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J. Biol. Chem., Vol. 275, Issue 50, 39482-39486, December 15, 2000
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From the Human Genome Research and
Received for publication, August 3, 2000, and in revised form, September 18, 2000
Neuromedin U is a neuropeptide prominently
expressed in the upper gastrointestinal tract and central nervous
system. Recently, GPR66/FM-3 (NmU-R1) was identified as a specific
receptor for neuromedin U. A BLAST search of the
GenBankTM genomic database using the NmU-R1 cDNA
sequence revealed a human genomic fragment encoding a G protein-coupled
receptor that we designated NmU-R2 based on its homology to NmU-R1. The
full-length NmU-R2 cDNA was subsequently cloned, stably expressed
in 293 cells, and shown to mobilize intracellular calcium in response
to neuromedin U. This response was dose-dependent
(EC50 = 5 nM) and specific in that other
neuromedins did not induce a calcium flux in receptor-transfected cells. Expression analysis of human NmU-R2 demonstrated its mRNA to
be most highly expressed in central nervous system tissues. Based on
these data, we conclude that NmU-R2 is a novel neuromedin U receptor
subtype that is likely to mediate central nervous system-specific neuromedin U effects.
The neuromedins (Nm)1
are a group of smooth muscle-stimulating peptides commonly divided into
four groups: bombesin-like (NmB, NmC), kassinin-like (NmL and -K or
neurokinins A and B, respectively), neurotensin-like (NmN), and
neuromedin U (NmU). Among this group of peptides, neuromedin U has been
the least well understood, in large part due to the lack of a known
receptor. Neuromedin U was first reported in 1985 by Minamino et
al. (1, 2) as a peptide isolated from porcine spinal cord. These
investigators isolated two active peptides, NmU-25 and an
additional cleavage product, NmU-8, and characterized them as
having smooth muscle contractile activity. Neuromedin U was
subsequently isolated from a variety of species including rat (3, 4),
guinea pig (5), dog (6), rabbit (7), chicken (7, 8), and frog (9).
The cDNAs for rat and human NmU have been cloned, and analysis of
the nucleic acid sequence suggests that NmU is produced as a 174-amino
acid precursor (10, 11). The precursor contains a signal peptide and
several dibasic cleavage sites that give rise to a number of possible
secreted peptides, including NmU, which is present near the carboxyl
terminus. Neuromedin U shows remarkable conservation throughout
evolution, and a core active peptide
(Phe-Leu-Phe-Arg-Pro-Arg-Asn-NH2) is absolutely conserved among mammalian species.
A variety of biological activities have been reported for NmU
although its role in normal physiology is unclear. The first biological
activity ascribed to NmU was smooth muscle contraction (1, 2). These
experiments have not been consistent among different species, however,
in regard to the specific tissues that respond to NmU (1, 12-15).
Neuromedin U has also been reported to increase arterial blood pressure
(16, 17) and modify ion transport in the intestinal tract (15).
Finally, NmU injected subcutaneously into rats has been reported to
result in a short term increase in circulating ACTH levels and a long
term increase in serum corticosterone levels (18, 19), suggesting a
role in regulation of the hypothalamo-pituitary-adrenal axis.
Recently, several groups, including our own, have reported that
GPR66/FM-3 is a specific receptor for NmU (20-23). Originally identified as a partial mouse expressed sequence tag residing in
GenBankTM, full-length mouse GPR66/FM-3 was cloned from a T
cell library and subsequently used as a probe to identify a human clone
(24). Comparison of GPR66/FM-3 to other known G protein-coupled
receptors (GPCRs) shows it is most similar to the human growth hormone
secretagogue and neurotensin receptors (33 and 29% amino acid
identity, respectively) as well as the recently described motilin
receptor (25). This receptor (NmU-R1) is expressed in peripheral
tissues, particularly in the upper gastrointestinal tract and lymphoid
tissues (20-24), but is essentially absent from central nervous system
tissues. In the present study we report the identification and
characterization of a second specific NmU receptor that is
predominantly expressed in the central nervous system.
Cloning and Expression of NmU-R2--
A genomic fragment
encoding NmU-R2 (GenBankTM accession number AC008571) was
identified using the deduced amino acid sequence of NmU-R1 as
"bait" for a TBLASTN search of the GenBankTM high
throughput genomic database. A putative NmU-R2 open reading frame was
assembled using Sequencher (Genecodes, Ann Arbor, MI). The
coding region of NmU-R2 was subsequently amplified by polymerase chain
reaction (PCR) employing a specific 5' forward primer containing a consensus Kozak sequence (underlined): 5'-GCCGCCACC
ATGTCAGGG ATGGAA AAACTTCAGAAT-3', a specific 3' reverse primer:
5'-AAGAATTCAGGTTTTGTTAAAGTGGAAGCTTT-3', and human testis
Marathon-Ready cDNA as a template (CLONTECH, Palo Alto, CA). The thermal cycling profile for PCR was 95 °C, 30 s; 63 °C, 30 s; and 68 °C, 2 min (35 cycles). The
resulting PCR product was cloned into pCR3.1 (Invitrogen, Carlsbad,
CA), and the insert was sequenced. Expression of NmU-R2 in 293 cells (American Type Culture Collection, CRL1573) was accomplished using LipofectAMINE 2000 (Life Technologies, Inc.) according to the manufacturer's protocol. Cells stably expressing NmU-R2 were selected in Dulbecco's modified Eagle's medium, 10% fetal calf serum
containing G418 at 1.0 mg/ml and subsequently maintained in Dulbecco's
modified Eagle's medium, 10% fetal calf serum containing G418 at 0.5 mg/ml.
Ligand Screening--
Ligand screening was accomplished using
the fluorometric imaging plate reader (FLIPR, Molecular Devices,
Sunnyvale, CA) (26, 27). Briefly, 48 h before screening, 293 cells
were transfected with NmU-R2 as described above. The cells were then
replated 24 h before screening into clear bottom, black-walled
96-well plates precoated with poly-D-lysine
(Becton-Dickinson, Franklin Lakes, NJ) at a density of 5 × 105 cells/well. On the day of screening cells were loaded
for 1 h with Fluo-3AM (Sigma) according to the FLIPR
manufacturer's protocol.
The peptide libraries that were used in the large scale screening of
NmU-R2 included more than 500 peptides that are known or suspected GPCR
ligands. These were obtained from various commercial sources (RBI,
Natick, MA; Bachem, King of Prussia, PA; Sigma) or were
custom-synthesized (Research Genetics, Huntsville, AL). In particular,
rat NmU-23, pig NmU-25, pig NmU-8, and human neuromedins B, C, L, K,
and N were obtained from Bachem (King of Prussia, PA) whereas high
performance liquid chromatography-purified human NmU-25 and nonamidated
NmU-8 were custom-synthesized (Research Genetics). A commercial small
molecule library (RBI) was also screened. This library covers various
pharmacological classes including adenosines, purinergics, adrenergics,
histaminergics, cholinergics, ion channel modulators, dopaminergics,
glutaminergics, opioids, serotonergics, and Single Cell Ca2+ Imaging--
293 cells stably
expressing NmU-R2 plated onto poly-D-lysine-coated
coverslips that subsequently formed the base of a perfusion chamber.
Cultures were loaded with fura-2 acetoxymethyl ester (5 µM, 45 min, 37 °C, Molecular Probes, Eugene, OR) in a
buffered salt solution (149 mM NaCl, 3.25 mM
KCl, 2.0 mM CaCl2, 2.0 mM MgCl2, 10 mM HEPES, 11 mM glucose).
The perfusion chamber was continuously perfused with the buffered salt
solution (3 ml/min), and drug additions were made using a pinch valve
arrangement with minimal dead space. Intracellular Ca2+
concentration was estimated using an Attofluor RatioVision digital fluorescence imaging system (Atto Instruments, Rockville, MD). Fura-2
was excited, alternately, at 334 and 380 nm, and the emission was
collected at 510 nm and 1.25-s intervals. Calibration of the 334/380 nm
fura-2 signal was performed in vitro with fura-2
pentapotassium salt in the presence of 1 mM
Ca2+ or 1 mM EGTA, and the 334/380 nm
excitation ratio was converted to Ca2+ concentration values
using the procedure of Grynkiewicz et al. (28).
Messenger RNA Expression Analysis--
Expression of NmUR-2 was
examined using dot blots and Northern blots obtained from a commercial
source (CLONTECH). Hybridization to blots was
carried out using PCR-generated DNA fragments encompassing 1200 base
pairs of the coding region of NmUR-2 beginning at the 3'-end and
including most of the coding region. The DNA fragments were
random-prime labeled with [32P]dCTP, and the blots were
hybridized for 14 h in ExpressHyb (CLONTECH) containing 2 × 106 cpm/ml of radiolabeled probe. The
following day the blots were washed and exposed to Kodak Biomax MS film
for 3 days at A peripheral NmU receptor (NmU-R1) has been recently identified
that is expressed in peripheral tissues (20-22). To identify potential
subtypes of this receptor we performed a TBLASTN search of the high
throughput genomic database subset of GenBankTM using the
deduced amino acid sequence of NmU-R1 as bait. One such search
identified a genomic fragment from chromosome 5 (GenBankTM
AC008571) containing a region of relatively high homology (~57%) to
NmU-R1. Although the genomic sequence was unordered, a predicted open
reading frame was assembled based on sequence homology to NmU-R1. PCR
primers were designed based upon this predicted open reading frame, and
a cDNA was subsequently obtained that was designated NmU-R2 based
on its homology to NmU-R1 (Fig. 1A). The genomic structure of
NmU-R2 differs significantly from that of NmU-R1 in that the predicted
open reading frame is encoded on four exons instead of the two found in
the NmU-R1 gene (Fig. 1). Interestingly, the intron 2-exon 3 boundary
of NmU-R2 coincides with the intron 1-exon 2 boundary of NmU-R1, and
the overall homology between the open reading frames is far higher
upstream of this point (TM1-TM6) than downstream. The conservation of
one of the intron/exon boundaries and the relatively high homology of
the two genes suggests that these two receptors arose from a
duplicative event sometime in the past. The position of exon 2 of
NmU-R2 is such that it encompasses only the third intracellular loop
and the very beginning of transmembrane domain six. The length of this
loop is also shortened in NmU-R2 as compared with NmU-R1 (Fig.
1A). Finally, both the amino- and carboxyl-terminal
sequences show considerable divergence with NmU-R2 having extended
termini.
In addition to the NmU-R2 genomic fragment, we also identified several
expressed sequence tag (EST) sequences in the GenBankTM
database that proved to have identity with NmU-R2. Four of five of
these ESTs originated from brain-related cDNA libraries with the
fifth coming from a heart library. Three of these ESTs appear to
represent an alternative NmU-R2 transcript. This transcript includes
exons 2 and 3 of the NmU-R2 gene but do not splice upstream to exon 1. Instead, two novel exons are spliced in, creating a presumably sterile
transcript with multiple stops in all reading frames (Fig.
1B). Although the "aberrant" transcripts are all from
fetal brain libraries, it is possible that such transcripts might exist
elsewhere and suggests caution in interpreting the results of
expression studies. The downstream splicing of exon 3 to exon 4 in
these alternate transcripts could not be assessed from the sequence
data present in GenBankTM. The expression data presented in
this study were confirmed with probes that do not hybridize to the
alternate NmU-R2 transcript (data not shown).
To gain some insight into the possible physiological role of a second
NmU receptor, the expression of this receptor was comprehensively assessed using dot blots and Northern blots of human tissues. The
expression of NmU-R2 on dot blots was highest in testis and central
nervous system tissues, particularly spinal cord (Fig. 2A). This is in contrast to
NmU-R1, which showed very little expression in central nervous system
tissues (20-22). Low levels of NmU-R2 expression were detected by dot
blot in stomach and duodenum (Fig. 2A), but unlike NmU-R1,
NmU-R2 expression was absent or very low in other gastrointestinal
tract tissues. Similarly, expression of NmU-R2 in lymphoid tissues was
either very low or undetectable (Fig. 2A). Aside from the
expression in central nervous system tissues and gastrointestinal
tract, expression of NmU-R2 mRNA was also observed in kidney, lung,
and thyroid (Fig. 2A). No expression of NmU-R2 was detected
in uterus, despite the fact that NmU binding has been reported in rat
uterus (29). This may reflect a species-specific difference as
discussed in the introduction. Alternatively, the expression of NmU
receptor was reported to be estrogen-dependent (30) and may
vary depending on when tissue was obtained. In any case, this is
clearly an area where further investigation is warranted.
Identification of a Novel Neuromedin U Receptor Subtype Expressed
in the Central Nervous System*
, Cardiovascular/Central Nervous System Research,
Schering-Plough Research Institute, Kenilworth, New Jersey 07033
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INTRODUCTION
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-aminobutyric acid
compounds (LOPAC Library, exact content available from manufacturer).
70 °C. The dot-blot films were analyzed for NmU-R2
expression levels using the MCID M4 image analysis system (Imaging
Research, Ontario, Canada), and the data were displayed as
absolute optical density.
RESULTS AND DISCUSSION
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View larger version (63K):
[in a new window]
Fig. 1.
Deduced amino acid sequence and genomic
structure of NmU-R2. A, the predicted amino acid
sequence of NmU-R2 is shown aligned with that of NmU-R1. Amino acid
identities and regions of high overall homology are boxed
while similar amino acids are indicated with shading.
Potential N-linked glycosylation sites are noted (*) as are
the predicted transmembrane domains (overscore). The
boundaries of exons for NmU-R2 are also indicated (solid
arrowheads) with the predicted splice junction nucleic acid
sequences written as exon/intron/exon. The position of the single exon
boundary in NmU-R1 is indicated for comparison (open
arrowhead). B, genomic structure and alternate splicing
of NmU-R2. The open reading frame of NmU-R2 and an assembly EST
(GenBankTM accession numbers R13353, R13890, H11359)
representing an alternatively spliced NmU-R2 transcript were exon
mapped onto an unordered genomic fragment (GenBankTM
accession number AC008571) using Sequencher. Exons unique to the
alternative (sterile) transcript are indicated as filled
boxes, exons shared by both the NmU-R2 transcript and the
alternative transcript are filled in gray, and exons unique
to the NmU-R2 transcript are unfilled. Solid
lines indicate the splicing pattern of NmU-R2 whereas splicing of
the alternative transcript is indicated with dashed lines.
Distances between exons are drawn to scale although exon sizes are not.
Numbers at either end of the schematic are distances (kilobases) not
including a gap of unknown length (indicated by a
break).
View larger version (54K):
[in a new window]
Fig. 2.
NmU-R2 expression analysis.
A, the expression of NmU-R2 was examined by dot blot using a
full-length NmU-R2 32P-labeled probe. The autoradiograph
was scanned, and the results are indicated as a bar graph of corrected
optical densities (units). The bar for testis expression is shown
broken (optical density = 70) to facilitate comparison
of the comparatively lower levels of NmU-R2 expression observed in
other tissues. Results shown are one of two experiments.
CNS, central nervous system; CV, cardiovascular
system; GI, gastrointestinal system. B, Northern
blot analysis of NmU-R2 expression in central nervous system tissues. A
commercial Northern blot (CLONTECH) was probed with
full-length, 32P-labeled NmU-R2 cDNA. Size markers are
indicated (left side), and a 32P-labeled
-actin probe was used to control for mRNA loading (bottom
of panel).
The expression of NmU-R2 in the central nervous system and gastrointestinal tract was examined in more detail by Northern blot analysis. Spinal cord and corpus callosum demonstrated the highest expression with a NmU-R2 message of 2.4 kilobases (Fig. 2B). In contrast, expression of NmU-R2 in the gastrointestinal tract as detected by Northern blot was very low and could not be detected even upon very long exposure (1 week, data not shown). Despite the inability to detect NmU-R2 in the Northern blot, the dot-blot results suggest that NmU-R2 may have some overlapping expression with NmU-R1 in the gastrointestinal tract. Thus, interpretation of the physiological effects of NmU in this tissue will need to be carefully considered in regard to which receptor is mediating a given event.
The identification of NmU-R2 as a neuromedin receptor was accomplished
in human embryonic kidney cells (293) that were transiently transfected
with NmU-R2 cDNA. The NmU-R2 transfected cells were assessed for
their ability to mobilize intracellular calcium when stimulated with
each of more than 1000 known or suspected GPCR ligands including small
molecules and various peptides, among them neuromedins B, C, K, L, N,
and U (human NmU-25, rat NmU-23, and pig NmU-8). From among this
library of potential ligands, only the NmUs generated a specific,
dose-dependent calcium flux in the transfected cells (Fig.
3A). This response was
dose-dependent with an EC50 of 5 nM
and a maximal response observed between 80 and 800 nM (Fig.
3B). Maximal intracellular calcium concentration reached
600-800 nM when cells were stimulated with 100 nM NmU-25 (Fig. 3C). We observed no significant
difference in the ability of NmU-25/23 to stimulate NmU-R2 when
compared with NmU-8 (data not shown). We did find amidation of NmU-8 to
be necessary for activity as a nonamidated form did not activate the
receptor even at concentrations in excess of 10 µM (data
not shown). In addition, we found that pertussis toxin (100 ng/ml,
overnight incubation) did not significantly alter NmU-R2 receptor
signaling, suggesting that in 293 cells this receptor couples to
calcium through the Gq subset of G proteins (Fig.
3A, inset).
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Recently, a number of other investigators have also reported characterization of NmU-R2 (23, 31, 32). All of these manuscripts report an NmU EC50 of 1-5 nM in functional assays, which is consistent with our findings. These reports agree also that coupling appears to be primarily through Gq although Hosoya et al. (31) reported some Gi coupling. The reports vary, however, in their findings regarding distribution of NmU-R2 mRNA. Hosoya et al. (31) reported only rat expression and provided no data regarding the expression pattern of human NmU-R2. Furthermore, these investigators performed their analysis using only quantitative PCR and provided no other confirmation of their results. Similarly, Raddatz et al. (32), while providing human expression data, relied solely on quantitative PCR for expression analysis. Howard et al. (23) discussed expression of human NmU-R2 but presented no data, reporting only that NmU-R2 expression was weakly observed in some tissues. Hosoya et al. (31) found rat NmU-R2 most prominently expressed in uterus (31); however, neither Raddatz et al. (32) nor our group found any significant expression of human NmU-R2 in uterus (Fig. 2). In contrast, expression of NmU-R2 was very high in human testis (Fig. 2) (32) but low in rat testis (31).
The recent reports on NmU-R2 do agree that this receptor is most prominently expressed in the brain of both human and rat; however, the exact nature of the message detected and whether it is translated remains unknown. This question becomes more important in light of our finding of alternative transcripts for NmU-R2 (Fig. 1B).
In particular, Raddatz et al. (32) reported expression of neuromedin U receptor in dorsal root ganglion; however, we have been unable to demonstrate NmU responsiveness in either mouse or rat dorsal root ganglion, even at doses as high as 1 µm, although this could be species-related (data not shown).2 Howard et al. (23) have shown that intracerebral injection of NmU altered feeding behavior in rats but did not demonstrate dose responsiveness of this effect, only that 1 µg of NmU was not sufficient to alter feeding behaviors, while 3 or 10 µg produced similar effects. These studies also failed to demonstrate that the effect observed was mediated by central nervous system receptors because no similar studies were reported using peripheral administration of NmU. Given that NmU-R1 is highly expressed in gastrointestinal tract tissue and that nothing is known about the ability of NmU to cross the blood-brain barrier, it is not unreasonable to assume that some of the observed effects upon feeding behavior might be mediated directly or indirectly via peripheral receptors. Finally, given the history of species-specific effects of NmU on muscle contraction (see introduction) and differences in the species-specific expression of the NmU receptors, it will be important to develop additional animal models for NmU function and to confirm any findings in human cells or tissues whenever possible.
In summary, we have demonstrated the existence of a second specific
neuromedin U receptor that we have designated NmU-R2. Cells transfected
with NmU-R2 show a dose-dependent intracellular Ca2+ mobilization in response to NmU stimulation. We also
show that NmU-R2 calcium signaling in 293 cells is pertussis toxin
insensitive, suggesting that NmU-R2, like NmU-R1, is coupled to the
Gq family of G proteins in these cells. Unlike NmU-R1,
NmU-R2 mRNA is highly expressed in the central nervous system and
suggests that NmU-R2 mediates the effects of NmU in these tissues.
However, given that NmU-R1 and NmU-R2 expression overlap in some
peripheral tissues and that NmU itself is broadly expressed it will be
necessary to conduct further studies to understand the precise role
each receptor plays in mediating the peripheral and central effects of
neuromedin U. Targeted gene knockouts of each of the NmU receptors, as
well as of NmU itself, would undoubtedly be invaluable in such studies.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF292402 (NmU-R2).
§ To whom correspondence should be addressed: Schering-Plough Research Institute, 2015 Galloping Hill Rd., K-15-1/1800, Kenilworth, NJ 07033. Tel.: 908-740-7408; Fax: 908-740-7101; E-mail: joseph. hedrick{at}spcorp.com.
Published, JBC Papers in Press, September 28, 2000, DOI 10.1074/jbc.C000522200
2 J. Crona, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: Nm, neuromedin; TM, transmembrane; GPCR, G protein-coupled receptor; ACTH, adrenocorticotropic hormone; PCR, polymerase chain reaction; EST, expressed sequence tag.
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