Transcript
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AXOR12: a novel human G protein-coupled receptor, activated by the peptide KiSS-1.

Alison I. Muir2, Larissa Chamberlain8, Nabil A. Elshourbagy6, David Michalovich5§, Darren J.

Moore8, Amy Calamari6, Philip G. Szekeres2, Henry M. Sarau7, Jon K. Chambers2, Paul

Murdock3, Klaudia Steplewski6, Usman Shabon6, Jane E. Miller2, Susan E. Middleton2, John G.

Darker4, Christopher G. C. Larminie5, Shelagh Wilson2, Derk J. Bergsma6 Piers Emson8, Richard

Faull9, Karen L. Philpott1, David C. Harrison1*

Depts of Neurology1, Discovery Biology2, Biotechnology and Genetics3, Discovery Chemistry4,

Bioinformatics5, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, CM19 5AW,

UK

Depts of Biotechnology and Genetics6, Pulmonary Biology7 GlaxoSmithKline, 709 Swedeland

Road, King of Prussia, PA, USA

Neurobiology Programme, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK8

Dept of Anatomy with Radiology, University of Auckland, New Zealand9

§Current Address: Inpharmatica Ltd., 60 Charlotte St, London, W1T 2NU, UK

*Corresponding author

Telephone: +44 (0)1279 622728, Fax: +44 (0)1279 622371

Email: [email protected]

Running Title: AXOR12, a novel receptor activated by KiSS-1

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on May 31, 2001 as Manuscript M102743200 by guest on A

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Summary

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 KiSS-1, a gene whose

expression prevents metastasis in melanoma cells. Quantitative reverse transcriptase-polymerase

chain reaction (RT-PCR) was used to study the expression of AXOR12 and KiSS-1 in a variety

of tissues. 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.

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Introduction

The G protein-coupled receptors (GPCRs) form a large family of membrane bound proteins

which share a unique structural feature comprising 7 transmembrane alpha helices. These

molecules act as receptors for a diverse range of extracellular signalling molecules including

small molecules (amino acids, biogenic amines), lipids, small bioactive peptides and large

polypeptides (1). They have been successfully used as drug targets by the pharmaceutical

industry for a number of years. Attention has focussed on a number of proteins which 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 which may act as endogenous ligands

for these receptors.

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. In order 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. While there

was no activity in response to galanin, we identified three peptides which 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 manuscript, a search of patent literature revealed the existence of

additional, high potency agonists with sequence similarities to the surrogate agonists identified

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from the screen. These peptides were derived from a precursor known as KiSS-1. The KiSS-1

gene was originally identified as being upregulated in melanoma cells which have lost the

potential to metastasize following microcell mediated transfer of human chromosome 6 (3).

Subsequent studies have shown that 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.

In order to gain insight into the physiological role of this receptor, we used quantitative RT-PCR

to localize the mRNA expression of AXOR12 and of 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 localisation which was further explored by

immunohistochemistry using an antibody raised to the extreme C-terminal tail of the receptor. by guest on April 16, 2020

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Experimental Procedures

Receptor Cloning

The human AXOR12 gene was initially identified within a genomic clone (AC005379) as five

coding exons interrupted by 4 introns. The full-length cDNA was obtained by a modification of

a previously described cDNA capture method (5). Briefly, 5µg of plasmid DNA from a human

brain cDNA library was screened with a biotinylated oligonucleotide (5'-biotin with 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 α subunit consisting of Gαq with the C-

terminal 5 amino acids substituted with the corresponding amino acids from Gαi2, which

facilitates GPCR signalling through phospholipase C) were prepared for functional studies. A

Ca2+ mobilization assay using the microtiter plate-based Ca2+ mobilization fluorometric imaging

plate reader (FLIPR) was performed as described previously (8). HEK293 cells co-transfected

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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.

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) at a DNA ratio of 10:1 (CHO AXOR12:Gqi5 cells). Forty eight hours later the

cells were seeded into 96 well plates at 103 cells per well and selected with G418 (Life

Technologies) (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 FLIPR calcium assay. The clone which 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, NPAF, RFRP1-3 and

galanin-like peptide were synthesised by conventional solid phase techniques using Fmoc (9-

fluorenylmethoxycarbonyl) 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 MS. 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 Incorporated, California, USA. Antho-RW-amides I and II, Peptide

F1 and galanin were purchased from Bachem.

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Taqman RT-PCR localisation 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 for the housekeeping

gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express

V1.0 software (Applied Biosystems). Primer and probe sequences (forward primer, reverse

primer, Taqman probe); AXOR12: 5'-TGGCACCCACGCAGCTA-3', 5'-

AGTTGCTGTAGGACATGCAGTGA-3', 5'-CCGCCTACGCGCTTAAGACCTGG-3'; KiSS-1:

5'-ACTCACTGGTTTCTTGGCAGCT-3', 5'-CAGAGGCCACCTTTTCTAATGG-3', 5'-

CTTTTCCTCTGTGCCACCCACTTTGG-3'; GAPDH: 5'-

CAAGGTCATCCATGACAACTTTG-3', 5'-GGGCCATCCACAGTCTTCTG-3', 5'-

ACCACAGTCCATGCCATCACTGCCA-3'. Levels of mRNA measured were calculated as

copies of mRNA detected per ng of reverse transcribed polyA+ 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 synthesised (Bio

Synthesis Incorporated). Polyclonal antibodies were produced as described in detail elsewhere

(13). In brief, New Zealand white rabbits were injected with peptide-PPD conjugate and were

boosted at regular intervals. Crude bleeds were tested for antibody titre using a standard ELISA

protocol. AXOR12 antiserum was purified from crude rabbit serum by immunoaffinity

chromatography on peptide-coupled Sulfolink gel (Pierce Chemical Company). Western

blotting was carried out essentially as described elsewhere (13). In brief, membranes were

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prepared from selected tissue regions of human brain (frontal cortex, hippocampus, basal

ganglia), and CHO AXOR12:Gqi5 cells or non-transfected CHO cells. Protein concentrations

were determined using the BCA protein assay kit (Pierce Chemical Company) according to the

manufactures instructions. For SDS-polyacrylamide gel electrophoresis, 10 µg of membrane

protein was denatured in Laemmli sample buffer (14). Following electrophoresis, proteins were

blotted onto 0.45µm 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 visualised by incubating the membrane with a horseradish

peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000, Promega, Madison, USA)

followed by chemiluminescent detection (ECL: Amersham Pharmacia Biotech). Controls

included pre-absorption of AXOR12 antibody with immunogenic peptide (10µM) prior to

incubation with the blot, and omission of the primary antibody.

Immunocytochemistry was performed on stably transfected CHO AXOR12:Gqi5 cells. Cells

were fixed in 4% paraformaldehyde/phosphate buffered saline (PBS) for 20 min at 4°C,

permeabilized for 5 min in 0.5% TritonX-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 3 times in PBS and incubated for 30 min in anti-rabbit

secondary antibody conjugated to FITC. Coverslips were mounted with vectashield containing

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

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or abnormalities following thorough pathological examination. The post-mortem delay from

death until tissue fixation ranged from 10 to 18 hours. Coronal 50 µm-thick brain sections were

cut on a freezing sledge microtome and collected in 0.1M PBS, pH7.4. Endogenous peroxidase

activity was quenched for 30 min. Sections were pre-incubated with normal goat serum (1%) in

buffer A (0.1M 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 visualised

using 0.5mg/ml 3’3-diaminobenzidine (DAB) 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 50µM of

peptide antigen overnight at 4°C prior to incubation, in addition to omission of the primary

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Results

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 receptor, 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 7 hydrophobic putative transmembrane domains. AXOR12 has

sequence homology with class I members of the GPCR superfamily, exhibiting 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.

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 measured in a microtiter plate-based (FLIPR)

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 1). 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 utilized. 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 1). Galanin and galanin-like peptide, when tested at concentrations up to 1 µM did not

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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 non-mammalian

peptides.

A range of peptides of different lengths were synthesised, corresponding to the C-terminal end of

the putative secreted segment of KiSS-1, and were tested in CHO AXOR12:Gqi5 cells using the

FLIPR assay as described above. 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 1) revealed that

these peptides were substantially more potent than the non-mammalian LRF- or LRW-amide

peptides tested (Fig 4, Table 1). 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 twenty–fold drop in functional potency. Chain elongation to the pentadecapeptide 107-121 or

the longer fragment 96-121 afforded smaller reductions in potency (Fig4). 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 1). Peptides

corresponding to the N-terminus of the putative secreted segment and also to putatively non-

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secreted segments of KiSS-1 were inactive (pEC50 <5) (Table 1). NPFF, NPAF 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 following activation of AXOR12 when transiently

transfected without additional G protein α-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 Gβγ-mediated activation of

phospholipase Cβ 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 CNS, 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 CNS,

although at much lower levels (Fig. 6).

To gain a greater understanding of the specific cellular localisation and level of protein

expression of AXOR12, we generated an antiserum directed against a C-terminal peptide from

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AXOR12. Western blot analysis of membranes from CHO AXOR12:Gqi5 cells revealed a broad

immunoreactive band of approximately 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 approximately 75 kDa (as well as a larger band at approximately 125

kDa), in hippocampus, basal ganglia and frontal cortex (Fig. 7B). These immunoreactive bands

were competed out by the immunising 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 localise 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 were immunoreactive for AXOR12

(Fig 7C, 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 and V and VI (Fig 8A). However in the basal ganglia (caudate nucleus,

putamen, globus pallidus and substantia nigra) staining was primarily localised to AXOR12

immunoreactive fibres and processes. In the cerebellum AXOR12 was strikingly localised 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 preabsorbed with 10 µM immunogenic

peptide (Fig 8D, E) or when the primary antibody was omitted (data not shown).

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Discussion.

In this paper 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 C. elegans which has at least twenty two

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 NPAF), (24, 25); prolactin releasing peptide, (26); and RF-amide related peptides

1 and 3 (RFRP1, -3) (21).

Some orphan receptors have been shown to be activated by naturally occurring peptides from

lower organisms which 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 non-mammalian 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 synthesised a number

of putative N-terminally truncated products of this peptide and tested them against AXOR12.

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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 and NPAF and RFRP1-3, indicating the selectivity of activity of

the KiSS-1 peptides.

Expression of KiSS-1 in normal tissues was initially detected only in placenta (3). Our own

observations have shown that, while 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 localisation of AXOR12,

which is also in placenta, several CNS regions and pituitary. Our data on AXOR12 localisation

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 CNS regions that were examined. Indeed, neuronal

localisation 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 CNS. The

prominent and widespread expression of AXOR12 throughout the CNS, 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 CNS

functions ranging from cognition through to movement and balance.

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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 ten is associated with the allelic mutations jittery and hesitant which have

neuropathic phenotypes characterised by ataxia, dystonia and seizures (31).

KiSS-1 peptide was originally identified as being differentially upregulated in C8161 melanoma

cells that have been rendered non-metastatic 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 (GenBank entries AI823800, AI819198, 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 so far suggests that they may have

important physiological roles both in the CNS and in tumor biology. As such, they represent an

intriguing target for novel therapies in the fields of both neurology and oncology.

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Footnotes

Abbreviations:

GPCR, G protein-coupled receptor; RT-PCR, reverse transcriptase-polymerase chain reaction;

FLIPR, fluorometric imaging plate reader; NPFF, neuropeptide FF; NPAF, neuropeptide AF;

RFRP, RF-amide-like peptide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS,

phosphate buffered saline.

EMBL entry:

Accession number AJ309020, Homo sapiens mRNA for G protein-coupled receptor (AXOR12gene)

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Figure legends

Fig 1. The deduced peptide sequence of human AXOR12, and alignment with the peptide

sequence of rat GPR54. Potential transmembrane domains are indicated (TM1-7) and predicted

N-linked glycosylation sites are shown with arrows.

Fig 2. AXOR 12 expressed in mammalian cells responds to a number of peptides with a

common C-terminally amidated sequence, with dose-dependent increases in intracellular Ca2+.

HEK293 cells transiently transfected with AXOR12 (circles), but not HEK293 cells transfected

with empty vector (triangles) respond to two peptides; Antho-RW-amide I (filled symbols) and

Antho-RW-amide II (open symbols) isolated from the sea anemone Anthopleura elegantissima

with potencies in the low micromolar range. Data are expressed as the change in fluorescent

intensity units (FIU) over background and are from a single experiment, representative of a total

of three such experiments. Each point was determined in triplicate and is given as a mean ± SE.

Fig 3. Peptide sequence of KiSS-1 showing the predicted signal peptide sequence (underlined)

and the putative secreted peptide (bold, underlined). Potential dibasic cleavage sites (RK/RR)

and cleavage/amidation sites (GKR) are indicated by arrows.

Fig 4. CHO AXOR12:Gqi5 cells responded to non-mammalian peptides containing the LRF- or

LRW-amide motif, but peptides deduced from KiSS-1, containing LRF-amide, were more potent

activators, as measured by mobilization of intracellular Ca2+. Data are expressed as the change in

fluorescent intensity units (FIU) over background and are from a single experiment,

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representative of three such experiments. Each point was determined in triplicate and is given as

a mean. ¡ KiSS-1(112-121), o KiSS-1(94-121), l KiSS-1(107-121), n KiSS-1(114-121), t

Antho-RW-amide I, ∆ Antho-RW-amide II, s peptide F1.

Fig 5. Localization by quantitative RT-PCR of AXOR12 and KiSS-1 in human tissues, together

with expression of the housekeeping gene GAPDH. Bars indicate mean number of copies

detected per ng of reverse transcribed polyA+ RNA from 2 male and 2 female individuals. Error

bars indicate standard error.

Fig 6. Localization by quantitative RT-PCR of AXOR12 and KiSS-1 in human central nervous

system, together with expression of the housekeeping gene GAPDH. Bars indicate mean number

of copies detected per ng of reverse transcribed polyA+ RNA from 2 male and 2 female

individuals. Error bars indicate standard error.

Fig 7. A, B: Western blot analysis of AXOR12 immunoreactivity A: Membrane proteins from

CHO AXOR12:Gqi5 cells (lane 1) or untransfected CHO cells (lane 2); B: Membrane proteins

from human frontal cortex (lane 3), hippocampus (lane 4) and basal ganglia (lane 5). A band of

approximately 75kDa reacted specifically with AXOR12 antiserum in all brain membranes and

with CHO AXOR12:Gqi5 cell membranes but this specific signal was absent in non-transfected

CHO cells.

C, D: Immunocytochemical analysis of AXOR12 immunoreactivity in CHO AXOR12:Gqi5

cells. C: AXOR12 immunoreactivity localised to the cell surface of CHO AXOR12:Gqi5 cells.

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D: Visualisation of the nuclear stain DAPI in the same field as C showing all cells present within

the field.

Fig 8. Immunohistochemical detection of AXOR12 in human brain.

A: In laminar layer III of the sensory-motor cortex striking immunoreactive pyramidal cells

(arrow) and their ascending processes (arrowhead) were stained. II and III indicate laminar

layers II and III respectively.

B: In the cerebellar cortex AXOR12 immunoreactivity was observed in Purkinje cell perikarya

(arrow) and their ascending apical dendrites (arrowhead). ML: molecular layer; PCL: Purkinje

cell layer; GCL: granule cell layer.

C: Throughout the medulla a number of neuronal cell types (arrowheads) were immunolabelled

with AXOR12.

Scale bars: 50 µm.

D: Low-power view of AXOR12 immunoreactivity present in the sensory-motor cortex grey

matter.

E: Immunoreactivity was abolished by pre-absorption of AXOR12 antiserum with 10µM of

peptide antigen.

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Table 1. pEC50 of peptides tested on CHO AXOR12:Gqi5 cells as determined from dose-

dependent changes in intracellular Ca2+. The peptide sequences and source species of the

peptides are shown along with the mean pEC50 and standard error derived from three

experiments.

peptide sequence species mean pEC50 SEM

Antho-RW-amide II pESLRW-amide sea anemone 5.73 0.28

Antho-RW-amide I pEGLRW-amide sea anemone 6.42 0.28

Peptide F1 TNRNFLRF-amide lobster 5.63 0.08

KiSS-1(114-121) WNSFGLRF-amide human 8.03 0.51

KiSS-1(112-121) YNWNSFGLRF-amide human 9.30 0.39

KiSS-1(107-121) KDLPNYNWNSFGLRF-amide human 8.92 0.37

KiSS-1(94-121) IPAPQGAVLVQREKDLPNYNWNSFGLRF-amide human 8.76 0.43

KiSS-1(68-121) GTSLSPPPESSGSRQQPGLSAPHSRQ---

---IPAPQGAVLVQREKDLPNYNWNSFGLRF-amide

human 8.00 0.16

KiSS-1(68-91) GTSLSPPPESSGSRQQPGLSAPHS human <4 N/A

KiSS-1(68-80) GTSLSPPPESSGS human <5 N/A

KiSS-1(58-65) PAATLRS human <4 N/A

KiSS-1(125-144) EAAPGNHGRSAGRGWGAGAGQ human <4 N/A

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Richard Faull, Karen L. Philpott and David C. HarrisonDarker, Christopher G. C. Larminie, Shelagh Wilson, Derk J. Bergsma, Piers Emson,

Murdock, Klaudia Steplewski, Usman Shabon, Jane E. Miller, Susan E. Middleton, John G.Moore, Amy Calamari, Philip G. Szekeres, Henry M. Sarau, Jon K. Chambers, Paul

Alison I. Muir, Larissa Chamberlain, Nabil A. Elshourbagy, David Michalovich, Darren J.AXOR12: A novel human G protein-coupled receptor, activated by the peptide KiSS-1

published online May 31, 2001J. Biol. Chem. 

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