| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 276, Issue 31, 28969-28975, August 3, 2001
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the Departments of j Neurology, a Discovery Biology, g Biotechnology and Genetics, h Discovery Chemistry, and d Bioinformatics, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex CM19 5AW, United Kingdom, the Departments of c Biotechnology and Genetics and f Pulmonary Biology, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania, 19406, the b Neurobiology Programme, The Babraham Institute, Babraham, Cambridge CB2 4AT, United Kingdom, and the i Department of Anatomy with Radiology, University of Auckland, P. O. Box 92019, Auckland, New Zealand
Received for publication, March 28, 2001, and in revised form, May 23, 2001
 |
ABSTRACT |
|---|
 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
A novel human G protein-coupled receptor named
AXOR12, exhibiting 81% homology to the rat orphan receptor GPR54, was
cloned from a human brain cDNA library. Heterologous expression of
AXOR12 in mammalian cells permitted the identification of three
surrogate agonist peptides, all with a common C-terminal amidated
motif. High potency agonism, indicative of a cognate ligand, was
evident from peptides derived from the gene KiSS-1,
the expression of which prevents metastasis in melanoma cells.
Quantitative reverse transcriptase-polymerase chain reaction was
used to study the expression of AXOR12 and KiSS-1 in a variety of
tissues. The highest levels of expression of AXOR12 mRNA were
observed in brain, pituitary gland, and placenta. The highest levels of
KiSS-1 gene expression were observed in placenta and brain.
A polyclonal antibody raised to the C terminus of AXOR12 was generated
and used to show localization of the receptor to neurons in the
cerebellum, cerebral cortex, and brainstem. The biological
significance of these expression patterns and the nature of the
putative cognate ligand for AXOR12 are discussed.
The G protein-coupled receptors
(GPCRs)1 form a large family
of membrane bound proteins that share a unique structural feature comprising seven transmembrane Here, we describe the cloning of a novel human orphan receptor, a class
I GPCR with sequence similarity to receptors for the neuropeptide
galanin. This receptor was given the name AXOR12 in accordance with its
position in a series of receptors identified in our organization.
AXOR12 has a high degree of homology to the rat orphan receptor GPR54
(2) (81% amino acid identity), which suggests that these two receptors
may be orthologs. To identify a ligand for AXOR12, we expressed this
receptor in mammalian cells and screened the transfected cells in a
functional assay against a library rich in known and putative peptide
transmitters. Although there was no activity in response to galanin, we
identified three peptides that acted as low potency agonists of AXOR12.
These peptides were all derived from invertebrates and shared a
C-terminal LRF- or LRW-amide motif.
During the preparation of this article, a search of patent literature
revealed the existence of additional high potency agonists with
sequence similarities to the surrogate agonists identified from the
screen. These peptides were derived from a precursor known as KiSS-1.
The KiSS-1 gene was identified originally as being
up-regulated in melanoma cells that have lost the potential to
metastasize after microcell-mediated transfer of human chromosome 6 (3). Subsequent studies have shown that the exogenous expression of
KiSS-1 in other tumor cell lines also prevents metastasis
(4). The mechanism by which this occurs remains largely unknown;
however, KiSS-1 has structural features that suggest that it may be the precursor of a secreted peptide with an LRF-amide motif at the C
terminus. We synthesized the putative processed products of KiSS-1 and
observed that they acted as high potency agonists of AXOR12.
To gain insight into the physiological role of this receptor, we used
quantitative RT-PCR to localize the mRNA expression of AXOR12 and
KiSS-1 in a range of human tissues. We observed high levels of AXOR12
expression in the brain. Further RT-PCR analysis of brain expression
revealed a distinctive pattern of mRNA localization that was
further explored by immunohistochemistry using an antibody raised to
the extreme C-terminal tail of the receptor.
Receptor Cloning--
The human AXOR12 gene
was identified initially within a genomic clone (GenBankTM
accession number AC005379) as five coding exons interrupted by four
introns. The full-length cDNA was obtained by a modification of a
previously described cDNA capture method (5). Briefly, 5 µg of
plasmid DNA from a human brain cDNA library was screened with a biotinylated oligonucleotide (5'-biotin with an 18-atom spacer)
corresponding to the 5'-coding region
(5'-GATGCGGACCGTGACCAACTTCTAC-3'). Two additional 40-mers
(5'-GGAACTCGCTGGTCATCTACGTCATCTGCCGCCACAAGCC-3' and
5'-ATCGCCAACCTGGCGGCCACGGACGTGACCTTCCTCCTGTG-3'), corresponding to the
immediate 5' and 3' regions of the biotinylated probe, were also used
as blocking oligos. Bacterial colonies from the second round of
selection were screened by PCR using AXOR12-specific primers. Five
positive clones were identified, and the entire inserts were sequenced
on both strands using an ABI sequencer. Two of the sequenced clones
were identical to each other and to the full-length AXOR12 cDNA
predicted from the genomic DNA sequence.
Heterologous Receptor Expression and Functional Analysis in
Mammalian Cells--
The AXOR12 cDNA was subcloned into the
mammalian expression vector pCDN (6) as described previously (7).
Transient transfections of HEK293 cells with AXOR12 alone or AXOR12
co-transfected with Gqi5 (a chimeric G protein
To obtain mammalian cells stably expressing AXOR12, the cDNA was
subcloned into the vector pCD (a derivative of pCDN lacking the gene
for neomycin resistance) and co-transfected with pCDN Gqi5 (10) into
Chinese hamster ovary cells using LipofectAMINE PLUS (Life
Technologies, Inc.) at a DNA ratio of 10:1 (CHO/AXOR12:Gqi5 cells).
48 h later the cells were seeded into 96-well plates at 103
cells/well and selected with G418 (Life Technologies, Inc.) (400 µg/ml) and in the absence of nucleosides. Doubly selected cells were
screened by Northern blotting to confirm AXOR12 expression, and
positive clones were screened functionally in the fluorometric imaging
plate reader calcium assay. The clone that responded most sensitively
to surrogate agonists was used in all future experiments.
Peptide Synthesis--
The peptides KiSS-1-(107-121),
KiSS-1-(112-121), KiSS-1-(114-121), NPFF, neuropeptide AF,
RF-amide-like peptide-1 and -3, and galanin-like peptide were
synthesized by conventional solid-phase techniques using Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry (11),
and purification was conducted by preparative reverse phase HPLC. All
final products showed a purity of >95% by analytical reverse phase
HPLC, and peptide identities were confirmed by electrospray mass
spectrometry. The peptides KiSS-1-(58-65), KiSS-1-(68-91), KiSS-1-(68-80), KiSS-1-(68-121), KiSS-1-(96-121), and
KiSS-1-(125-144) were prepared by California Peptide Research Inc.,
CA. Antho-RW-amides I and II, Peptide F1, and galanin were purchased
from Bachem.
TaqMan RT-PCR localization of AXOR12 and KiSS-1--
RNA
purification and TaqMan RT-PCR analysis of human tissue were performed
as described previously (12). TaqMan primer and probe sets for AXOR12,
KiSS-1, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase were designed using Primer Express V1.0 software (Applied
Biosystems). Primer and probe sequences (forward primer, reverse
primer, TaqMan probe) were; AXOR12, 5'-TGGCACCCACGCAGCTA-3', 5'-AGTTGCTGTAGGACATGCAGTGA-3', 5'-CCGCCTACGCGCTTAAGACCTGG-3'; KiSS-1,
5'-ACTCACTGGTTTCTTGGCAGCT-3', 5'-CAGAGGCCACCTTTTCTAATGG-3', 5'-CTTTTCCTCTGTGCCACCCACTTTGG-3'; and glyceraldehyde-3-phosphate dehydrogenase, 5'-CAAGGTCATCCATGACAACTTTG-3',
5'-GGGCCATCCACAGTCTTCTG-3', 5'-ACCACAGTCCATGCCATCACTGCCA-3'. The levels
of mRNA measured were calculated as copies of mRNA detected per
ng of reverse transcribed poly(A)+ RNA.
Receptor Protein Localization Studies--
A unique synthetic
peptide (CVLGEDNAPL) located at the extreme C terminus of the human
AXOR12 receptor sequence, corresponding to amino acids 389-398, was
synthesized (Bio Synthesis Inc.). Polyclonal antibodies were produced
as described in detail elsewhere (13). In brief, New Zealand White
rabbits were injected with a peptide-purified protein derivative of
tuberculin conjugate and boosted at regular intervals. Crude
bleeds were tested for antibody titer using a standard enzyme-linked
immunosorbent assay protocol. AXOR12 antiserum was purified from crude
rabbit serum by immunoaffinity chromatography on peptide-coupled
SulfolinkTM gel (Pierce). Western blotting was carried out essentially
as described elsewhere (13). In brief, membranes were prepared from
selected tissue regions of human brain (frontal cortex, hippocampus, and basal ganglia) and CHO AXOR12:Gqi5 cells or nontransfected CHO
cells. Protein concentrations were determined using the BCA protein
assay kit (Pierce) according to manufacturer instructions. For
SDS-polyacrylamide gel electrophoresis, 10 µg of membrane protein was
denatured in Laemmli sample buffer (14). After electrophoresis, the
proteins were blotted onto 0.45-µm nitrocellulose membranes, blocked
in 5% milk solution in Tris-buffered saline/0.1% Tween 20, and
incubated with affinity-purified AXOR12 antiserum (1:5000). Immunoreactivity was detected and visualized by incubating the membrane
with a horseradish peroxidase-conjugated goat anti-rabbit secondary
antibody (1:5000, Promega, Madison, WI) followed by chemiluminescent
detection (ECL, Amersham Pharmacia Biotech). Controls included
pre-absorption of AXOR12 antibody with immunogenic peptide (10 µM) prior to incubation with the blot and omission of the
primary antibody.
Immunocytochemistry was performed on stably transfected CHO AXOR12:Gqi5
cells. The cells were fixed in 4% paraformaldehyde/PBS for 20 min at 4 °C, permeabilized for 5 min in 0.5% Triton X-100/PBS, and
blocked for 30 min in 50% fetal calf serum. The fixed cells were
incubated in a 1:250 dilution of affinity-purified AXOR12 antiserum
overnight at 4 °C, washed three times in PBS, and incubated for 30 min in anti-rabbit secondary antibody conjugated to fluorescein isothiocyanate. Coverslips were mounted with vectashield containing 4',6-diamidino-2-phenylindole (DAPI, Vector Laboratories).
Immunohistochemistry was carried out on perfusion-fixed post-mortem
human brain tissue obtained from the New Zealand Neurological Foundation Human Brain Bank (University of Auckland) with consent from
the University of Auckland Human Subjects Ethics Committee. All tissues
used in this study were from cases with no previous history of
neurological disorders or abnormalities after thorough pathological
examinations. The post-mortem delay from death until tissue fixation
ranged from 10 to 18 h. Coronal 50-µm-thick brain sections were
cut on a freezing sledge microtome and collected in 0.1 M
PBS, pH 7.4. Endogenous peroxidase activity was quenched for 30 min.
Sections were pre-incubated with normal goat serum (1%) in buffer A
(0.1 M PBS/0.3% (v/v) Triton X-100) for 1 h followed by incubation with affinity-purified AXOR12 antiserum (1:2000) overnight at 4 °C. Sections were then incubated with biotinylated goat anti-rabbit secondary antibody (1:200, Vector Laboratories) for
2 h followed by incubation with ABC reagent (1:200, Vector Laboratories) for 45 min. Sections were visualized using 0.5 mg/ml 3'3-diaminobenzidine as a substrate and 0.03%
H2O2. Stained tissue sections were mounted onto
microscope slides, air-dried, and coverslipped with Depex (BDH
Laboratory Supplies). Controls included pre-absorption of primary
antibody with 50 µM peptide antigen overnight at 4 °C
prior to incubation in addition to the omission of the primary antibody
or the use of pre-immune serum.
As part of an ongoing effort to identify ligands for novel human
orphan GPCRs, we cloned a novel GPCR, originally identified within a
genomic clone, using a cDNA capture method. As shown in Fig.
1 this gene, AXOR12, encodes a
398-amino acid protein. TMHMM, a program that uses a hidden Markov
model for predicting transmembrane helices (15), predicted that the
protein contained seven hydrophobic putative transmembrane domains.
AXOR12 has sequence homology with class I members of the GPCR
superfamily, exhibiting the highest sequence homology (81% amino acid
identity) to GPR54, a rat receptor previously characterized as a
galanin receptor-like orphan (2). The human GPCRs to which AXOR12 has
closest homology are members of the galanin receptor family, sharing
28, 30, and 30% amino acid identity with human GalR1, GalR2, and
GalR3, respectively.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices. These molecules act as
receptors for a diverse range of extracellular signaling molecules including small molecules (amino acids and biogenic amines), lipids, small bioactive peptides, and large polypeptides (1). They have been
used successfully as drug targets by the pharmaceutical industry for a
number of years. Attention has focused on a number of proteins that are
known to be GPCRs through structural homology but for which no ligand
has been identified: so-called orphan receptors. At the same time as
the recent discovery of new GPCRs, there has been a renewed focus on
discovering potential novel peptides that may act as endogenous ligands
for these receptors.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit consisting
of G
q with the C-terminal five amino acids substituted
with the corresponding amino acids from Gi2, which
facilitates GPCR signaling through phospholipase C) were prepared for
functional studies. A Ca2+ mobilization assay using the
microtiter plate-based Ca2+ mobilization fluorometric
imaging plate reader was performed as described previously (8). HEK293
cells co-transfected with AXOR12 and Gqi5 were screened against a large
library of over 1500 known and putative GPCR agonists including all
available mammalian neuropeptides as described previously (9). Peptides in this library were tested at a final concentration of >100
nM and other potential small molecule agonists at >1
µM.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (59K):
[in a new window]
Fig. 1.
The deduced peptide sequence of human
(h) AXOR12 and alignment with the peptide sequence of
rat (r) GPR54. Potential transmembrane domains
are indicated (TM1-7), and predicted N-linked
glycosylation sites are shown with arrows.
We adopted a "reverse pharmacological" strategy (16) to identify agonists for AXOR12. Thus, HEK293 cells co-transfected with AXOR12 and Gqi5 were challenged with a large library of more than 1500 putative ligands, and responses were measured in a microtiter plate-based (fluorometric imaging plate reader) calcium mobilization assay. We observed that specific responses were elicited by two neuropeptides originally isolated from the sea anemone, Anthopleura elegantissima (antho-RW-amide I (17) and antho-RW-amide II (18)), and a neuropeptide from the lobster, peptide F1 (19) (sequences shown in Table I). Further experiments demonstrated that these responses were not dependent upon co-transfection of the recombinant chimeric G protein and were concentration-dependent with EC50 values in the low micromolar range (Fig. 2). For further studies, CHO AXOR12:Gqi5 cells were used. The CHO parental cells did not respond to the peptides studied here, but the transfected cell line responded to the three surrogate agonists (Fig. 4 and Table I). Galanin and galanin-like peptide, when tested at concentrations up to 1 µM, did not activate CHO AXOR12:Gqi5 cells (data not shown). The common feature of all activating peptides is the presence of an amidated LRF or LRW motif at the C terminus. This suggests that the cognate ligand for this receptor is likely to have a similar structure at the C terminus.
|
|
From a search of the patent literature, KiSS-1, a potential ligand for
AXOR12, was identified (patent number WO200024890, Takeda Chemical
Industries, Ltd). An analysis of the peptide sequence of KiSS-1 showed
it to have features typical of secreted neuropeptides including a
signal sequence, as predicted by signalP (20), several potential
dibasic cleavage sites, and a cleavage/amidation site (Fig.
3). This would result in a putative
54-amino acid-secreted peptide product corresponding to residues
68-121 of the full-length KiSS-1. Most interestingly, this contains a
C-terminal LRF-amide sequence, as predicted from our studies with
nonmammalian peptides.
|
A range of peptides of different lengths were synthesized,
corresponding to the C-terminal end of the putative secreted segment of
KiSS-1, and were tested in CHO AXOR12:Gqi5 cells using the fluorometric
imaging plate reader assay as described above. A comparison of the
peptides derived from the putative secreted segment of KiSS-1 (68-121,
94-121, 107-121, 112-121, and 114-121) (Table I) revealed that
these peptides were substantially more potent than the nonmammalian
LRF- or LRW-amide peptides tested (Fig.
4, Table I). Of the four truncated KiSS-1
sequences, the decapeptide 112-121 possessed the highest potency.
Further N-terminal deletion of this peptide resulted in nearly a
20-fold drop in functional potency. Chain elongation to the
pentadecapeptide 107-121 or the longer fragment 96-121 afforded
smaller reductions in potency (Fig. 4). Likewise, elongation to the
54-amino acid peptide corresponding to the entire putative secreted
segment of KiSS-1, tested in separate experiments, resulted in a
further reduction in potency (Table I). Peptides corresponding to the N
terminus of the putative secreted segment and also to putatively
nonsecreted segments of KiSS-1 were inactive (pEC50 < 5)
(Table I). NPFF, neuropeptide AF, and the RF-amide like peptides active
at the type I neuropeptide FF receptor, NPFFR1 (21), were also tested,
and all failed to generate a response (pEC50 < 5, data not
shown).
|
The calcium mobilization response seen after the activation of
AXOR12 when transiently transfected without additional G protein -subunits into HEK293 cells (Fig. 2) suggests that this receptor is
coupled to G proteins of the Gq/11 subfamily. In agreement with this hypothesis, KiSS-1-(112-121) caused identical calcium mobilization in both control and pertussis toxin-treated HEK293 cells
transiently expressing AXOR12 (data not shown), suggesting that
activation of G proteins from the Gi/o family and
subsequent G
-mediated activation of phospholipase
C
does not contribute to the functional response observed. In
addition, neither basal nor forskolin-elevated levels of intracellular
cAMP were modulated by KiSS-1-(112-121) in HEK293 cells transiently
expressing AXOR12 (data not shown), suggesting that this receptor does
not couple strongly to G proteins of the Gs and/or
Gi/o subfamilies.
To characterize the expression pattern of both the receptor
AXOR12 and its putative ligand KiSS-1 we carried out quantitative RT-PCR analysis on a broad range of human tissues. AXOR12 was widely
expressed in human tissues including the placenta, brain, and pituitary
at high levels (Fig. 5), with lower
levels detected in lymphocytes, pancreas, and adipose tissue. Within
the human central nervous system, AXOR12 mRNA was widespread in its
expression including the amygdala, nucleus accumbens, hippocampus, and
cingulate gyrus (Fig. 6). KiSS-1 mRNA
was detected predominantly in the placenta (Fig. 5) but was also
widespread throughout the central nervous system, although at
much lower levels (Fig. 6).
|
|
To gain a greater understanding of the specific cellular
localization and level of protein expression of AXOR12, we generated an
antiserum directed against a C-terminal peptide from AXOR12. Western
blot analysis of membranes from CHO AXOR12:Gqi5 cells revealed a broad
immunoreactive band of ~75 kDa, which was absent in membranes from
untransfected CHO cells (Fig.
7A). Similarly, analysis of
human brain membrane proteins revealed a specific band of ~75 kDa (as
well as a larger band at ~125 kDa) in hippocampus, basal ganglia, and
frontal cortex (Fig. 7B). These immunoreactive bands were
competed out by the immunizing peptide (data not shown). In both cases,
the 75-kDa protein detected agrees with the predicted size of the
native AXOR12 receptor together with a probable degree of glycosylation
at extracellular consensus sites (see Fig. 1).
|
The AXOR12 receptor-specific antiserum was used to localize receptor immunoreactivity in CHO AXOR12:Gqi5 cells. Expression of the receptor was detected both at the membrane and within the cells (Fig. 7C), although only a subset of the cells was immunoreactive for AXOR12 (Fig. 7, C and D).
Immunohistochemical analysis of human brain sections revealed prominent
neuronal expression in the regions sampled including the cerebral
cortex, thalamus, pons-medulla, and cerebellum. In the cerebral cortex,
AXOR12-specific staining was present on a large number of pyramidal
neurones of layers III, V, and VI (Fig. 8A). However, in the basal
ganglia (caudate nucleus, putamen, globus pallidus, and substantia
nigra), staining was primarily localized to AXOR12-immunoreactive
fibers and processes. In the cerebellum AXOR12 was strikingly localized
to the surface of the Purkinje cells and their apical dendrites (Fig.
8B) and to a lesser extent the cells of the deep cerebellar
nuclei. In the pons medulla AXOR12 immunoreactivity was widespread in a
number of nuclei including the raphe nuclei, inferior olive, and
hypoglossal nuclei (Fig. 8C). AXOR12 staining was abolished
when the antibody was pre-absorbed with 10 µM immunogenic
peptide (Fig. 8, D and E) or when the primary antibody was omitted (data not shown).
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
In this article we describe the cloning of a potential human ortholog of the rat G protein-coupled receptor GPR54, which we term AXOR12. A number of peptides possessing an LRW-amide or an LRF-amide motif at the C terminus are identified as surrogate ligands of AXOR12.
The first RF-amide peptide to be described was FMRFamide, a cardioexcitatory neuropeptide isolated from the bivalve mollusk Macrocallista nimbosa (22). FMRFamide and related RF-amides are widespread among invertebrates including Caenorhabditis elegans, which has at least 22 such peptides. These are expressed in the central and peripheral nervous systems and have a diverse range of functions including control of defecation, feeding, and reproduction (23). More recently, several RF-amides have been discovered in mammalian species: neuropeptides FF and AF (NPFF and neuropeptide AF (NPAF)) (24, 25), prolactin releasing peptide (26), and RF-amide related peptides-1 and -3 (21).
Some orphan receptors have been shown to be activated by naturally occurring peptides from lower organisms that have structural similarity to the cognate neuropeptide ligand for these receptors (27). For this reason we screened AXOR12 against a library with a wide diversity of mammalian and nonmammalian peptides. On the basis of our initial screen we reasoned that the natural ligand for AXOR12 was likely to contain a LRF- or LRW-amide motif at its C terminus. KiSS-1, a putative human neuropeptide with an LRF-amide motif identified from the patent literature, was therefore a candidate cognate ligand for this receptor.
Structurally, KiSS-1 bears all of the hallmarks of a secreted neuropeptide, with a putative signal sequence and several sites amenable to cleavage including an amidation/cleavage site that would result in a number of amidated peptide fragments of various lengths. We synthesized a number of putative N-terminally truncated products of this peptide and tested them against AXOR12. The results showed that the C-terminal decapeptide from the putative full-length secreted segment of KiSS-1 possessed sub-nanomolar activity at AXOR12. A reduction in chain length to the octapeptide 114-121 resulted in a significant drop in functional activity, and it is postulated that Tyr112 and Asn113 may each play an important role in receptor interaction and activation. Chain elongation resulted in minor decreases in activity, suggesting that the most relevant pharmacophore resides in the C-terminal fragment 112-121. AXOR12 failed to be activated by other RF-amide peptides, NPFF, neuropeptide AF, and RF-amide-like peptide-1 and -3, indicating the selectivity of activity of the KiSS-1 peptides.
Expression of KiSS-1 in normal tissues was detected initially only in placenta (3). Our own observations have shown that although KiSS-1 mRNA is extremely abundant in the placenta, low levels of message are also found in the brain and more specifically in the hypothalamus and basal ganglia. This distribution pattern is consistent with the mRNA localization of AXOR12, which is also in placenta, several central nervous system regions, and pituitary. Our data on AXOR12 localization broadly correspond to the published data for the putative rat ortholog GPR54 (2).
Immunohistochemical data indicate that expression of the receptor occurs specifically on a number of neuronal cell types in the human central nervous system regions that were examined. Indeed, neuronal localization of AXOR12 in many regions of the cerebral cortex, cerebellum, and medulla fits well with the observed mRNA expression pattern in similar regions of the human central nervous system. The prominent and widespread expression of AXOR12 throughout the central nervous system, especially on a number of projection neurones including pyramidal cells in the cerebral cortex and cerebellar Purkinje cells, indicates that ligands acting on AXOR12 would be able to influence a wide range of central nervous system functions ranging from cognition through to movement and balance.
The mapping of AXOR12 to human chromosome 19p13.3 (2) corresponds with a number of inherited neurological diseases including familial febrile convulsions (28), vacuolar neuromyopathy (29), and cayman-type cerebellar ataxia (30). Furthermore, the syntenic region on mouse chromosome 10 is associated with the allelic mutations jittery and hesitant, which have neuropathic phenotypes characterized by ataxia, dystonia, and seizures (31).
The KiSS-1 peptide was identified originally as being differentially up-regulated in C8161 melanoma cells that have been rendered nonmetastatic by microcell-mediated transfer of human chromosome 6 (3). Transfection of KiSS-1 into human breast carcinoma cells also prevents these cells from metastasizing (4). The role of AXOR12 in these systems has not been explored, although expressed sequence tags corresponding to AXOR12 have been identified in a number of tumor cDNA libraries (GenBankTM accession numbers AI823800, AI819198, and AA887801). Interestingly, several neuropeptides are known to have functional roles in tumor biology including galanin, vasopressin, cholecystokinin, and neurotensin by autocrine and paracrine mechanisms (32, 33). The high levels of expression of both KiSS-1 and AXOR12 in placenta are also noteworthy in this respect. The placenta is also an invasive tissue, and there are similarities in the behavior of invading cancer cells and that of invading placenta cells (34). It is possible that KiSS-1 and AXOR12 may form part of a mechanism that is common to both of these processes.
In conclusion, AXOR12 constitutes a new human G protein-coupled
receptor that has now been paired with its neuropeptide ligand, KiSS-1.
Although there is still much to be discovered about both the receptor
and its ligand, the biological evidence thus far suggests that they may
have important physiological roles both in the central nervous system
and in tumor biology. As such, they represent an intriguing target for
novel therapies in the fields of both neurology and oncology.
| |
FOOTNOTES |
|---|
* 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) AJ309020.
e Current Address: Inpharmatica Ltd., 60 Charlotte St., London W1T 2NU, United Kingdom.
k To whom correspondence should be addressed. Tel.: 44-0-1279-622728; Fax: 44-0-1279-622371; E-mail: David_C_Harrison@gsk.com.
Published, JBC Papers in Press, May 31, 2001, DOI 10.1074/jbc.M102743200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: GPCR, G protein-coupled receptor; RT, reverse transcriptase; PCR, polymerase chain reaction; CHO, Chinese hamster ovary; NPFF, neuropeptide FF; HPLC, high pressure liquid chromatography.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Wilson, S., Bergsma, D. J., Chambers, J. K., Muir, A. I., Fantom, K. G. M., Ellis, C., Murdock, P. R., Herrity, N. C., and Stadel, J. M. (1998) Br. J. Pharmacol. 125, 1387-1392 |
| 2. | Lee, D. K., Nguyen, T., O'Neill, G. P., Cheng, R., Liu, Y., Howard, A. D., Coulombe, N., Tan, C. P., Tang-Nguyen, A. T., George, S. R., and O'Dowd, B. F. (1999) FEBS Lett. 446, 103-107 |
| 3. | Lee, J.-H., Miele, M. E., Hicks, D. J., Phillips, K. K., Trent, J. M., Weissman, B. E., and Welch, D. R. (1996) J. Natl. Cancer Inst. 88, 1731-1737 |
| 4. | Lee, J.-H., and Welch, D. R. (1997) Cancer Res. 57, 2384-2387 |
| 5. | Shepard, A. R., and Rae, J. L. (1997) Nucleic Acids Res. 25, 3183-3185 |
| 6. | Trill, J. J., Shatzman, A. R., and Ganguly, S. (1995) Curr. Opin. Biotech. 6, 553-560 |
| 7. | Aiyar, N., Disa, J., Stadel, J. M., and Lysko, P. G. (1999) Mol. Cell. Biochem. 197, 179-185 |
| 8. | Chambers, J., Ames, R. S., Bergsma, D., Muir, A., Fitzgerald, L. R., Hervieu, G., Dytko, G. M., Foley, J. J., Martin, J., Liu, W. S., Park, J., Ellis, C., Ganguly, S., Konchar, S., Cluderay, J., Leslie, R., Wilson, S., and Sarau, H. M. (1999) Nature 400, 261-265 |
| 9. | Szekeres, P. G., Muir, A. I., Spinage, L. D., Miller, J. E., Butler, S. I., Smith, A., Rennie, G. I., Murdock, P. R., Fitzgerald, L. R., Wu, H., McMillan, L. J., Guerrera, S., Vawter, L., Elshourbagy, N. A., Mooney, J. L., Bergsma, D. J., Wilson, S., and Chambers, J. K. (2000) J. Biol. Chem. 275, 20247-20250 |
| 10. | Conklin, B. R., Farfel, Z., Lustig, K. D., Julius, D., and Bourne, H. R. (1993) Nature 363, 274-276 |
| 11. | Chan, W. C., and White, P. D. (2000) Fmoc Solid Phase Peptide Synthesis: A Practical Approach , Oxford University Press, Oxford |
| 12. | Sarau, H. M., Ames, R. S., Chambers, J., Ellis, C., Elshourbagy, N., Foley, J. J., Schmidt, D. B., Muccitelli, R. M., Jenkins, O., Murdock, P. R., Herrity, N. C., Halsey, W., Sathe, G., Muir, A. I., Nuthulaganti, P., Dytko, G. M., Buckley, P. T., Wilson, S., Bergsma, D. J., and Hay, D. W. P. (1999) Mol. Pharmacol. 56, 657-663 |
| 13. | Moore, D., Chambers, J., Waldvogel, H., Faull, R., and Emson, P. (2000) J. Comp. Neurol. 421, 374-384 |
| 14. | Laemmli, U. K. (1970) Nature 227, 680-685 |
| 15. | Sonnhammer, E. L. L., von Heijne, G., and Krogh, A. (1998) Proceedings of the Sixth International Conference on Intelligent Systems for Molecular Biology , pp. 175-182, American Association for Artificial Intelligence Press, Menlo Park, CA |
| 16. | Stadel, J. M., Wilson, S., and Bergsma, D. J. (1997) Trends Pharmacol. Sci. 18, 430-437 |
| 17. | Graff, D., and Grimmelikhuijzen, C. J. (1988) Brain Res. 442, 354-358 |
| 18. | Graff, D., and Grimmelikhuijzen, C. J. (1988) FEBS Lett. 239, 137-140 |
| 19. | Trimmer, B. A., Kobierski, L. A., and Kravitz, E. A. (1987) J. Comp. Neurol. 266, 16-26 |
| 20. | Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Protein Eng. 10, 1-6 |
| 21. | Hinuma, S., Shintani, Y., Fukusumi, S., Iijima, N., Matsumoto, Y., Hosoya, M., Fujii, R., Watanabe, T., Kikuchi, K., Terao, Y., Yano, T., Yamamoto, T., Kawamata, Y., Habata, Y., Asada, M., Kitada, C., Kurokawa, T., Onda, H., Nishimura, O., Tanaka, M., Ibata, Y., and Fujino, M. (2000) Nat. Cell Biol. 2, 703-708 |
| 22. | Price, D., and Greenberg, M. (1977) Science 197, 670-671 |
| 23. | Li, C., Kim, K., and Nelson, L. S. (1999) Brain Res. 848, 26-34 |
| 24. | Yang, H. Y., Fratta, W., Majane, E. A., and Costa, E. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 7757-7761 |
| 25. | Perry, S. J., Huang, E. Y.-K., Cronk, D., Bagust, J., Sharma, R., Walker, R. J., Wilson, S., and Burke, J. F. (1997) FEBS Lett. 409, 426-430 |
| 26. | Hinuma, S., Habata, Y., Fujii, R., Kawamata, Y., Hosoya, M., Fukusumi, S., Kitada, C., Masuo, Y., Asano, T., Matsumoto, H., Sekiguchi, M., Kurokawa, T., Nishimura, O., Onda, H., and Fujino, M. (1998) Nature 393, 272-276 |
| 27. | Elshourbagy, N. A., Ames, R. S., Fitzgerald, L. R., Foley, J. J., Chambers, J. K., Szekeres, P. G., Evans, N. A., Schmidt, D. B., Buckley, P. T., Dytko, G. M., Murdock, P. R., Milligan, G., Groarke, D. A., Tan, K. B., Shabon, U., Nuthulaganti, P., Wang, D. Y., Wilson, S., Bergsma, D. J., and Sarau, H. M. (2000) J. Biol. Chem. 275, 25965-25971 |
| 28. | Johnson, E. W., Dubovsky, J., Rich, S. S., O'Donovan, C. A., Orr, H. T., Anderson, V. E., Gil-Nagel, A., Ahmann, P., Dokken, C. G., Schneider, D. T., and Weber, J. L. (1998) Hum. Mol. Genet. 7, 63-67 |
| 29. | Servidei, S., Capon, F., Spinazzola, A., Mirabella, M., Semprini, S., de Rosa, G., Gennarelli, M., Sangiuolo, F., Ricci, E., Mohrenweiser, H. W., Dallapiccola, B., Tonali, P., and Novelli, G. (1999) Neurology 53, 830-837 |
| 30. | Nystuen, A., Benke, P. J., Merren, J., Stone, E. M., and Sheffield, V. C. (1996) Hum. Mol. Genet. 5, 525-531 |
| 31. | Kapfhamer, D., Sweet, H. O., Sufalko, D., Warren, S., Johnson, K. R., and Burmeister, M. (1996) Genomics 35, 533-538 |
| 32. | Seufferlein, T., and Rozengurt, E. (1996) Cancer Res. 56, 5758-5764 |
| 33. | Ormandy, C. J., Lee, C. S. L., Ormandy, H. F., Fantl, V., Shine, J., Peters, G., and Sutherland, R. L. (1998) Cancer Res. 58, 1353-1357 |
| 34. | Murray, M. J., and Lessey, B. A. (1999) Semin. Reprod. Endocrinol. 17, 275-290 |
This article has been cited by other articles:
|
|
 |
|
  C. Zhang, T. A. Roepke, M. J. Kelly, and O. K. Ronnekleiv Kisspeptin Depolarizes Gonadotropin-Releasing Hormone Neurons through Activation of TRPC-Like Cationic Channels J. Neurosci., April 23, 2008; 28(17): 4423 - 4434. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Pielecka-Fortuna, Z. Chu, and S. M. Moenter Kisspeptin Acts Directly and Indirectly to Increase Gonadotropin-Releasing Hormone Neuron Activity and Its Effects Are Modulated by Estradiol Endocrinology, April 1, 2008; 149(4): 1979 - 1986. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Smith, A. Rao, A. Pereira, A. Caraty, R. P. Millar, and I. J. Clarke Kisspeptin Is Present in Ovine Hypophysial Portal Blood But Does Not Increase during the Preovulatory Luteinizing Hormone Surge: Evidence that Gonadotropes Are Not Direct Targets of Kisspeptin in Vivo Endocrinology, April 1, 2008; 149(4): 1951 - 1959. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R A Silvestre, E M Egido, R Hernandez, and J Marco Kisspeptin-13 inhibits insulin secretion without affecting glucagon or somatostatin release: study in the perfused rat pancreas J. Endocrinol., February 1, 2008; 196(2): 283 - 290. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L Filby, R. v. Aerle, J. Duitman, and C. R Tyler The Kisspeptin/Gonadotropin-Releasing Hormone Pathway and Molecular Signaling of Puberty in Fish Biol Reprod, February 1, 2008; 78(2): 278 - 289. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Nakamura, S. Aoki, Yewei Xing, H. Sasano, and W. E. Rainey Metastin Stimulates Aldosterone Synthesis in Human Adrenal Cells Reproductive Sciences, December 1, 2007; 14(8): 836 - 845. [Abstract] [PDF]
|
|
|
|
|
|
C. L. Roth, C. Mastronardi, A. Lomniczi, H. Wright, R. Cabrera, A. E. Mungenast, S. Heger, H. Jung, C. Dubay, and S. R. Ojeda Expression of a Tumor-Related Gene Network Increases in the Mammalian Hypothalamus at the Time of Female Puberty Endocrinology, November 1, 2007; 148(11): 5147 - 5161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Caraty, J. T. Smith, D. Lomet, S. Ben Said, A. Morrissey, J. Cognie, B. Doughton, G. Baril, C. Briant, and I. J. Clarke Kisspeptin Synchronizes Preovulatory Surges in Cyclical Ewes and Causes Ovulation in Seasonally Acyclic Ewes Endocrinology, November 1, 2007; 148(11): 5258 - 5267. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. S. Dhillo, O. B. Chaudhri, E. L. Thompson, K. G. Murphy, M. Patterson, R. Ramachandran, G. K. Nijher, V. Amber, A. Kokkinos, M. Donaldson, et al. Kisspeptin-54 Stimulates Gonadotropin Release Most Potently during the Preovulatory Phase of the Menstrual Cycle in Women J. Clin. Endocrinol. Metab., October 1, 2007; 92(10): 3958 - 3966. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Lapatto, J. C. Pallais, D. Zhang, Y.-M. Chan, A. Mahan, F. Cerrato, W. W. Le, G. E. Hoffman, and S. B. Seminara Kiss1 / Mice Exhibit More Variable Hypogonadism than Gpr54 / Mice Endocrinology, October 1, 2007; 148(10): 4927 - 4936. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. Luque, R. D. Kineman, and M. Tena-Sempere Regulation of Hypothalamic Expression of KiSS-1 and GPR54 Genes by Metabolic Factors: Analyses Using Mouse Models and a Cell Line Endocrinology, October 1, 2007; 148(10): 4601 - 4611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Li, D. Mitchell, J. Luo, Z. Yi, S.-G. Cho, J. Guo, X. Li, G. Ning, X. Wu, and M. Liu Estrogen Regulates KiSS1 Gene Expression through Estrogen Receptor {alpha} and SP Protein Complexes Endocrinology, October 1, 2007; 148(10): 4821 - 4828. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Marot, I. Bieche, C. Aumas, S. Esselin, C. Bouquet, S. Vacher, G. Lazennec, M. Perricaudet, F. Kuttenn, R. Lidereau, et al. High tumoral levels of Kiss1 and G-protein-coupled receptor 54 expression are correlated with poor prognosis of estrogen receptor-positive breast tumors Endocr. Relat. Cancer, September 1, 2007; 14(3): 691 - 702. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Kauffman, J. H. Park, A. A. McPhie-Lalmansingh, M. L. Gottsch, C. Bodo, J. G. Hohmann, M. N. Pavlova, A. D. Rohde, D. K. Clifton, R. A. Steiner, et al. The Kisspeptin Receptor GPR54 Is Required for Sexual Differentiation of the Brain and Behavior J. Neurosci., August 15, 2007; 27(33): 8826 - 8835. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Luan, Y. Zhou, W. Wang, H. Yu, P. Li, X. Gan, D. Wei, and J. Xiao Association study of the polymorphisms in the KISS1 gene with central precocious puberty in Chinese girls Eur. J. Endocrinol., July 1, 2007; 157(1): 113 - 118. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ramaswamy, S. B. Seminara, C. R. Pohl, M. J. DiPietro, W. F. Crowley Jr., and T. M. Plant Effect of Continuous Intravenous Administration of Human Metastin 45-54 on the Neuroendocrine Activity of the Hypothalamic-Pituitary-Testicular Axis in the Adult Male Rhesus Monkey (Macaca mulatta) Endocrinology, July 1, 2007; 148(7): 3364 - 3370. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. d'Anglemont de Tassigny, L. A. Fagg, J. P. C. Dixon, K. Day, H. G. Leitch, A. G. Hendrick, D. Zahn, I. Franceschini, A. Caraty, M. B. L. Carlton, et al. Hypogonadotropic hypogonadism in mice lacking a functional Kiss1 gene PNAS, June 19, 2007; 104(25): 10714 - 10719. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Kauffman, M. L. Gottsch, J. Roa, A. C. Byquist, A. Crown, D. K. Clifton, G. E. Hoffman, R. A. Steiner, and M. Tena-Sempere Sexual Differentiation of Kiss1 Gene Expression in the Brain of the Rat Endocrinology, April 1, 2007; 148(4): 1774 - 1783. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Smith, C. M. Clay, A. Caraty, and I. J. Clarke KiSS-1 Messenger Ribonucleic Acid Expression in the Hypothalamus of the Ewe Is Regulated by Sex Steroids and Season Endocrinology, March 1, 2007; 148(3): 1150 - 1157. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G Nicolle, E Comperat, N Nicolaiew, G Cancel-Tassin, and O Cussenot Metastin (KISS-1) and metastin-coupled receptor (GPR54) expression in transitional cell carcinoma of the bladder Ann. Onc., March 1, 2007; 18(3): 605 - 607. [Full Text] [PDF] |
,
KiSS-1-(112-121); 
, KiSS-1-(94-121); 
, KiSS-1-(107-121); 
,
KiSS-1-(114-121); 
, Antho-RW-amide I; 
, Antho-RW-amide II; 
,
peptide F1.
