BK
(10 FM)
activity.
addition
down
was preserved,
rise
CHO
a
to BK in
was followed
of BK, the accumulation as expected
it was a weak
,uM)
response
able
30 pM,
went
of the response
in transfected after
around
that gradually
initial
phase
effect
PM) the response
of IP3 in the BK dependent
as 15 seconds
agonists. (10
S,8,D-Phe7]-BK
devoid
this
accumulation
after
(~30
of (Ca2+]i,
concentration
in the [Ca’+]i
were
increase
Ca 2+, the transient
as early
tested
over 1 VM (10
completely
basal
higher
up to 15 minutes
Adamantaneacetyl-D-Arg,[Hyp3,Thi Arg9,[Leu8]-BK
At
than for IP2 and IP3 (2 fold increase),
[D-Phe7]-BK
PM)
4D).
of extracellular
as low as 1 pM were
and the half maximal
At low concentrations
by a rapid
concentrations
to accumulate level)
at 300 pM,
Kd value.
of inositol-phosphate
in IP3
at concentrations and
(fig.
was abolished
the BK analogs
(1 nM),
was obtained
In the absence phase
to BK (fig. 4). BK concentrations
was characterized
2 to 3 minutes
increase
in response
effect
measurement
IPl (10 fold the basal Amongst
#14
than the apparent Ca2+
of [Caz+]i.
by the direct
promoted
maximal lower
the sustained
verified
in clone
0
of the human BZ receptor. Displacement curves were obtained for a (A) or antagonists (B) of B2 receptors. The ordinate is expressed as
characterization
variety of analogs reported as agonists percentage of the total specific binding.
[Ca2+]i
-5
.
[D-Phe’]-BK
0
-6
.
D-Arg.[Hyp3.D-Phe’]-BK
0
Des-Arg’-BK
-7 (Id)
D-Arg.[Hyp3.ThiS”.D-Ph~‘]-EK
.
BK
-10
Log(analog] HOE
[Hyp*]-BK
-1,
(M)
(fig.
[Ca2+]i at much
their
agonistic
not shown)
activity, were
D-
able to
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BIOCHEMICAL
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EGTA
Bk.
I
900 BOO 700
AND
BIOPHYSICAL
RESEARCH
C
ATP 900
I rl
I
2, ,(
400 300 200 100
12
.” 500 400 300 “2 zoo i
1000 900 800 700 600 . 500 400 ^ 300 200 100 O-
02
lo:t 0
6
12 Time
100
,000
(PM)
0
+-
JP
10
[Elradykinine]
1000 900 800 700 600
s E
1
0
18 ATP 10 UM
Bk. 333 DM
t3
mean peak [CE?+]~
500
2
6
r
.
E
700 600
5z 9 -t
0
COMMUNICATIONS
0
10
1
2
3
4
5
6
7
Time (min.)
(min.)
F~J& Functional coupling of the human BZ receptor in CHO ceils. A: A CHO clonal cell Line stably transfected with pSVL-HGIO (done #14) was assayed for [Ca’+]i in response to BK and ATP, in presence (plain line) or absence (dashed line) of extracellular Ca*+. B: The JP02 line, transfected with pSV2neo alone was used as control. Vertical bars indicate the addition of agents. C: dose response curve of [Ca*+], values averaged at the peak of the response to BK (about 1 min after the agonist addition). Error bars represent the standarddeviation on the mean. The data from 3 independent experiments were pooled. D: [Ca*+]i response elicited by the addition of BK (3,30, 100, and 300 PM) in transfected CHO cells (clone #14). Each trace in panels A, B and D is representative of at least 3 independent experiments.
antagonize
partially
Phe7]-BK
were
Subtyping
HGlO
of the analog
“muscular”
guinea
receptor
pig
(7,8).
trachea,
as
Ms,Ww3,Thi
S,8D-Phe7]-BK ,
subsequently
demonstrated
(22),
suggesting
Moreover, other
in many
from In our
early
retained
an
that
tissues agents,
the biological transfected
used
Bl antagonist
receptor.
B2 receptors
[Th@,D-Phe7]-BK Similarly,
have been which
Farmer
as well as [D-Phe7]-BK
is agonist
to
and
[Hyp3,D-Phe7]-BK,
D-Arg,
other
the
preparations
observation
taken
in agonistic
or antagonistic
as bioassays,
the biological
and receptors response
for these
and [Th&*,D-
agonistic
Adamantaneacetyl-D-Arg,[Hyp3,Thi5,*,D-Phe7]-BK
properties.
guinea properties
response
BK receptor,
actually
the acted
two
classical
recently
as antagonists
1311
this
species
involving
in
as
D-
It
was
profile differences.
the release
interaction
of
is therefore
(23). B2 agonists agonists. were
developped with
such
preparation.
reflect
on
subtype
pharmacological
indirectly,
act as full or partial
and D-Arg,[Hyp3,D-Phe7]-BK Only
on
pig have a similar
reduced
and antagonist
antagonists,
The kinin-receptor
of the bioassay
the human
82
as agonists
is evoked
mediators.
on the basis
of a B3 receptor
classical
could
essentially
subtype
the existence
act
and [Th?,*,D-Phe7]-BK,
into subtypes,
on the “neuron&
that
from
secondary
and the precision
cell line expressing [D-Phe’]-BK
subdivided
et al. (2) proposed
explanation
D-Arg,[Hyp3,Th&*,D-Phe7]-BK moderate
des-Arg9,[Leu8]-BK
as antagonists.
differences
B2 antagonists,
B2 antagonists but
that
endogenous
remote
of BK. The
ineffective
of the human
of the properties the
the effect
totally
were
the most
The
second
able to inhibit antagonists
no residual
agonistic
active.
The
generation
a BK response, Hoe140 property.
and
N-
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DP-Ek 10 ,LM Bk 100 $4
Fig, Agonistic and antagonistic properties of BK analogs. A-D: The ago&tic properties of des-Arg9-BK (dABK), Lys-BK, [D-Phe’J-BK (DP-BK), and [ThiS%s,D-Phe7]-BK (TDP-BK) were assayed on CHO cells expressing the human recombinant BK receptor (clone #14). E-H: The antagonistic properties of Hoe140 and DArg,[Hyp3,This~E,D-Phe7]-BK (DAHTDP) were assayed upon addition either before (E,G) or after (F,H) the BK addition. The presence of the agents in the incubation medium is represented by straight lines over the trace. The control response to BK (100 PM) is indicated as a dashed trace. Each trace is representative of duplicate coverslips from at least two independent experiments. The results obtained for [Hyp3]-BK (1 nM), N-Adamantaneacety-DArg [Hyp3,Thi5,*,D-Phe’]-BK (10 PM) and D-Arg,(Hyp3,D-Phe’]-BK (10 PM) were identical to those displayed reqkxtively for Lys-BK (B), Hoe140 (E,F) and D-Arg,[Hyp3,Thi5,8,D-Phe7j-BK (G,H).
Altogether,
binding
and functional
assays
on CHO
cells would
class our human
receptor
as a B2 receptor
of
with some properties of a B3 receptor the neuronal subtype (agonistic properties of [Tlu ‘5s8,D-Phe7]-BK), (agonistic properties of D-Arg,[Hyp3,Thi5)8,D-Phe7]-BK and D-Arg,[Hyp3,D-Phe7]-BK). However, when the same response. receptor was expressed in xenopus oocytes, [Thl‘5,8,D-Phe7]-BK was unable to generate a biological Similarly, the rat ortholog of our human receptor has been reported as a B2 receptor of the muscular type, based on the antagonistic properties of the same analog [Thiss8,D-Phe7]-BK (7-8). We therefore feel that the respective properties of the so-called neuronal, muscular and B3 subtypes could rather be the reflect of species differences, and/or of the characteristics of G protein-coupling in different cell types. Future work dealing with the expression of different levels of the cloned human receptor in various cell types could be of great help in the clarification of the BK receptor classification.
Acknowledgments We are grateful to Dr. J.E. Dumont for continual support, and to Dr S. Swillens for computer work. This work was supported by the Belgian programme on Interuniversity Poles of attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. It was also supported by the For&s de la Recherche Scientijique Mkdicale of Belgium, Boehringer Ingelheim and the Association Recherrhe Biomkiicale et Diagnostic. 1312
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The scientific responsibility is assumed by the authors. D.E. and E.R. are respectively fellows of the IRSfA and Televie. M.P. is Chercheur Qua@! of the Fonds National de la Recherche Scientifique.
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