Departments of Pulmonary Biology (H.M.S., D.B.S., J.J.F., P.T.B.,
D.W.P.H.), Gene Expression Sciences (D.Y.W., J.A.L.), and Molecular
Biology (J.L.M.), SmithKline Beecham Pharmaceuticals, King of Prussia,
Pennsylvania; and Department of Medicinal Chemistry, Via Zambeletti,
Milan, Italy (G.A.M.G.)
There have been proposals that the tachykinin receptor classification
should be extended to include a novel receptor, the "neurokinin-4"
receptor (NK-4R), which has a close homology with the human NK-3
receptor (hNK-3R). We compared the pharmacological and molecular
biological characteristics of the hNK-3R and NK-4R. Binding
experiments, with 125I-[MePhe7]-NKB binding
to HEK 293 cell membranes transiently expressing the hNK-3R (HEK
293-hNK-3R) or NK-4R (HEK 293-NK-4R), and functional studies
(Ca2+ mobilization in the same cells) revealed a similar
profile of sensitivity to tachykinin agonists and antagonists for both
receptors; i.e., in binding studies with the hNK-3R,
MePhe7-NKB > NKB > senktide 
NKA = Substance P; with the NK-4R, MePhe7-NKB > NKB = senktide Substance P = NKA; and with antagonists, SB
223412 = SR 142801 > SB 222200 SR 48968 CP 99994 for
both hNK-3R and NK-4R. Thus, the pharmacology of the two receptors was
nearly identical. However, attempts to isolate or identify the NK-4R
gene by using various molecular biological techniques were
unsuccessful. Procedures, including nested polymerase chain reaction studies, that used products with restriction
endonuclease sites specific for either hNK-3R or NK-4R, failed to
demonstrate the presence of NK-4R in genomic DNA from human, monkey,
mouse, rat, hamster, or guinea pig, and in cDNA libraries from human lung, brain, or heart, whereas the hNK-3R was detectable in the latter
libraries. In view of the failure to demonstrate the presence of the
putative NK-4R it is thought to be premature to extend the current
tachykinin receptor classification.
 |
Introduction |
The
mammalian tachykinins, or neurokinins (NKs), are a family of small
peptides, notably, Substance P, NKA, and NKB that share the common
carboxy-terminal region
Phe-X-Gly-Leu-Met-NH2 (Maggio, 1988
;
Maggi et al., 1993). The tachykinins are localized in both the central
and peripheral nervous systems, in particular in capsacin-sensitive primary afferent neurons (unmyelinated C fibers) that innervate many
regions, including the airways, gastrointestinal and urinary tracts,
and the skin (Otsuka and Yoshioka, 1993; Maggi et al., 1995; Maggi,
1996). The biological effects of the tachykinins are mediated via three
tachykinin receptor subtypes, NK-1, NK-2, and NK-3, which are members
of the superfamily of G-protein-coupled, seven transmembrane
(TM)-spanning receptors (Maggio, 1988; Nakanishi, 1991; Maggi et al.,
1993). The human variants of the three tachykinin receptors have been
cloned and expressed (Gerard et al., 1990; Buell et al., 1992;
Huang et al., 1992), and in the past few years potent and selective
nonpeptide antagonists for the individual receptors have been
identified (Snider et al., 1991; Maggi et al., 1993; McLean et al.,
1993; Emonds-Alt et al., 1995; Maggi, 1996; Sarau et al., 1997; Gao and
Peet, 1999).
In 1992 Xie et al. (1992), in an expression-cloning search for the
-opiate receptor, reported on the cloning and expression of a novel
human orphan receptor that was highly homologous (81% sequence
identity at the amino acid level) to the human NK-3 receptor (hNK-3R).
Despite this close homology, no specific binding of the peptide
tachykinin agonist [3H]eledoisin was detected
in Cos-7 cells transfected with the receptor, and it was concluded to
be an atypical opiate receptor (Xie et al., 1992). In a subsequent
study, NKB, the natural ligand with the highest affinity for the hNK-3R
(Maggio, 1988; Nakanishi, 1991; Maggi et al., 1993), produced a
concentration-dependent response in Xenopus ooctyes, and
also in clonally selected NIH 3T3 fibroblasts, expressing this orphan
receptor (Donaldson et al., 1996). The sensitivity of the orphan
receptor, which was designated "NK-4", to tachykinin agonists was
similar to that of the hNK-3R, except that the latter did not respond
to Substance P (1 µM). Northern blot analysis revealed low-level
expression of the NK-4R in some, but not all, human tissues examined
(Donaldson et al., 1996), with a pattern of expression reportedly
different from that observed previously for the hNK-3R (Buell et al.,
1992). More extensive pharmacological characterization of the hNK-3R and the putative NK-4R, was reported later by using radioligand binding
and arachidonic mobilization studies in Chinese hamster ovary cells
stably expressing the two receptors. The authors concluded that the
pharmacological profiles of the receptors were very similar in many
respects, and it was proposed that the novel receptor may represent an
NK-3R homolog or an NK-4R (Krause et al., 1997). Unfortunately, in
neither of these studies were the effects of tachykinin receptor
antagonists, in particular hNK-3R antagonists, explored.
The major goals of this study were as follows: 1) to compare the
pharmacological profiles of the hNK-3R and the NK-4R by using a
standard binding (inhibition of
125I-[MePhe7]-NKB in cell
membranes) assay and an intact cellular functional assay
(Ca2+ mobilization), with several tachykinin
agonists and also tachykinin receptor-selective antagonists; and 2) to
gain additional information about the expression and molecular
biological characteristics of the NK-4R; a comparison was made with the
hNK-3R.
| |
Experimental Procedures |
Materials.
125I-[MePhe7]-NKB
(specific activity, 2200 Ci/mmol) was obtained from New England Nuclear
(Boston, MA). NKA, NKB, Substance P, and
[MePhe7]-NKB were purchased from Peninsula
Laboratories (Belmont, CA) and senktide
{succinyl-[Asp9
MePhe8]-SP(6-13)} from California Peptide
Research, Inc. (Napa, CA). Taq polymerase and buffer were
purchased from Perkin-Elmer (Branchburg, NJ). Restriction enzymes and
buffers were obtained from Promega (Madison, WI). SB 222200, SB 223412, SR 142801, SR 48964, and CP 99994 were synthesized in the Department of
Medicinal Chemistry, SmithKline Beecham S.p.A, Milan, Italy.
Receptor Cloning and Expression.
The cDNA encoding the
putative NK-4R was kindly provided by Dr. G.-X. Xie. Polymerase
chain reaction (PCR) primers were designed according to the published
sequence (Xie et al., 1992): 5'-GGGCTCCGGGCACTC-3' and
5'-GACCCCAGAGAGAAATCAAGAGCC-3'. The GX coding region was amplified by
PCR in standard buffer conditions with 1.5 mM
MgCl2 and 4% dimethyl sulfoxide by using 35 cycles of 94, 54, and 72°C at 1 min/step. The 1.4-kb PCR product was
initially subcloned into pCRII vector (Invitrogen, Carlsbad, CA) and
subsequently subcloned into the expression vector pCDNA3 (Invitrogen).
Correct orientation of the cDNA insert encoding the GX receptor was
confirmed by restriction analysis.
The sequences of the PCR amplicon and coding region of the GX plasmid
obtained from Dr. Xie were shown to be identical by automated DNA
sequencing but differed from the published sequence (Xie et al., 1992).
Three silent point mutations were noted in the codons for Leu-22,
Gly-68, and Ile-184; however, two nucleotide changes results in the
conversion of Ala to Arg at position 59. These differences are not in
the binding domains of the receptor and are not in the restriction
sites or regions amplified in subsequent PCR studies (see below).
The GX/pCDNA3 plasmid was purified (Qiagen Maxiprep; Qiagen Inc.,
Valencia, CA) and used to transfect human embryonic kidney cells (HEK
293 cells) with a calcium phosphate transfection method as described in
Lee et al. (1994). Cells were harvested 48 h after transfection
and used for binding or functional assays.
hNK-3R and NK-4R PCR.
Nested PCR primers
corresponding to regions of nucleotide identity between hNK-3R and
NK-4R within exons 1 and 4 were designed to amplify intervening regions
that contain receptor-specific restriction endonuclease sites (Fig.
1). Primers exon 1: forward (1)
5'-GCGCTCTGGTC(C/G)CTGGC(C/G)GGA-3'; forward (2)
5'-CTGGCCCACAAGCGCATG-3'; reverse (1) 5'-CCACCGC(A/G)ATGGCCGTGATGG-3';
reverse (2) 5'-ATGGCCGTCATGGAGTAG-3'. Primers exon 4: forward (1)
5'-TGAC(A/C)TTTGC(T/C)ATCTGCTGGC-3'; forward (2)
5'-ATCTGCTGGCTGCCCTATCA-3'; reverse (1) 5'-TTATTCAGACAGCAGTAGATGATG-3'; reverse (2) 5'-CAGACAGCAGTAGATGATGGG-3'.
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Fig. 1.
Priming sites for nested PCR reactions for the hNK-3R
and NK-4R and unique restriction site location for the amplified
region.
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PCR reactions were carried out in a Perkin-Elmer 2400. All templates
were amplified in standard buffer conditions with 1.5 mM
MgCl2 and 4% dimethyl sulfoxide by using 35 cycles of 94, 54, and 72°C at 1 min/step. For genomic PCR, 100 ng of
CsCl-purified cellular DNA was used as the initial template in a
reaction volume of 100 µl. One microliter from the initial PCR
reaction was used as template for the nested reaction. For the cDNA
library PCR, 109 colony-forming units or
plaque-forming units from the indicated plasmid or phage libraries were
screened in the initial reaction. One microliter from the initial
reaction was then used for the nested reaction; all reaction volumes
were 100 µl. Products from the nested PCR reaction were purified by
using QIAquick spin columns (Qiagen Inc.), digested with the indicated
endonuclease, and resolved by acrylamide gel electrophoresis (5%
native gel).
Human cDNA for the hNK-3R, with sequence identical to published
reports, was isolated from human placenta
poly(A)+ RNA using reverse transcription-PCR
technology and site-directed mutagenesis. The hNK-3R and NK-4R were
transiently expressed in HEK 293 cells, as outlined previously (Sarau
et al., 1997).
Radioligand Binding Assays.
Receptor binding assays were
performed with membranes from HEK 293 cells transiently expressing the
hNK-3 receptors (HEK 293-hNK-3R) or the NK-4R (HEK 293-NK-4R), as
detailed previously (Sarau et al., 1997).
125I-[MePhe7]-NKB
competition binding studies with HEK 293-hNK-3R or HEK 293-NK-4R membranes were conducted with approximately 15 µg of membrane protein
and 0.15 nM
125I-[MePhe7]-NKB in a
total of 150 µl of 50 mM Tris, pH 7.4, 4 mM
MnCl2, 1 µM phosphoramidon, and 0.1%
ovalbumin, with or without various concentrations of antagonist, for 90 min at 25°C. Incubations were stopped by rapid filtration through
Whatman GF/C filters that were presoaked for 60 min in 0.5% BSA, with
a Brandell tissue harvestor (Gaithersburg, MD). Membranes were washed
with 10 ml of ice-cold 20 mM Tris, pH 7.4, containing 0.1% BSA and
then placed in vials with 10 ml of Beckman Ready Safe and counted in a
liquid scintillation counter. Concentration-response curves for each compound were run with duplicate samples in at least three independent experiments. Nonspecific binding was assessed as the binding in the
presence of 0.5 µM cold MePhe7-NKB. The
IC50 for ligands, defined as the concentration
required to inhibit 50% of the specific binding, was determined from
concentration-response curves. Values presented are the apparent
inhibition constant (Ki), which was
calculated from the IC50 as described by Cheng and Prusoff (1973).
Ca2+ Mobilization Assay.
Ca2+ mobilization experiments in HEK 293-hNK-3R
or HEK 293-NK-4R cells were conducted with Fluo 3-loaded cells and a
Fluorescence Imaging Plate Reader (Molecular Devices, Sunnyvale,
CA) as outlined previously (Sarau et al., 1999). Briefly, cells
(approximately 80% confluent) were harvested and plated in 96-well
black wall/clear bottom plates (Biocoat plates from Becton Dickinson
Labs, Bedford, MA) at approximately 40,000 cells/well and grown
in the incubator for 18 to 24 h. On the day of assay the medium
was aspirated and replaced with 100 µl of Earls' mimimal essential
medium with Earls' salts, L-glutamine 0.1% BSA, 4 µM
Fluo 3 acetoxymethyl ester (Fluo 3 AM; Molecular Probes, Eugene, OR),
and 2.5 mM probenecid. Plates were incubated for 60 min at 37°C, and
then the medium was aspirated and replaced with the same medium without
Fluo-3 AM, and incubated for 10 min at 37°C in 100 µl of buffer
[120 mM NaCl, 4.6 mM KCl, 103 mM
KH2PO4, 25 mM
NaHCO3, 1.0 mM CaCl2, 11 mM
glucose, 20 mM HEPES (pH 7.4) with 2.5 mM probenecid]. Plates were
placed into a Fluorescence Imaging Plate Reader where cells were
exposed to excitation (488 mm) from a 6-W argon laser. Fluorescence was
monitored at 566-mm emission for all 96 wells simultaneously, and data
points were collected every second. The maximal change in emission
after agonist addition was quantitated. The percentage of maximal
NKB-induced Ca2+ mobilization was determined for
each concentration of antagonist and the IC50,
defined as the concentration of test compound that inhibits 50% of the
maximal response induced by 1 nM NKB, assessed. Values presented are
generally the mean IC50 ± S.E. of three
individual experiments unless stated otherwise.
| |
Results |
Pharmacological Characterization
Binding Experiments.
Initially, studies were performed to
assess the binding characteristics of
125I-[MePhe7]-NKB to
membranes prepared from transiently expressed HEK 293-NK-4R cells.
Binding of the radioligand was saturable, specific, and of high
affinity; the Kd and
Bmax were determined to be 1.0 ± 0.2 nM and 2.1 ± 0.7 pmol/mg, respectively (n = 3;
data not shown). The binding of
125I-[MePhe7]-NKB to HEK
293-hNK-3R cell membranes was saturable, specific, and of high
affinity; the Kd and
Bmax values were 0.9 ± 0.2 nM and
0.6 ± 0.1 pmol/mg, respectively (n = 3; data not shown).
A comparison was made of the effects of tachykinin receptor agonists
and antagonists on the binding of
125I-[MePhe7]-NKB to HEK
293-hNK-3R or HEK 293-NK-4R cell membranes; the results are summarized
in Fig. 2 and Table
1. The natural ligand with the highest
affinity for the NK-3 receptor, NKB, and the NK-3R-selective agonists
senktide and [MePhe7]-NKB (0.1 nM-0.1 µM)
produced concentration-dependent inhibition of
125I-[MePhe7]-NKB binding
to HEK 293-NK-4R cell membranes, with respective IC50 values of 18.3 ± 3.3, 15.1 ± 2.6, and 3.3 ± 0.5 nM (n = 3) (Fig. 2A; Table 1).
In contrast, Substance P (NK-1R-preferring natural ligand) and NKA
(NK-2R-preferring natural ligand) demonstrated much lower affinity for
125I-[MePhe7]-NKB binding
to HEK 293-NK-4R cell membranes with IC50 values of 2130 ± 540 and 3530 ± 180 nM, respectively
(n = 3) (Fig. 2A; Table 1). Thus, the relative rank
order potency was [MePhe7]-NKB > NKB = senktide Substance P = NKA. The NK-3R-selective antagonists
SB 223412 (Sarau et al., 1997), SB 222200 (Sarau et al., 2000), and SR
142801 (Emonds-Alt et al., 1995) also produced potent,
concentration-dependent inhibition of
125I-[MePhe7]-NKB binding
to HEK 293-NK-4R cell membranes with respective IC50 values of 4.1 ± 0.7, 11.0 ± 2.7, and 2.6 ± 0.6 nM (n = 3; Table 1). The
NK-1R-selective antagonist CP 99994 (McLean et al., 1993) was a weak
competitor, with an IC50 of 8500 ± 2400 nM,
whereas the NK-2R antagonist SR48968 (Emonds-Alt et al., 1992) showed
significant inhibition with an IC50 of 711 ± 120 nM (n = 3) (Fig. 2C; Table 1).
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Fig. 2.
Competition binding of
125I-[MePhe7]-NKB to (A, C) HEK 293-hNK-4Rs
and (B, D) HEK 293-NK-3Rs by tachykinin agonists (A, B) and antagonists
(C, D). HEK 293-hNK-3R or HEK 293-NK-4R membranes were incubated with
0.15 nM 125I-[MePhe7]-NKB in the absence and
presence of various concentrations of agonists (A, B) senktide ( ),
Substance P ( ), NKB ( ), [MePhe7]-NKB ( ), or NKA
( ), and antagonists (C, D) SB 223412 (), SB 222200 (), SR
142801 (), SR 48968 (), or CP 99994 () as described under
Experimental Procedures. Values presented are the mean
of two or three experiments. Standard errors are omitted for clarity,
but are given for the EC50 values (agonists) and
IC50 values (antagonists) in Table 1.
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TABLE 1
Effects of tachykinin receptor ligands on
125I-[MePhe7]-NKB binding and Ca2+
mobilization in (hNK-4R) and hNK-3R cells
The experimental protocols for these studies are given under
Experimental Procedures. Results are presented as
EC50 or IC50 values and are the mean ± S.E. or
the mean; n = 3 unless indicated in parentheses.
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The rank order for the agonist displacement of
125I-[MePhe7]-NKB binding
to HEK 293-hNK-3R cell membranes was not very different from that
obtained with the NK-4R membranes:
[MePhe7]-NKB > NKB > senktide
NKA = Substance P (Fig. 2B; Table 1). Furthermore, the antagonists
also showed a similar rank order potency for inhibition of
125I-[MePhe7]-NKB binding
to HEK 293-hNK-3R cell membranes: SB 223412 = SR 142801 > SB
222200 SR 48968 >CP 99994 (Fig. 2D; Table 1).
Ca2+ Mobilization Studies.
Cellular functional
activity of agonists and antagonists was determined by assessment of
their effects on Ca2+ mobilization in HEK
293-NK-4R or HEK 293-hNK-3R cells (Fig.
3). In HEK 293-NK-4R cells, the rank
order potencies of agonists was senktide = NKB = [MePhe7]-NKB > NKA > Substance P. All agonists demonstrated the same efficacy, eliciting a similar
maximum response (Fig. 3A). A similar rank order of potencies for the
agonists was demonstrated for their ability to produce
Ca2+ mobilization in HEK 293-hNK-3R. However,
senktide was more potent (3-fold) than NKB or
[MePhe7]-NKB in HEK 293-hNK-3R cells (Fig. 3B).
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Fig. 3.
Tachykinin agonist-induced Ca2+
mobilization (A, B) and effects of tachykinin receptor antagonists SB
223412, SB 222200, SR 142801, SR 48968, and CP 99994 on NKB-induced
Ca2+ mobilization (C, D) in HEK 293-NK-4R (A, C) or HEK
293-hNK-3R cells (B, D). A and B, Fluo 3-loaded HEK 293-hNK-3 or HEK
293-NK-4R cells were stimulated with increasing concentrations of
senktide ( ), Substance P (), NKB (), MePhe7-NKB
(), or NKA (). The results are expressed as Ca2+
mobilized (optical units) and are the mean for three experiments.
Standard errors are omitted for clarity but given for the
EC50 values in Table 1. C and D, Fluo-3 loaded HEK
293-hNK-3 or HEK 293-NK-4R cells were stimulated with NKB after the
addition of increasing concentrations of SB 223412 (), SB 222200 (), SR 142801 (), SR 48968 (), or CP 99994 (). The results
are expressed as a percentage of the maximum Ca2+
concentrations elicited by 1 nM NKB in the absence of any antagonist.
Values presented are the mean of three experiments. Standard errors are
omitted for clarity but given for the IC50 values in Table
1.
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In studies examining the effects of antagonists against NKB-induced
Ca2+ mobilization an NKB concentration of 1 nM
was used; this represents approximately 60 to 70% of the maximum
response. SB 223412, SB 222200, and SR 142801 possessed similar
potencies for inhibition of NKB (1 nM)-induced
Ca2+ mobilization in HEK 293-NK-4R cells, with
IC50 values of 50 ± 34 nM
(n = 3), 108 ± 52 nM (n = 3), and
254 ± 180 nM (n = 3), respectively. SR 48968 and
CP 99994 had IC50 values of 1212 ± 660 nM
(n = 3) and >10 µM (n = 3),
respectively (Fig. 3C). A similar profile for the antagonists was
evident against NKB-induced Ca2+ mobilization in
the HEK 293-hNK-3R cells (Fig. 3D). Thus, SB 223412, SR 142801, and SB
222200 had similar potencies, with respective IC50 values = 125 ± 46 nM
(n = 3), 231 ± 110 nM (n = 3),
and 264 ± 73 nM (n = 3). Significant inhibition
was demonstrated with SR 48968 (IC50 = 14,600 nM;
n = 2), but not with CP 99994, in concentrations up to
10 µM (n = 2) (Fig. 3D).
Molecular Biological Characterization
Isolation and Expression of NK-4R.
A comparison of the
alignment of the human tachykinin receptor family, including NK-4R, is
indicated in Fig. 4. The predicted organization of the NK-4R is based on the sequence alignment.
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Fig. 4.
Alignment of human tachykinin receptor family. The
predicted organization of the sequence of NK-4R is based on the
sequence alignment.
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PCR and nested PCR procedures were used to isolate the NK-4R cDNA from
human lung, brain, or heart cDNA libraries and to detect the NK-4R gene
with multiple primers directed to the 5'- or 3'-untranslated region
(UTR) and internal-coding regions of NK-4R. No evidence for an
NK-4R-specific signal was detected from human genomic DNA and cDNA
libraries that are abundant in NK-4R mRNA (G.-X. Xie, personal
communication), although multiple primer sets were used under a variety
of PCR reaction conditions (data not shown). In addition, the NK-4R
gene was not identified via Northern or Southern blot analysis by using
5'-UTR- or 3'-UTR hNK-4R-specific probes derived from either GX/pCDNA3
or the published sequence (Xie et al. 1992; data not shown). These
unexpected results are inconsistent with the reported human origins of
NK-4R (Xie et al., 1992).
Nested PCR and Restriction Site Analysis of hNK-3R and NK-4R.
In view of the unsuccessful isolation and detection of the NK-4R cDNA,
a series of experiments, with nested PCR and restriction endonucleases
specific for the hNK-3R and NK-4R, were performed to verify that the
NK-4R is a human gene. Specifically, nested PCR primers were designed
to amplify a DNA fragment from exon 1 (containing TM1-TM3) and
separate primers to amplify a DNA portion from exon 4 (containing TM6
and TM7) (Fig. 1); see Experimental Procedures for the
primers used. The PCR primers were designed to simultaneously amplify
both hNK-3R and NK-4R sequences from regions of 100% nucleotide
identity, but contained hNK-3R- or NK-4R-specific restriction
endonuclease sites within the intervening sequences (Fig. 1).
Therefore, if both NK-3R or NK-4R genes are present in either human
genomic DNA or cDNA libraries, the characteristic restriction patterns
for both genes should be observed. The results of these studies are
summarized in Fig. 5. With the exon 1 amplicon (Fig. 5) and the exon 4 amplicon (data not shown) only
NK-3R-specific restriction patterns were identified in PCR products
derived from human genomic DNA for both exon 1 and exon 4 ampicons
(Fig. 5) and cDNA libraries from human placenta, lung, heart, or brain, tissues reported to contain NK-4R mRNA (Xie et al. 1992; G.-X. Xie,
personal communication). For example, the results from placenta cDNA
library and exon 1 fragments (lanes 9-11) indicate that a single band
was detected when no endonuclease digest was used (lane 9), two bands
were obtained by using the hNK-3R-specific digest (PvuII;
lane 10), and only one band was demonstrated by using the
NK-4R-specific digest (MboI; lane 11). These data indicate the presence of hNK-3R, but not NK-4R, in this cDNA library. There was
no evidence for the presence of either hNK-3R or NK-4R in human liver
or skeletal muscle cDNA libraries because no band was demonstrated in
lanes 22 and 23, respectively.
To investigate whether the putative NK-4R may be a nonhuman gene
product, the same PCR amplification and restriction digest experiments
were conducted with mouse, rat, hamster, guinea pig, and monkey (Cos
cell) genomic DNA. Results similar to that obtained from human genomic
DNA (Fig. 5) were obtained; there was no evidence of an NK-4R gene in
any of the species tested (data not shown). Furthermore, no positive
hybridization was detected, by using a synthetic oligonucleotide
specific to the 3'-UTR of NK-4R, in a genomic DNA blot from Clontech
(Palo Alto, CA), which includes human, rat, dog, rabbit, yeast, monkey,
mouse, bovine, and chicken (data not shown).
| |
Discussion |
The existence of a novel tachykinin receptor, designated NK-4,
which has a close structural and functional homology with the hNK-3R,
has been proposed (Donaldson et al., 1996; Krause et al., 1997). The
major goals of this study were, first, to conduct a comprehensive
comparison of the pharmacological profiles of the hNK-3R and the NK-4R,
with radioligand binding and Ca2+ mobilization
experiments. For the first time the effects of tachykinin receptor
antagonists in NK-4R were explored. These studies were performed with
the hNK-3R and NK-4R transiently expressed in HEK 293 cells. This
should normalize the responses of the receptor-expressing cells and
avoid differential expression of stable clonal cell lines. Second,
attempts were made to compare the distribution and molecular biological
characteristics of both receptors. Binding and functional studies
confirmed the similar pharmacological characteristics of the hNK-3R and
NK-4R, although some small differences were apparent, e.g., senktide
appeared to have lower affinity for hNK-3R than NK-4R, but this was not
observed in the functional analysis where the three hNK-3R-selective
ligands showed equal potency. Surprisingly, despite the use of several
independent approaches, no evidence was obtained that the putative
NK-4R is a gene product of human or many nonhuman species.
It has been demonstrated that high primary sequence identity exists
within TM regions of hNK-3R and NK-4R, ranging from 83 to 100% with an
average of 92% identity. It is known that the residues that are
important for the interaction of tachykinin receptors (in particular,
NK-1R) with potent and selective antagonists are located in TM regions
(Fong et al., 1993; Gether et al., 1993a,b). This information, in
addition to the very similar pharmacological profile of the hNK-3R and
NK-4R to various tachykinin ligands, including NK-3R-selective agonists
(Donaldson et al., 1996; Krause et al., 1997; current study) and NK-3R
antagonists (current study) highlights the importance of determining
whether this putative hNK-3R-like receptor is in fact a human
tachykinin receptor subtype or is a species variant of the hNK-3R. To
address this question several approaches were used: Northern and
Southern analysis with NK-4R-specific probes, PCR cloning from various
human tissues reported to express NK-4R, and restriction analysis of
PCR amplicon derived from genomic and cDNA libraries. Overall, these
experiments did not provide evidence that NK-4R is a human gene
product; furthermore, it does not appear to be a gene product of the
several nonhuman species investigated: mouse, rat, monkey, bovine, dog,
chicken, yeast, rabbit, or guinea pig.
To investigate whether the NK-4R sequence corresponds to a human gene,
regions of either exon 1 (containing TM1-TM3) or exon 4 (containing
TM6 and TM7) from NK-3R and NK-4R were simultaneously amplified by
nested PCR and then differentiated by the presence of either hNK-3R- or
NK-4R-specific restriction sites. PCR amplification with human genomic
DNA and various human cDNA libraries revealed amplicons with
restriction endonuclease sites corresponding to the hNK-3R but not the
NK-4R; the tissues explored included placenta, from which the NK-4R was
originally cloned (Xie et al., 1992). It is unlikely that the PCR
primers selectively amplify the hNK-3R amplicon over the NK-4R amplicon
because each primer set corresponds to a region of nucleotide identity
between the two receptor cDNAs and thus serves as internal PCR control.
It is possible that the PCR primers may have failed to amplify NK-4R
from the human genomic DNA if the intron-exon organization of the NK-4R
gene differs from that of the hNK-3R gene. However, this appears
unlikely because the NK-4R and hNK-3R sequences are very similar (75 and 82% sequence identity at the nucleotide and amino acid level,
respectively) and the gene organization of the known tachykinin
receptors NK-1, NK-2, and NK-3 are identical (Fig. 4; Donaldson et al.,
1996). These studies are consistent with the failure to amplify an
NK-4R fragment by using various sets of PCR primers, PCR conditions, and cDNA synthesis conditions from human placenta RNA, and the inability to amplify an NK-4R fragment of the 3'-UTR from human genomic
DNA with various primer sets and PCR conditions. Collectively, the
results from the PCR analysis of human cDNA and genomic DNA suggest
strongly that the NK-4R is not represented in the human genome.
The current results contrast with those from a previous study in which
NK-4R transcripts were found in skeletal muscle, liver, lung, and heart
(Donaldson et al., 1996); the reason(s) for this discrepancy is not
apparent. However, given the 75% nucleotide identity between hNK-3R
and NK-4R the specificity of a hybridization probe generated from a
full-length NK-4R cDNA may be in question; in contrast, the PCR
technique used in this study does not depend on the generation of a
specific hybridization probe.
Finally, attempts were made to associate the novel receptor to a
nonhuman species. However, 3'-UTR probes synthesized from the published
sequence (Xie et al., 1992) or cloned from the plasmid provided by Dr.
Xie failed to hybridize with Southern blots of human, rat, dog, rabbit,
yeast, monkey, mouse, bovine, and chicken genomic DNAs. In addition, no
NK-4R amplification was observed when PCR analysis of mouse, rat,
monkey, guinea pig, and hamster genomic DNAs was undertaken. Thus, the
NK-4R cDNA does not appear to originate from a number of common
laboratory species.
In summary, based on binding and functional studies with tachykinin
ligands, including NK-3R-selective agonists and antagonists, the
reported hNK-3R-like receptor (putative NK-4R) has pharmacological characteristics that are very similar to those of the hNK-3R. This
similarity in profiles is not unexpected, given the close homology
between the two receptors (overall, 82% at the amino acid level).
Unfortunately, attempts to identify and localize the NK-4R, by using
genomic and cDNA libraries from human and nonhuman species and standard
molecular biological techniques, were unsuccessful. The results suggest
that the NK-4R is not represented in the human genome, and highlight
the caution that should be exercised in using this gene product in
studies related to characterization of tachykinin receptors.
Furthermore, it would appear to be inappropriate and premature to
extend the current human tachykinin receptor classification beyond the
present NK-1R, NK-2R, and NK-3Rs. Additional experiments, with
libraries from species not used in the current study, are required to
explore further whether this receptor is a species variant of the
hNK-3R.
We thank John Adamou, Bob Ames, Mary Brawner, Nabil Elshourbagy,
and Parvathi Nuthulaganti for the cloning and expression efforts; Mario
Grugni, Roberto Rigolio, and Karl F. Erhard for the synthesis of SR
142801; Luca F. Raveglia for the synthesis of CP 99994; and Mark
Luttmann for assistance in the preparation of the manuscript.
NK, neurokinin;
TM, transmembrane;
hNK-3R, human neurokinin-3 receptor;
NK-4R, putative human NK-4 receptor;
SB 222200, (S)-(
)-N-(
-ethylbenzyl)-3-methyl-2-phenylquinoline-4-carboxamide;
SB 223412, (S)-()-N-(-ethylbenzyl)-3-hydroxy-2-phenylquino line-4-carboxamide;
SR 142801, (S)-(+)-N-{{3-[1-benzoyl-3-(3,4-dichlorophenyl)piperidine-3-yl]prop-1-yl}-4-phenylpiperidin-4-yl}-N- methylacetamide, SR 48964, (S)-N-methyl-N[4-(4-acetylamino-4-phenyl
piperidino)-2-(3,4-dichlorophenyl)butyl]benzamide;
CP 99994, (+)-(2S,3S)-cis-(2-methoxybenzylamino)-2-phenylpiperidine
dihydrochloride;
PCR, polymerase chain reaction;
HEK, human embryonic
kidney;
HEK 293-hNK-3R, HEK 293 cells transiently expressing the human
NK-3 receptor;
HEK 293-NK-4R, HEK 293 cells transiently expressing the
human NK-4 receptor;
UTR, untranslated region.
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Abstract
Introduction
