The Three Subfamilies of Leucine-Rich Repeat-Containing G Protein-Coupled Receptors (LGR): Identification of LGR6 and LGR7 and the Signaling Mechanism for LGR7
Sheau Yu Hsu,
Masataka Kudo,
Thomas Chen1,
Koji Nakabayashi,
Alka Bhalla,
Peter J. van der
Spek,
Marcel van Duin and
Aaron J.
W. Hsueh
Division of Reproductive Biology (S.Y.H., M.K., T.C. K.N.,
A.B., A.J.W.H.) Department of Gynecology and Obstetrics
Stanford University School of Medicine Stanford, California
94305-5317
Scientific Development Group (P.J. v.d.S.,
M.v.D.) N.V. Organon Oss, The Netherlands
5340
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ABSTRACT
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Glycoprotein hormone receptors, including LH
receptor, FSH receptor, and TSH receptor, belong to the large G
protein-coupled receptor (GPCR) superfamily but are unique in having a
large ectodomain important for ligand binding. In addition to two
recently isolated mammalian LGRs (leucine-rich repeat-containing, G
protein-coupled receptors), LGR4 and LGR5, we further identified two
new paralogs, LGR6 and LGR7, for glycoprotein hormone receptors.
Phylogenetic analysis showed that there are three LGR subgroups: the
known glycoprotein hormone receptors; LGR4 to 6; and a third subgroup
represented by LGR7. LGR6 has a subgroup-specific hinge region after
leucine-rich repeats whereas LGR7, like snail LGR, contains a low
density lipoprotein (LDL) receptor cysteine-rich motif at the N
terminus. Similar to LGR4 and LGR5, LGR6 and LGR7 mRNAs are expressed
in multiple tissues. Although the putative ligands for LGR6 and
LGR7 are unknown, studies on single amino acid mutants of LGR7, with a
design based on known LH and TSH receptor gain-of-function mutations,
indicated that the action of LGR7 is likely mediated by the protein
kinase A but not the phospholipase C pathway. Thus, mutagenesis of
conserved residues to allow constitutive receptor activation is a novel
approach for the characterization of signaling pathways of selective
orphan GPCRs. The present study also defines the existence of three
subclasses of leucine-rich repeat-containing, G protein-coupled
receptors in the human genome and allows future studies on the
physiological importance of this expanding subgroup of GPCR.
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INTRODUCTION
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In vertebrates, gonadotropins and TSH are essential for the
differentiation and growth of gonads and thyroid gland, respectively
(1, 2, 3, 4). Among these glycoprotein hormones with common
- and
specific ß- subunits, LH, FSH, and TSH are secreted by the
anterior pituitary whereas the human chorionic gonadotropin (hCG) is
secreted by placenta cells. These heterodimers bind specific plasma
membrane receptors on target cells and signal mainly through the
cAMP-dependent pathway. The receptors for these glycoprotein hormones
belong to the large G protein- coupled, seven-transmembrane protein
superfamily but are unique in having a large N-terminal extracellular
(ecto-) domain important for interaction with the large glycoprotein
hormone ligands (5, 6). Hallmarks of this subgroup of G protein-coupled
receptors (GPCRs) are the leucine-rich repeats in the ectodomain that
have been postulated to form a horseshoe-shaped interaction motif for
ligand binding (7, 8, 9). Recently, putative receptors homologous to the
mammalian glycoprotein hormone receptors were found in sea anemone (10, 11), nematode (12), pond snail Lymnaea stagnalis (13), and
Drosophila (14), suggesting that this subgroup of GPCR
evolved early during evolution and that these invertebrate
receptors represent ancient homologs of mammalian glycoprotein hormone
receptors.
Based on the conserved sequences of mammalian glycoprotein hormone
receptors and invertebrate homologs, we and others have recently
isolated two novel mammalian leucine-rich repeat-containing, G
protein-coupled receptors (LGRs) based on a homologous sequence search
of the expressed sequence tags (ESTs) database (15, 16, 17). Because
phylogenetic analysis showed that sea anemone LGR shares a closer
relatedness to mammalian glycoprotein hormone receptors than to an LGR
isolated from pond snail Lymnaea stagnalis (13, 15), one can
predict that there are additional LGRs in mammalian genomes. Indeed, a
recent search of the EST and genomic databases and subsequent
characterization revealed that there are at least two additional
mammalian LGRs. These two genes were named as LGR6 and LGR7 based on
the chronological order of discovery. Analysis of primary sequences and
domain arrangement in these LGRs showed that LGR6 is closely related to
LGR4 and LGR5; whereas LGR7 and snail LGR are likely derived from a
common ancestor. Together with the three known glycoprotein hormone
receptors, these studies define the existence of three subgroups of
LGRs in mammals. Based on the conserved mechanisms identified in
constitutively activated LH and TSH receptors (18, 19, 20), studies of
putative gain-of-function point mutants of LGR7 showed that this orphan
receptor could mediate signaling through the protein kinase A-dependent
pathway. Thus, site-directed mutagenesis of key residues in the
functional domains of seven-transmembrane receptors provided a novel
approach to reveal the signal transduction pathway of selective orphan
GPCRs and to facilitate future identification of their cognate
ligands.
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RESULTS
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cDNA Cloning of Two Novel Leucine-Rich Repeat-Containing GPCR, LGR6
and LGR7
Using the known invertebrate and mammalian LGRs as queries to
search for homologous EST sequences, two candidate sets of sequences
with homology to known LGRs were selected. cDNAs corresponding to these
two novel genes, designated as LGR6 and LGR7, were obtained by reverse
transcription-PCR using gene-specific primers and mRNA from human ovary
and testis as templates. Using 5'- and 3'-rapid amplification of cDNA
ends (RACE), a full-length LGR6 cDNA encoding an open reading frame
(ORF) of 846 amino acids with a calculated molecular mass of 91.6 kDa
was obtained (Fig. 1
). Analysis of
multiple cDNA clones showed that the deduced methionine translation
start site is preceded with an in-frame stop codon at position -177.
Using a similar approach, two splicing variants of LGR7 were obtained
which differ in the N terminus of the coding region. The long form of
LGR7, LGR7(1), is 3759 nucleotides long and encodes an ORF of 757 amino
acids with a calculated molecular mass of 87 kDa; whereas the short
form of LGR7, LGR7(2), encodes an ORF of 723 amino acids with a
corresponding mass of 82.9 kDa (Fig. 2
).
The LGR7(2) variant is 34 amino acids shorter than the LGR7(1), and
these two variants could be derived from alternative splicing events at
the N terminus of the coding region.

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Figure 1. Comparison of Deduced Amino Acid Sequences for
LGR6, LGR4, and LGR5
Sequence alignment for different regions of LGR6 with that of two
closely related LGRs, LGR4 and LGR5. A, The ectodomain includes the
multiple leucine-rich repeats (LRR) and a C-flanking cysteine-rich
hinge region. Individual leucine-rich repeats are indicated by
arrowheads. Putative N-glycosylation sites in the
ectodomain are in bold letters and
underlined. Conserved PYAYQCC and GXFKPCE motifs
preceding transmembrane I are in bold italics. B, The
seven-transmembrane region and the C-terminal tail. The transmembrane
(TM) domain, intracellular loop (IL), and extracellular loop (EL) are
indicated by arrowheads. Potential protein kinase C
phosphorylation sites in the intracellular C-terminal region are in
bold letters and underlined. The two
conserved cysteines found in extracellular loops 1 and 2, and believed
to form a disulfide bond, are marked by solid circles.
Residue numbers are shown on the right and
asterisks indicate the position of the stop codon.
Shaded residues are identical in the three receptor
proteins aligned, and gaps indicated by dashes are
included for optimal protein alignment.
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Figure 2. Comparison of Deduced Amino Acid Sequences between
Two Human LGR7 Variants and Snail LGR
Sequence alignment for different regions of two splicing variants (1 2 ) of LGR7 and snail LGR (LsLGR) was performed. A, Ectodomain with
LDL receptor cysteine-rich motifs and leucine-rich repeats (LRR).
Residues that are identical in LGR7(1 ) and LGR7(2 ) are shown as
double lines, and residues that are missing in LGR7(2 )
are indicated by bold asterisks. The consensus LDL
receptor cysteine-rich motif in LGR7 and the corresponding region of
snail LGR are indicated by bold italics. C-terminal
cysteine-rich hinge regions in the two receptors are
overlined. Putative N-linked glycosylation sites are in
bold (N). B, The seven-transmembrane region
and the C-terminal tail. Different structural motifs including
transmembrane (TM) region, intracellular loop (IL), and extracellular
loop (EL), are indicated by arrowheads; asterisks
indicate the stop codon. Potential phosphorylation sites for protein
kinase C are in bold letters and underlined.
Shaded residues are identical in the LGR7 and snail LGR
sequences. Residue numbers are shown on the right and gaps
are included for optimal protein alignment.
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Comparison with known sequences in the GenBank showed that LGR6 and
LGR7 are novel LGRs with closest relatedness to the newly isolated LGR4
and LGR5, and snail LGR, respectively. A motif search indicated that
LGR6 and LGR7 contain an N terminus ectodomain composed of variable
leucine-rich repeats and a seven-transmembrane region followed by
unique C-terminal intracellular tail. The similarity between these LGRs
and other known LGRs extends from the ectodomain to the
seven-transmembrane region, and LGR6 shares approximately 4547%
identity with LGR4 and LGR5, as compared with 33% identity with the
glycoprotein hormone receptors. However, LGR6 is distinct from LGR4 and
LGR5 in having only 13 leucine-rich repeats instead of the 17 repeats
found in LGR4 and LGR5 (Fig. 1A
). Among the 13 leucine-rich repeats of
LGR6, the first seven repeats align perfectly with leucine-rich repeats
13 and leucine-rich repeats 811 of LGR4 and LGR5, but the remaining
leucine-rich repeats showed disparity in these receptors. In addition,
distinctive sequence motifs, including a PYAYQCC motif and a GXFKPCE
motif in the hinge region of the ectodomain, and the highly conserved
cysteine residues for disulfide bond formation in extracellular loops 1
and 2, were completely conserved in LGR4, LGR5, and LGR6 (Fig. 1
, A and
B). A motif search using the ScanProsite program showed that both LGR6
and LGR7 contain N-glycosylation sites at the ectodomain and that LGR7
carries a protein kinase C phosphorylation site at the intracellular
C-terminal sequences [STR, residues 750752 of LGR7(1)]. Unlike LGR4
and LGR5 (15), both LGR6 and LGR7 lack protein kinase A phosphorylation
sites in their C termini.
Among all known LGRs, LGR7 shares the highest identity with snail LGR
(33%) (13) and less with the three mammalian glycoprotein hormone
receptors (24%). As shown in Fig. 2
, LGR7 and snail (Ls) LGR shared
similar primary sequences and common domain arrangement as shown by the
presence of the N-terminal low density lipoprotein (LDL)
receptor cysteine-rich motif followed by leucine-rich repeats and the
seven transmembrane region. However, the predicted tertiary structure
of the LGR7 ectodomain differed from that of snail LGR; the ectodomain
of snail LGR is bulkier and contains approximately 760 amino acids
instead of the 410 amino acids found in LGR7(1) (Fig. 2A
). In addition
to the 10 leucine-rich repeats at the C terminus of the ectodomain,
snail LGR contains 12 LDL-receptor cysteine-rich motifs at the N
terminus. In contrast, both LGR7 variants have only one such motif
[residue 4062 of LGR7(1), CLPQLLHCNGVDDCGNQADEDNC] preceding the
conserved leucine-rich repeat domain. In addition, these two receptors
are distinct in their hinge region of the ectodomain. In LGR7 and snail
LGR, the hinge regions are approximately 30 amino acids long as
compared with 72123 amino acids found in other LGRs. The unique
PYAYQCC and GXFKPCE motifs found in this region of other LGRs are
absent in these two receptors. Thus, the overall structural features of
LGR7 are similar to that of snail LGR.
Mammalian LGRs Have Diverged into Three Distinct Subtypes
Phylogenetic analysis using the neighbor-joining and parsimony
methods showed that the 11 known LGRs from vertebrates and
invertebrates can be divided into three distinct subgroups (Fig. 3A
). The first subgroup contains the
mammalian gonadotropin and TSH receptors, and LGRs from sea anemone,
Caenorhabditis elegans, and Drosophila. The
second branch contains only vertebrate receptors including LGR4, LGR5,
and LGR6, whereas snail LGR and LGR7 belong to the third subgroup. To
gain insight into the evolution of LGRs, phylogenetic analysis was
performed together with diverse GPCRs with a polypeptide or
neurotransmitter ligand using full-length receptor sequences. As shown
in Fig. 3B
, LGRs share a branch with diverse GPCRs known to have a
peptide ligand, including a subgroup of angiotensin receptor-like GPCRs
[angiotensin receptor, platelet-activating factor (PAF)
receptor, and formyl-methionyl-leucyl-phenylalanine (FMLP)
receptor] and another subgroup of bombesin receptor-like GPCRs
(bombesin, gastrin, thrombin, and neuropeptide Y receptors). In
contrast, the relatedness to other family 1 GPCRs (21), such as
receptors for various kinins, amine derivatives, and somatostatin
(SST), is more remote.

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Figure 3. Three Subgroups of LGRs from Diverse
Species and the Evolutionary Relatedness between LGRs and Family 1
GPCRs with a Peptide or Neurotransmitter Ligand
A, Phylogenetic relatedness of diverse LGRs from mammals and
invertebrates. Full-length amino acid sequences of 11 LGRs from mammals
(LH, FSH, and TSH receptors plus LGR4 to LGR7), sea anemone, nematode,
pond snail, and Drosophila were analyzed using the
Blocks program. Sequence blocks predicted from this alignment were then
used to construct the neighbor-joining tree for establishing possible
subfamily relationships. Comparable results were obtained using the
parsimony method in the Phylogeny Inference Package. In the three major
branches of LGR evolution, the three classical glycoprotein hormone
receptors as well as LGRs from sea anemone, nematode, and
Drosophila belong to the first subgroup whereas LGR6 is
most closely related to the second subgroup containing LGR4 and LGR5.
In contrast, LGR7 belongs to a third subgroup together with snail LGR.
B, Diagrammatic representation of phylogenetic relatedness between
mammalian LGRs and diverse family 1 GPCRs (21 ). Different subgroups of
family 1 GPCRs with known ligands were first grouped based on sequence
similarity, and full-length consensus sequences for each subgroup of
family 1 GPCRs were generated using the Blocks program. Subsequently,
the individual full-length amino acid sequences of different LGRs, and
the consensus sequences of different subtypes of family 1 GPCRs with a
peptide or neurotransmitter ligand, were analyzed together. The GPCRs
included in the phylogenetic tree are adenosine receptors,
cholecystokinin receptors, tachykinin receptors, opsins, neurotensin
receptors, TSH-releasing hormone receptor, angiotensin receptors, PAF
receptor, FMLP receptor, bombesin receptors, gastrin receptors,
thrombin receptors, neuropeptide Y receptors, 5-HT type 1 serotonin
receptors, dopamine receptors, ß-adrenergic receptors, GH secretogue
receptor (GHS receptor), galanin receptors, somatostatin (SST)
receptors, and endothelin receptors. R, Receptor.
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Tissue Expression Pattern of LGR6 and LGR7
To investigate the tissue expression pattern of LGR6 and LGR7,
Northern blot analyses of different tissues were performed using
multiple tissue blots containing rat poly (A)+ RNA and specific
radiolabeled probes for LGR6 or LGR7. Specific LGR6 transcripts of 4.0
kb were detected in multiple tissues including testis, ovary, oviduct,
uterus, thymus, small intestine, colon, spleen, kidney, adrenal, brain,
and heart (Fig. 4A
). In addition, a minor
LGR6 transcript of >5.5 kb was also detected in the oviduct and heart,
whereas a smaller transcript was found in the spleen. Likewise, LGR7
transcripts were also found in multiple tissues. As shown in Fig. 4B
, a
major LGR7 transcript of 5.5 kb was detected in all tissues tested with
the exception of spleen. Furthermore, multiple smaller
transcripts (2 or 3 kb) were detected in oviduct, uterus, colon,
and brain.

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Figure 4. Tissue Expression Pattern of LGR6 and LGR7
For Northern blot analysis, 2 µg of poly (A)+-selected RNA from
different tissues of immature rats were probed with a
32P-labeled LGR6 or LGR7 cRNA probe. After washing under
highly stringent conditions (0.1 x SSC, 1% SDS at 75 C), the
blots were exposed to x-ray films with intensifying screens at -80 C.
Subsequent hybridization with a ß-actin cDNA probe was performed to
estimate nucleic acid loading. A, LGR6 Northern blot. A major message
of 4.0 kb was found in testis, ovary, oviduct, uterus, small intestine,
colon, spleen, kidney, adrenal, brain, and heart. A transcript of
higher mol wt (>5.5 kb) was also detected in the oviduct and heart. B,
LGR7 Northern blot. A major message of 5.5 kb was found in multiple
tissues while multiple transcripts of lower sizes were detected in
oviduct, uterus, colon, and brain. Lower panels show the
expression of ß-actin in the same blots. The sizes of mol wt markers
are shown on the left.
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Isolation of Human LGR6 and LGR7 Genes and their Chromosomal
Localization
Using an LGR6 cDNA fragment corresponding to the transmembrane
region as a probe, genomic clones for LGR6 were isolated from a
bacterial artificial chromosome-based human genomic DNA library. For
LGR7, a genomic DNA clone (AQ053279) from the genomic survey sequence
database was found to contain the C terminus of the LGR7 gene and was
obtained from Genome Systems (St. Louis, MO). To identify
the chromosomal localization of LGR6 and LGR7 genes in the human
genome, genomic fragments (>50 Kb) derived from these BAC clones were
used as probes in fluorescence in situ hybridization (FISH)
analysis. As shown in Fig. 5
, A and B,
LGR6 and LGR7 genes were localized to banded DNA in chromosome 1q32 and
4q32, respectively.

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Figure 5. Chromosomal Localization of LGR6 and LGR7 in
Humans
Using DNA fragments of bacterial artificial chromosome containing human
genomic fragments of LGR6 and LGR7 as probes, LGR6 (upper
panel) and LGR7 (lower panel) genes were
localized to chromosome 1q32 and 4q32 regions, respectively, by the
fluorescence in situ hybridization method. Denatured
chromosomes from synchronous cultures of human lymphocytes were
hybridized with biotinylated probes for localization. Assignment of the
mapping data was achieved by superimposing fluorescence signals with
the 4,6-diamdino-2-phenylindole-banded chromosome. Specific signals on
the banded chromosome are indicated by arrows.
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Signal Transduction by Mutant LGR7 as well as by Chimeric Receptors
with the Ectodomain from the LH Receptor and the Transmembrane Region
from LGR6 or LGR7
To investigate the signal transduction pathway of LGR6 and LGR7,
wild-type and mutated receptors were constructed to analyze receptor
binding and signal transduction. Earlier studies using mutant LH and
TSH receptors found in patients with male-limited precocious puberty
and nonimmune hyperthyroidism, respectively, indicated that these
diseases are associated with constitutive receptor activation as a
result of point mutations of key residues in the transmembrane VI
region of these receptors (18, 19, 20, 22, 23, 24, 25). To investigate the
possibility that the orphan LGRs could be rendered constitutively
active in the absence of their unknown ligands, detailed comparisons of
this region were conducted between gain-of-function LH and TSH
receptors and the two new receptors. It was found that a tripeptide
motif (FTD, residue 576578 in the LH receptor) is completely
conserved in the three glycoprotein hormone receptors and LGR7, but not
in LGR6. Previous studies have shown that mutations of D578 in the LH
receptor and the corresponding D633 in the TSH receptor cause
constitutive increases of basal cAMP production in cells expressing
these mutant receptors (20, 22, 26). Accordingly, we constructed
mutants for the two LGR7 variants, LGR7(1) and LGR7(2), by generating a
point mutation at the corresponding residue in transmembrane VI. These
mutants were named as LGR7(1) D637Y and LGR7(2) D603Y.
As shown in Fig. 6
, transfection of 293T
cells with increasing concentrations of the expression plasmid encoding
the mutant LGR7 receptors [LGR7(1) D637Y or LGR7(2) D603Y], similar
to those transfected with the plasmid encoding the D578Y
gain-of-function mutant LH receptor, resulted in dose-dependent
increases of basal cAMP production by transfected cells. In contrast,
cAMP levels in cells transfected with different amounts of wild-type LH
receptor or LGR7 did not show an increase in basal cAMP production. To
allow quantitative comparison of basal cAMP production by different
receptors, cAMP levels in transfected cells were normalized based on
the level of cell surface expression of an N-terminally tagged FLAG
epitope in different LGRs (Fig. 6
, shown as percentage changes
vs. wild-type LH receptor). Although the D637Y mutation
caused significant increases of basal cAMP levels in both LGR7(1) and
LGR7(2) constructs, cells expressing mutant LGR7(1) consistently showed
greater levels of cAMP increase as compared with those expressing the
mutant LGR7(2) construct. To evaluate the specificity of the activating
LGR7 mutation for the Gs protein, the effect of this mutation on a
different signal transduction pathway, phosphatidyl inositol (PI)
turnover, was measured. As shown in Table 1
, the Gs-activating mutants, LGR7(1)
D637Y and LGR7(2) D603Y, did not stimulate inositol phosphate (IP)
turnover by transfected 293T cells whereas hCG treatment significantly
increased the IP content in cells expressing either wild-type or
constitutively active mutant LH receptors.

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Figure 6. Gain-of-Function Mutants of LGR7(1 ) and LGR7(2 )
Leads to Constitutive Increases of Basal cAMP Production by Transfected
293T Cells
Based on the gain-of-function point mutation (LHR D578Y) found in the
LH receptor gene of patients with familial male-limited precocious
puberty, LGR7(1 ) and LGR7(2 ) with homologous point mutation [LGR7(1 )
D637Y and LGR7(2 ) D603Y] were generated. The mutated residues in the
TM VI of these two forms of LGR7 were identical, but the numbering of
this mutation differs in these two LGR7 variants [D637 in LGR7(1 )
vs. D603 in LGR7(2 )] due to dissimilar length in their
N-termini. After transfection of expression constructs encoding
wild-type or mutant receptors into 293T cells, basal cAMP levels were
monitored using an RIA. Transfection of 293T cells with increasing
concentrations (0500 ng/well) of expression vectors encoding LGR7(1 )
D637Y, LGR7(2 ) D603Y, or LHR D578Y led to increases in basal cAMP
levels in transfected cells. In contrast, cAMP levels in cells
transfected with wild-type (WT) receptors were negligible. Production
of cAMP by different receptors was normalized based on cell surface
expression of FLAG epitope tagged at the N terminus of all receptor
cDNAs determined by immunodetection. Numbers in
parentheses denote percentage of receptor protein expression by
individual constructs as compared with expression in cells transfected
with the wild-type LH receptor construct (LHR WT), which was
arbitrarily set as 100% (n = 3; mean ±
SE).
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Because chimeric receptors among the known glycoprotein hormone
receptors have been successfully used to study signal transduction by
these proteins (27), we further generated chimeric LH/LGR6 and LH/LGR7
receptors by fusing the ectodomain of the human LH receptor with the
transmembrane region and C-terminal tail of either LGR6
[LH(EC)/LGR6(TM)] or LGR7(1) [LH(EC)/LGR7(TM)] to further
investigate the putative signal transduction pathway of these orphan
receptors. Although the chimeric LH(EC)/LGR6(TM) receptor is expressed
on the cell surface as reflected by FLAG epitope expression, and is
binding to 125I-hCG as detected by the LH
receptor binding assay (data not shown), no stimulation of cAMP was
detected after incubation with hCG. In contrast, the same hCG treatment
increased cAMP production by cells expressing the wild-type LH
receptor. Because activation of the LH receptor by hCG elicits
increased PI turnover (28, 29), hydrolysis of PI in cells transfected
with the chimeric receptor was also performed. Measurement of PI
turnover showed a negligible increase of IP accumulation in cells
expressing the chimeric LH(EC)/LGR6(TM) receptor after stimulation with
hCG, whereas wild-type LH receptor-expressing cells showed an
approximately 1.4-fold increase in PI hydrolysis after hCG treatment.
In contrast to the chimeric receptor LH(EC)/LGR6(TM), the LH receptor
ectodomain and LGR7 transmembrane region appeared to be incompatible
because the chimeric LH(EC)/LGR7(TM) receptor was not found on the cell
surface based on the assay of FLAG epitope expression.
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DISCUSSION
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Based on database analysis, we have isolated two novel GPCR genes
belonging to the mammalian leucine-rich repeat-containing GPCRs. In
addition to the subgroup of classical glycoprotein hormone receptors,
LGR6 is evolutionarily related to two other mammalian LGRs (LGR4 and
LGR5) whereas LGR7 shares a similar ancestry with snail LGR, thus
indicating the presence of three subgroups of LGRs in the mammalian
genome. The LGR7 was found to signal through the cAMP-dependent
pathway, as reflected by increases in basal cAMP production in cells
transfected with mutant LGR7 constructs carrying a point mutation in
transmembrane VI. These orphan receptors are expressed in diverse
tissues, and the identification of the signaling mechanism for LGR7
could facilitate the identification of its cognate ligand(s).
Recent expansion of the nucleic acid sequence database for diverse
organisms have provided opportunities to identify novel mammalian gene
paralogs through pairwise sequence comparison (30). Although the
physiological roles of most new genes are not known, alignment of their
primary or secondary structures have allowed their preliminary
grouping. Studies on the entire nematode genome indicated that the GPCR
protein superfamily represents one of the most abundant signaling
molecules that allows the cell to communicate with its environment (31, 32), and the GPCR family proteins could account for more than 1% of
human genes. Among the known mammalian GPCRs, the glycoprotein hormone
receptors represent a unique group of family 1 GPCRs with a large
ectodomain for interaction with their ligands. Based on modeling with a
prototypic leucine-rich repeat- containing polypeptide,
ribonuclease inhibitor (8, 9), it was envisioned that the multiple
leucine-rich repeats in the ectodomain of mammalian glycoprotein
hormone receptors are arranged in a horseshoe-shaped binding motif and
the inwardly ß-sheet structures interact with the specific ligand
(7, 8, 9, 33). Unlike the glycoprotein hormone receptors with nine
leucine-rich repeats, newly isolated mammalian LGRs contain varying
numbers of repeats, indicating that the binding domain in these
receptors could have the same configuration, but with distinct
curvature and size. The glycoprotein hormones have been widely used in
the treatment of diverse diseases; the current finding of new LGRs and
the possibility of finding additional hormones as ligands for these
orphan LGRs could reveal novel endocrine regulatory mechanisms.
The physiological and pathophysiological actions of glycoprotein
hormone receptors are mediated mainly through interaction with the Gs
protein. Recent characterization of constitutively activated LH and TSH
receptors, based on patients with specific etiology (18, 19, 20, 25), has
allowed analyses of structural requirements for signaling by these
receptors. Taking advantage of these observations, mutant LGR7
constructs were made to explore the putative signaling pathways of
LGR7. Similar to LH and TSH receptors, mutation of a single
structure-determining amino acid in the transmembrane VI region of LGR7
caused constitutive signaling, as reflected by increases in basal cAMP
levels in transfected cells. It has been proposed that mutations
causing constitutive activation alter the receptor conformation from an
inactive to an active state, mimicking the ligand stimulation of these
receptors (34, 35, 36). Thus, the increase of basal cAMP production by
mutant LGR7 receptors could be the result of conformational changes as
found for constitutively active LH and TSH receptors. The present
findings suggest that wild-type LGR7 may signal through the
cAMP-protein kinase A pathway, a mechanism similar to the related
glycoprotein hormone receptors. Although the Gs activating mutants
LGR7(1) D637Y and LGR7(2) D603Y do not stimulate IP turnover by
transfected 293T cells, it is important to note that the coupling of
LGR7 to other G proteins cannot be excluded. Other examples of
signaling by orphan GPCRs include a wild-type orphan ACCA (adenylate
cyclase constitutive activator) receptor found to constitutively
activate adenylate cyclase in transfected cells (37).
Recently, endogenous ligands for several orphan receptors have been
isolated by monitoring the stimulation of downstream signaling
transduction (38, 39, 40, 41, 42, 43, 44). With no knowledge of the G protein-signaling
mechanism, several of these studies were made possible by coexpressing
the orphan receptor with chimeric G proteins or G proteins that are
promiscuous in receptor coupling. The present study demonstrates that
mutagenesis of key residues in the transmembrane domain of GPCR is a
novel approach to characterize signaling pathways for orphan GPCRs.
While the present study demonstrated that mutagenesis of conserved
residues is a useful approach for the characterization of signaling by
the orphan LGR7, previous studies indicated that an FSH receptor mutant
equivalent to D578Y in the LH receptor does not lead to receptor
activation (27), suggesting this approach is only applicable to
selective orphan GPCRs.
Because chimeric receptors with the ectodomain and transmembrane region
from different glycoprotein hormone receptors have been shown to be
functional (5, 27), we have attempted to characterize the potential
coupling of LGR6 to Gs or Gq pathways by fusing the transmembrane
region of LGR6 with the ectodomain of the LH receptor in a chimeric
construct. Although the chimeric receptor could be expressed on the
cell surface, no activation of the chimeric receptor by the LH receptor
ligand was observed. Previous studies on similar chimeric receptors
containing the ectodomain domain of the LH receptor and the
transmembrane domain of LGR4 or LGR5 also suggest that such chimeric
receptors do not react to ligand stimulation (15). Thus, either the
ectodomain of the LH receptor and the transmembrane region of these
orphan receptors are incompatible, or they could signal through other
unknown mechanisms. Indeed, a similar construct of the LH receptor
ectodomain plus the LGR7 transmembrane domain failed to allow
expression of the chimeric receptor on the cell surface of transfected
cells.
The newly identified LGR7 shows close sequence homology with the only
known snail LGR (13). Interestingly, the putative ligand-binding
domains of these two receptors appear to have diverged. The most
obvious difference is the number of LDL receptor cysteine-rich motifs
in the N terminus of these two receptors. In the ectodomain of snail
receptor, there are 12 LDL receptor cysteine-rich motifs, each encoding
three conserved cysteine residues (13, 45) important for disulfide bond
formation and ligand binding (46). Unlike snail LGR, LGR7 contains only
one typical LDL receptor cysteine-rich motif in its N terminus.
Assuming the motifs in these two related receptors comprise part of
their ligand-binding domain, the respective ligands for these two
receptors could have diverged during evolution. Future studies on the
LGR7 gene could reveal additional LGR7 variants that are closer to
snail LGR, and uncover the evolutionary relationship of these two
receptors.
Analysis of LGR6 sequences showed that LGR6 belongs to a subgroup of
LGRs which includes LGR4 and LGR5, suggesting that these receptors may
share similar ligand binding and signal transduction characteristics.
However, the ectodomain of LGR6 is unique and contains only 13
leucine-rich repeats instead of the 17 repeats found in LGR4 and LGR5.
It is unclear whether the cloned LGR6 cDNA is the only transcript
encoded by the LGR6 gene. Observed differences in the number of
leucine-rich repeats in the ectodomain of these receptors may be the
result of alternative splicing. Recent studies on GPCRs have shown that
within a given subfamily functional diversity is most often conferred
by the existence of multiple receptor subtypes, each encoded by a
distinct gene. Additional diversity results from alternative splicing
of a given gene to form receptor variants (47). Indeed, our unpublished
results showed that the LGR4 gene encodes multiple splicing variants,
including one with only 14 leucine-rich repeats. Likewise, glycoprotein
hormone receptor genes also encode multiple splicing variants with
distinct functional characteristics (48), and two splicing variants of
LGR7 were isolated here. Alternative splicing of these receptors,
especially in the ectodomain, could result in alternative binding
characteristics or specificity.
Sequence alignment showed that the three subgroups of LGRs could be
distinguished merely by the amino acid sequences in their hinge regions
between leucine-rich repeats and the transmembrane domain. In the
glycoprotein hormone receptors, this region is flanked by the conserved
YPSHCC and DXFNPCED motifs whereas LGR4, LGR5, and LGR6 contain YAYQCC
and GXFKPCEX sequences in the corresponding regions, respectively. In
contrast, these two motifs are absent in LGR7. Interestingly, recent
studies on the TSH receptor have shown that point mutation of the
serine residue in the conserved YPSHCC motif resulted in constitutive
activation of the TSH receptor, leading to severe congenital
hyperthyroidism in patients (49, 50, 51). Because LGR4, LGR5, and LGR6
share similar structural determinants in the hinge region,
investigations on this region of the orphan receptors could provide
insights toward the activation mechanisms of different LGRs.
Analysis using 11 known LGRs from diverse organisms indicated that LGRs
from sea anemone, nematode, and Drosophila grouped under a
single branch with mammalian glycoprotein hormone receptors while the
newly isolated LGR4, LGR5, LGR6, and LGR7 diverged early during
evolution. Assuming the evolutionary pressures on these receptors are
constant, the sequence divergence in mammalian LGRs suggests that the
ancestral gene giving rise to modern LGRs could have evolved before the
emergence of cnidarians for cell-cell communication. Also of interest,
analysis of the completely sequenced C. elegans genome
showed that this nematode contains only one GPCR with LGR
characteristics (12), suggesting a possible gene loss during the
evolution of modern nematodes and that different LGRs in present day
organisms evolved to serve adaptive functions in different
phylogenies.
Findings of multiple LGRs allow a better comparison of the relatedness
of the LGR subfamily with other GPCRs in the superfamily. Previous
studies have shown that known GPCRs can be divided into six major
families with distinct evolutionary origins, and the majority of GPCRs
with a peptide or neurotransmitter ligand belong to family 1 (21).
Phylogenetic analysis of LGRs with other GPCRs in family 1 indicated
that LGRs belong to a distinct branch and share the closest
relatedness with a subgroup of GPCRs including receptors for bombesin,
gastrin, thrombin, neuropeptide Y, angiotensin, PAF, and FMLP. This
classification would allow a better understanding of the ligand
signaling mechanisms for these receptors through comparison of their
structure-function relationship.
In conclusion, we have cloned two novel mammalian LGRs (LGR6 and LGR7)
and identified the putative signal transduction pathway for LGR7. The
constitutive activation of cAMP production by the mutant LGR7 suggests
that this orphan receptor could signal through the cAMP-dependent
pathway. This study represents the first demonstration of the
elucidation of the signaling mechanism for an orphan GPCR based on
single-point mutations to allow constitutive activation of the protein.
The present study also defines the existence of three subclasses of
leucine-rich repeat-containing GPCRs in the human genome and possibly
other metazoans. Identification and functional characterization of
these novel LGRs allow elucidation of the evolutionary relationship of
this subfamily of GPCRs with leucine-rich repeats. It also facilitates
future studies on the physiology of this expanding subgroup of GPCRs
and the identification of their cognate ligands.
 |
MATERIALS AND METHODS
|
---|
Hormones and Reagents
Purified hCG (CR-129) was supplied by the National Hormone and
Pituitary Program (NIDDK, NIH, Bethesda, MD). FLAG M1 antibody and FLAG
peptide were purchased from Sigma (St. Louis, MO).
125I-Na and myo-[3H]
inositol were purchased from Amersham Pharmacia Biotech
(Arlington Heights, IL). Dowex AG1-X8 was from Bio-Rad Laboratories, Inc. (Hercules, CA).
Computational Analysis
cDNA sequences related to invertebrate LGRs and mammalian
glycoprotein hormone receptors were identified from the EST and genomic
survey sequences (GSS) database at the National Center for
Biotechnology Information and an Incyte EST database using the
BLAST and Gapped BLAST server with the BLOSUM62 comparison matrix (52).
Initially, four independent human DNA entries (three ESTs and one GSS
AQ053279) were found to encode sequences homologous, but not identical,
to the known mammalian LGRs. Based on these sequences, RACE experiments
were used to clone the full-length cDNA sequences. After RACE, the four
original DNA entries were found to encode different portions of two
novel LGRs. The alignment of primary sequences for genes in the LGR
family was carried out by the Blocks WWW server
(http://blocks.fhcrc.org/blocks/blockmkr). The Block Maker program also
calculated the branching order and phylogenetic relatedness of aligned
sequences by the Cobbler and Gibbs algorithms. To compare the
phylogenetic relationship of LGRs with diverse peptide and
neurotransmitter GPCRs, neighbor-joining
(http://www.biophys.kyoto-u.ac.jp/maketree2.html) and parsimony methods
(Phylogeny Inference Package) were used. In all studies, the
full-length amino acid sequences of different GPCRs were used for
phylogenetic analyses to provide the maximum possible information
within families and subfamilies.
Additionally, the analyses of primary and secondary structures of
these novel LGRs were conducted using the BCM search launcher
(http://gc.bcm.tmc.edu:8088/search-launcher/launcher.html), Biology
Workbench (http://gc.bcm.tmc.edu:8088/search-launcher/launcher.html),
Blocks WWW server (http://www.blocks.fhcrc.org/blocks/), the eMotif
maker server (http://dna.stanford.edu/emotif/), or the ExPASy Molecular
Biology Server (http://expasy.hcuge.ch/).
Identification and Isolation of the Full-Length cDNAs for LGR6
and LGR7
For the isolation of full-length cDNA fragments, specific
primers with a design based on EST sequences were used to prepare cDNA
pools enriched with the candidate cDNAs derived from human ovary and
testis mRNA. Two micrograms of mRNA were reverse transcribed by using
25 U of avian myoblastosis virus reverse transcriptase with
oligo(dT) primer, 0.5 mM deoxynucleoside
triphosphate (dNTP), and 20 U of RNAse inhibitor. After second
strand synthesis with T4 DNA polymerase, the cDNA pool was tailed at
both ends with adaptor sequences to allow PCR amplification using
specific primers. The tailed cDNA products were then employed as a
template for 5'- and 3'-RACE with adaptor- and gene-specific
primers. All PCR amplifications were performed under highly stringent
conditions (annealing temperature >67 C) using Advantage DNA
polymerase (CLONTECH Laboratories, Inc., Palo Alto,
CA) or Pfu DNA polymerase (Stratagene, La Jolla, CA) to
minimize mismatching and infidelity during PCR amplification. PCR
products were fractionated using agarose electrophoresis, and specific
bands showing hybridization with radiolabeled cDNA probes were
subcloned into the pUC18 vector (Invitrogen, San Diego,
CA) to further identify candidate clones. The PCR products were
phenol/chloroform-extracted, precipitated with ethanol, phosphorylated
with T4 polynucleotide kinase, and blunt-ended with the Klenow enzyme.
The PCR products were then subcloned into the SmaI site in
the pUC18 vector. At least two independent PCR clones were sequenced to
verify the authenticity of the coding sequences of each cDNA. The
resulting sequences were assembled into contigs using the Blast2
sequences server (http://www.ncbi.nlm.nih.gov/gorf/bl2.html) and
ClustalW 1.7 at the BCM Search Launcher
(http://dot.imgen.bcm.tmc.edu:9331/multi-align). After the initial
round of RACE, it was determined that the multiple homologous sequences
belong to two independent LGR genes, LGR6 and LGR7. The full-length
sequences were obtained and confirmed after three rounds of RACE.
Northern Blot Analysis
For mRNA analysis, membranes containing poly(A)+ RNA from
various rat tissues were hybridized with specific cRNA probes. The rat
mRNAs were extracted from 27-day-old rats using Trizol solution
(Life Technologies, Inc., Gaithersburg, MD) followed by
the Oligotex mRNA purification columns (QIAGEN Inc.
Chatsworth, CA) to select for poly(A)+ RNA. The cRNA probes were
synthesized using a Riboprobe Combination System (Promega Corp., Madison, WI). For hybridization using cRNA probes,
membranes were prehybridized for 1 h at 60 C in the ExpressHyb
solution (CLONTECH Laboratories, Inc.). This was followed
by hybridization under the same conditions for 2 h but with 1
x 106 cpm/ml of
32P-labeled LGR6 or LGR7 cRNA probes. After
hybridization, the membranes were washed twice in 0.2 x sodium
chloride/sodium citrate (SSC), 0.5% SDS at 60 C, followed by
two washes under high stringency conditions (0.1 x SSC, 1% SDS
at 75 C) before exposure to RX film (Eastman Kodak Co.,
Rochester, NY) with intensifying screens (Amersham Pharmacia Biotech, Buckinghamshire, UK). To monitor the loading of mRNA
samples from different tissues, membranes were stripped and
rehybridized with a 32P-labled ß-actin cDNA
probe. The cDNA probe was generated by random priming (Life Technologies, Inc.). For hybridization using cDNA probes,
membranes were washed to a stringency of 0.1 x SSC, 1% SDS at 60
C.
Expression of LGR6 and LGR7 in Mammalian Cells
Wild-type and mutant LGR6 and LGR7 cDNAs were constructed by
sequential PCR amplification and standard restriction digest and
ligation procedures. To allow efficient targeting of receptors to the
cell surface and immunodetection in vitro, a lead cDNA
sequence containing a PRL signal peptide for cell surface expression
(MNIKGSPWKGSLLLLL-VSNLLLCQSVAP) and a FLAG (DYKDDDDK)
epitope were added to the N terminus of the mature region of LGRs in
all expression constructs (53). To construct chimeric LH/LGR6 and
LH/LGR7 receptors, junctional amino acid sequences were designed to be
CAPEPPDAFN/PCEYLFESWGIRL and CAPEPPDAFN/SCEDLMSNHVLRVS, respectively.
For expression in eukaryotic cells, the receptor cDNAs were subcloned
into the eukaryotic cell expression vector pcDNA3.1 Zeo
(Invitrogen, San Diego, CA), and the plasmids were
purified using the Maxi plasmid preparation kit
(QIAGEN, Inc.). Each construct was sequenced on both
strands using vector-derived primers and gene-specific primers before
use in transfection experiments for the analyses of signal transduction
and/or receptor binding.
Mammalian 293T cells derived from human embryonic kidney fibroblast
were maintained in DMEM/Hams F-12 (Life Technologies, Inc.) supplemented with 10% FBS, 100 µg/ml penicillin, 100
µg/ml streptomycin, and 2 mM L-glutamine. The
cells were transfected with expression plasmids using the calcium
phosphate precipitation method (54). After 1824 h of incubation with
the calcium phosphate-DNA precipitates, media were replaced with
DMEM/F12 containing 10% FBS. Forty-eight hours after transfection,
cells were washed twice with Dulbeccos PBS (D-PBS), harvested from
culture dishes, and centrifuged at 400 x g for 5 min.
Cell pellets were then resuspended in DMEM/F12 supplemented with 1
mg/ml of BSA. Cells (2 x 105/ml) were
placed on 12-well tissue culture plates (Corning, Inc.
Corning, NY) and preincubated at 37 C for 30 min in the presence of
0.25 mM 3-isobutyl-1-methyl xanthine (IBMX;
Sigma) to prevent hydrolysis of cAMP before hormonal
treatment for 16 h. To study basal cAMP production mediated by
increasing numbers of receptors, each well was transfected separately
with different amounts of the expression plasmid. To detect signaling
by the wild-type and mutated LGRs, levels of cAMP production by
transfected cells were measured by specific RIA using
[125I] cAMP (Amersham Pharmacia Biotech) (27). Cells transfected with the empty plasmid (mock)
were routinely used as negative controls. At the end of incubation,
cells and medium in each well were frozen and thawed once before
heating at 95 C for 3 min to inactivate phosphodiesterase activity.
Total cAMP in each well was measured in triplicate. For IP measurement,
transfected cells were labeled for 24 h with
myo-[3H]-inositol at 4 µCi/ml in
inositol-free DMEM supplemented with 5% FBS. After washing three times
with D-PBS, 2 x 105cells were preincubated
for 30 min in D-PBS containing 20 mM LiCl, and
treated with or without hormones at 37 C for 1 h. Total IPs were
extracted and separated as previously described (29). All experiments
were repeated three times using cells from independent transfections.
To monitor transfection efficiency, 0.5 µg of RSV-ß-gal plasmid was
routinely included in the transfection mixture, and the
ß-galactosidase activity in the cell lysate was measured as
previously described (55). Statistical analysis was performed using
Students t test.
Radioligand Binding Assays
For ligand binding analysis of the wild-type and mutant
receptors, human CG (CR-129) was iodinated by the lactoperoxidase
method (56) and characterized by a radioligand receptor assay using
human LH receptors stably expressed in 293T cells. Specific activity
and maximal binding of the labeled hCG were 100,000150,000 cpm/ng and
4050%, respectively. To estimate ligand binding to the cell surface,
transfected cells were washed twice with D-PBS and collected in D-PBS
before centrifugation at 400 x g for 5 min. Pellets
were resuspended in D-PBS containing 1 mg/ml BSA (binding assay
buffer). Resuspended cells (2 x 105/tube)
were incubated with increasing doses (or a saturating dose) of labeled
hCG at room temperature for 22 h in the presence or absence of
unlabeled hCG (Pregnyl, 100 IU/tube; Organon, West Orange,
NJ). At the end of incubation, cells were centrifuged and washed
twice with the binding assay buffer. Radioactivity in the pellets was
determined with a
-spectrometer (53).
Determination of FLAG Epitope-Tagged Receptors on the Cell
Surface
Transfected cells were washed twice with D-PBS, and resuspended
cells (2 x 106/tube) were incubated with
FLAG M1 antibody (50 µg/ml) in Tris-buffered saline (pH 7.4)
containing 5 mg/ml BSA and 2 mM CaCl2
(assay buffer) for 4 h at room temperature in siliconized
centrifuge tubes. Cells were then washed twice with 1 ml of assay
buffer after centrifugation at 14,000 x g for 15 sec.
The 125I-labeled second antibody (antimouse IgG
from sheep:
400,000 cpm/tube) was added to the resuspended cell
pellet and incubated for 1 h at room temperature. Cells were again
washed twice with 1 ml of assay buffer by repeated centrifugation
before determination of radioactivity in cell pellets. Background
binding was determined by adding excess amounts of the synthetic FLAG
peptide at a concentration of 100 µg/ml.
Genomic Analysis and Chromosomal Localization of LGR6 and
LGR7
To isolate genomic clones for LGR6, a human bacterial artificial
chromosome (BAC) genomic DNA library was screened using the
transmembrane region of LGR6 cDNA as a probe. The LGR7 genomic clone
was identified by a sequence search of the GSS database and obtained
from Genome Systems. These genomic fragments were then
confirmed by Southern blot hybridization. For the identification of the
chromosomal localization of LGR6 and LGR7 genes, genomic fragments
(>50 kb) were used as probes for FISH of human metaphase chromosomes.
Denatured chromosomes from synchronous cultures of human lymphocytes
were hybridized with biotinylated probes for signal localization.
 |
ACKNOWLEDGMENTS
|
---|
We are thankful to R. Slater (Department of Cytogenetics,
Erasmus University, Rotterdam, The Netherlands) for the chromosomal
localization of LGRs. We also thank Caren Spencer for editorial
assistance. The GenBank accession numbers for LGR6 and LGR7 are
AF190501 and AF190500, respectively.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317. E-mail:aaron.hsueh{at}stanford.edu
This study was supported by NIH Grant HD-31566. S.Y.H. was supported by
NIH Training Grant T32 DK-07217. The GenBank accession numbers for LGR6
and LGR7 are AF190501 and AF190500, respectively.
1 On sabbatical leave from the Department of Biochemistry and Cellular
and Molecular Biology, The University of Tennessee, Knoxville,
Tennessee 37996. 
Received for publication November 9, 1999.
Revision received May 4, 2000.
Accepted for publication May 9, 2000.
 |
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