Characterization of Two LGR Genes Homologous to Gonadotropin and Thyrotropin Receptors with Extracellular Leucine-Rich Repeats and a G Protein-Coupled, Seven-Transmembrane Region
Sheau Yu Hsu1,
Shan-Guang Liang1 and
Aaron J. W. Hsueh
Division of Reproductive Biology Department of Gynecology and
Obstetrics Stanford University Medical School Stanford,
California 94305-5317
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ABSTRACT
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The receptors for LH, FSH, and TSH
belong to the large G protein-coupled, seven-transmembrane (TM) protein
family and are unique in having a large N-terminal extracellular
(ecto-) domain containing leucine-rich repeats important for
interaction with the glycoprotein ligands. We have identified two new
leucine-rich repeat-containing, G protein-coupled receptors and named
them as LGR4 and LGR5, respectively. The ectodomains of both receptors
contain 17 leucine-rich repeats together with N- and C-terminal
flanking cysteine-rich sequences, compared with 9 repeats found in
known glycoprotein hormone receptors. The leucine-rich repeats in LGR4
and LGR5 are arrays of 24 amino acids showing similarity to repeats
found in the acid labile subunit of the insulin-like growth
factor (IGF)/IGF binding protein complexes as well as slit, decorin,
and Toll proteins. The TM region and the junction between ectodomain
and TM 1 are highly conserved in LGR4, LGR5, and seven other LGRs from
sea anemone, fly, nematode, mollusk, and mammal, suggesting their
common evolutionary origin. In contrast to the restricted tissue
expression of gonadotropin and TSH receptors in gonads and thyroid,
respectively, LGR4 is expressed in diverse tissues including ovary,
testis, adrenal, placenta, thymus, spinal cord, and thyroid, whereas
LGR5 is found in muscle, placenta, spinal cord, and brain.
Hybridization analysis of genomic DNA indicated that LGR4 and LGR5
genes are conserved in mammals. Comparison of overall amino acid
sequences indicated that LGR4 and LGR5 are closely related to each
other but diverge, during evolution, from the homologous receptor found
in snail and the mammalian glycoprotein hormone receptors. The
identification and characterization of new members of the LGR subfamily
of receptor genes not only allow future isolation of their ligands and
understanding of their physiological roles but also reveal the
evolutionary relationship of G protein-coupled receptors with
leucine-rich repeats.
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INTRODUCTION
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Proteins in the large seven-transmembrane (TM), G protein-coupled
receptor (GPCR) superfamily are functionally diverse and include
receptors ranging from the cAMP receptor in slime mold to mammalian
neurotransmitter and glycoprotein hormone receptors (1, 2, 3, 4, 5). Agonist
occupancy of these plasma membrane proteins leads to the activation of
different G proteins, which in turn modulate the activity of different
effector enzymes and ion channels (6, 7). Gonadotropins (LH, FSH, CG)
and TSH are essential for the growth and differentiation of the gonads
and thyroid gland, respectively. These glycoprotein hormones bind
specific membrane-bound GPCRs on target cells to activate the
Gs-cAMP-protein kinase A pathway (8, 9, 10, 11). The glycoprotein hormone
receptors represent a subgroup of GPCRs that have a large
N-terminal extracellular (ecto-) domain containing leucine-rich repeats
important for interaction with glycoprotein hormones from
adenohypophysis and placenta, which leads to cAMP production in target
cells.
Based on the conserved sequences of putative glycoprotein hormone
receptors in Drosophila and sea anemone (12, 13), the
expression sequence tags (EST) in the GenBank were searched, and
fragments of two new mammalian receptors in this subfamily of
leucine-rich repeat-containing, G-protein-coupled receptor
(LGR) were
identified.2 We report here
the molecular cloning of these putative mammalian receptors with a
protein architecture that is similar to the known glycoprotein hormone
receptors and their invertebrate homologs in both ectodomains and TM
segments. In addition to the three known receptors, the ectodomains of
LGR4 and LGR5 show high homology with the acid labile subunit (ALS)
(14, 15, 16), slit (17), decorin (18), and Toll proteins (19) containing
leucine-rich repeats, suggesting a common evolutionary origin. In
contrast to the restricted tissue expression pattern of known
gonadotropin and TSH receptors, these new receptors were found in
multiple tissues. Identification of this expanding family of LGRs has
implications for future studies to identify putative ligands for these
orphan receptors and for the understanding of the evolutionary origin
of proteins in this expanding subfamily of leucine-rich
repeat-containing seven-TM receptors.
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RESULTS
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Conserved Architecture of Ectodomain, TM Region, and C-Terminal
Tail of LGR4 and LGR5
Human sequences related to the sea anemone and
Drosophila glycoprotein hormone receptors (12, 13) were
identified from the EST database based on their similarities to
receptors found in the lower species and nonidentity to the three known
human glycoprotein hormone receptors. The full-length cDNAs for novel
receptors were isolated using RT-PCR and repeated screening of
sublibraries from rat ovary or human placenta enriched with each
receptor cDNA. Positive clones with long inserts were sequenced and
aligned to identify the open reading frames (ORFs) of individual
receptors. The prototypic LGR consists of an ectodomain with
leucine-rich repeats and a C-terminal half with seven-TM domains
similar to other GPCRs. Because three known glycoprotein hormone
receptors have the same leucine-rich repeat-containing ectodomain and G
protein-coupled TM region, the new mammalian receptors were named LGR4
and LGR5, respectively.
LGR4 cDNA from rat ovary consists of 3,504 bp with a predicted
ORF of 951 amino acids, whereas LGR5 from human placenta has 4,208 bp
with a 907-amino acid ORF (Fig. 1
). The
ectodomains of LGR4 and LGR5 are more closely related to each other
(54% identity; 67% similarity) than to the three known LGRs (1823%
identity; 3335% similarity). Similar to three known glycoprotein
hormone receptors, LGR4 and LGR5 are characterized by multiple
leucine-rich repeat sequences (Fig. 1C
, Table 1
, and Ref. 18). Six and four consensus
N-linked glycosylation sites (Fig. 1C
, underlined N; and
Table 1
) were found in the ectodomains of LGR4 and LGR5, respectively,
and two of these sites were conserved between LGR4 and LGR5.



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Figure 1. Comparison of Deduced Amino Acid Sequence of LGR4
and LGR5 cDNAs and Those Encoding FSH, LH, and TSH Receptors and Other
Leucine-Rich Repeat-Containing Proteins
Sequence alignment for different regions of LGR4, LGR5, and three human
glycoprotein hormone receptors were performed. Heavily
shaded residues are identical in at least four of the five
receptor proteins shown, whereas the lightly shaded residues show
identity in LGR4 and LGR5. Amino acid numbers are shown on the
right. Asparagine residues in the potential N-linked
glycosylation sites are underlined and shown in bold.
Gaps introduced for optimal alignment are indicated as
dashes. A, Signal peptide. B, N-flanking cysteine-rich
sequence. C, Leucine-rich repeats. Seventeen leucine-rich repeat
sequences in LGR4 and LGR5 are indicated by arrows at
the top of the sequences. Because there are only nine
repeat sequences in the three glycoprotein hormone receptors, they were
arbitrarily aligned with the first nine repeats in LGR4 and LGR5. D,
C-flanking cysteine-rich sequence. E, TM region. Seven putative TM (TM)
domains are indicated at the top of the sequence.
Intracellular loops (IL) and outside loops (OL) are also indicated. F,
C-terminal tail. Conserved protein kinase A phosphorylation sites are
underlined. The
GenBank accession numbers for LGR4 and LGR5 are
AF061443 and AF061444, respectively. G, Comparison of leucine-rich
repeats found in LGRs and diverse other proteins with typical type
leucine-rich repeats. Consensus leucine-rich repeats in LGR4 and LGR5
were compared with those found in three human glycoprotein hormone
receptors (LHR/FSHR/TSHR) and LGRs from lower species. In addition, the
leucine-rich repeats of several other secretory proteins (ALS of
IGF/IGF binding protein complexes, slit, and decorin) and single-TM
domain receptors (Toll and Tartan) (36 62 ) with homologous repeats are
shown. The number of repeats found in each protein is shown in
parenthesis, whereas consensus amino acid residues are
shaded. Uppercase letters indicate more
than half of the leucine-rich repeats found in a given protein are
conserved, whereas lowercase letters denote less than
50% consensus. a, Aliphatic residues, c, charged residues; r, rat; h,
human; Ae, Anthopleura elegantissima (sea anemone); Ce,
Caenorhabditis elegans (nematode); Dm,
Drosophila melanogaster (fly); Ls: Lymnaea
stagnalis (snail).
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Although there are 17 leucine-rich repeats in LGR4 and LGR5 as
compared with 9 repeats in the glycoprotein hormone receptors,
alignment of the N-terminal 9 repeats of the five mammalian LGRs showed
that the third potential N-glycosylation site in LGR4 (Asn 199) and the
second potential N-glycosylation site in LGR5 (Asn 208) align perfectly
with the N-glycosylation site found in the sixth repeat of gonadotropin
and TSH receptors. In addition, clusters of cysteines (cysteine-rich
sequences) are present in the N-terminal region and the junction
between the ectodomain and TM 1. Because the four N-flanking cysteine
residues are conserved in all of the mammalian LGRs and other
leucine-rich repeat proteins (Fig. 1B
), these residues likely form
disulfide bonds essential for maintaining the conformation of the large
ectodomain of these receptors. In the C-flanking region, LGR4 and LGR5
also contain a cysteine-rich, chemokine-like region similar to the
consensus CF3 subtype domain recently identified in 45 glycoprotein
hormone receptors isolated from different mammals (20, 21), further
confirming the similar protein architecture of these receptors. In
particular, the core sequences of this consensus CF3 domain (CCAF and
FK/NPCE sequences) are completely conserved (Fig. 1D
). However, the
length of residues between conserved cysteines in LGR4 and LGR5
(CC-4X-C-4/54X-C) in this region is different from that found in the
three known glycoprotein hormone receptors (CC-15/23X-C-31/88X-C). In
addition, the junctional insertion of about 50 amino acids unique for
the TSH receptor (22) was missing in LGR4 and LGR5.
Seven membrane-spanning regions were predicted based on stretches
of hydrophobic amino acids forming
-helices (SOUSI server,
www.tuat.ac.jp/cgi/
mitaku/& NAKAI server,
http://psort.nibb.ac.jp/cgi-bin; Fig. 1E
). They are believed to
delimit a barrel-like cylinder structure with the apolar face of the
helices turned toward the membrane lipids. Similar to their
ectodomains, the TM helices of LGR4 and LGR5 are more homologous to
each other (49% identity; 64% similarity) than to the known LGRs
(2527% identity; 4852% similarity). In contrast to the TM
helices, the sequences in intracellular loop 3, an area believed to be
important for G protein coupling for adrenergic receptors (23), are
similar between the two new receptors (54% identity; 73% similarity)
but distinct from the three known glycoprotein hormone receptors (18%
identity; 36% similarity). Likewise, three outside and two other
intracellular loops of LGR4 and LGR5 show closer homology to each other
as compared with gonadotropin and TSH receptors. The highly conserved
cysteine residues in the first and second outside loops, predicted to
form an intramolecular disulfide bridge to constrain protein
conformation, were conserved in all LGRs (24, 25). In addition, proline
residues in the fourth, sixth, and seventh TM segments, believed to be
necessary for proper insertion of the receptor
proteins into the membrane (26), were also conserved. Among the outside
loops of these receptors, the highest homology was found in the second
loop exhibiting a unique ß-strand structure.
Although minimal conservation could be found for the five
receptors in the C-terminal tail (Fig. 1F
), multiple potential
phosphorylation sites were found in LGR4 and LGR5 as in
glycoprotein hormone receptors. For the two new receptors, a
consensus protein kinase A phosphorylation site was conserved (Fig. 1F
, underlined and italic letters), suggesting possible
regulation through cAMP-regulated phosphorylation (27). In LGR5,
potential SH2 and SH3 interacting sequences (amino acids
878881 and 888891, respectively) were also found (28).
Comparison of Leucine-Rich Repeats in LGR4 and LGR5 with Similar
Repeats in Glycoprotein Hormone Receptors and Other Leucine-Rich
Repeat-Containing Proteins
The ectodomains of LGR4 and LGR5 are composed
of 17 imperfect leucine-rich repeat motifs of 2224 amino acids in
length (Fig. 1
, A and G). The new consensus repeat sequences derived
from LGR4 and LGR5 are similar to each other with the exception that
glycine 18 is more common in LGR4, whereas serine 21 is more common in
LGR5 (Fig. 1G
). In addition, repeats 10, 11, 12, and 17 in both
receptors are distinct from the remaining repeats and show greater
deviation from the consensus leucine-rich repeat sequence (18). Of
interest, leucine-rich repeats found in LGR4 and LGR5 are closely
related to comparable repeats in the three glycoprotein hormone
receptors and LGRs from lower species (Fig. 1G
). These repeats are also
present in ALS of the insulin-like growth factor (IGF)/IGF binding
protein complexes, the proteoglycan decorin, the Drosophila
and mammalian Toll receptors, the Drosophila-secreted
protein slit, and the Drosophila Tartan receptor (Fig. 1G
).
A consensus asparagine in residue 6 is present in the repeats of all
these proteins, a feature unique to the typical type repeats (18).
These findings suggest a close evolutionary origin of the leucine-rich
repeats in these proteins of diverse structural arrangement and
function (20, 21).
Tissue Expression Pattern of LGR4 and LGR5
Northern blot hybridization was performed to analyze the
expression pattern of LGR4 and LGR5 mRNAs in diverse human tissues. As
shown in Fig. 2A
, a major transcript of
5.5 kb for LGR4 is expressed in multiple steroidogenic tissues
(placenta, ovary, testis, and adrenal). The mRNA for this putative
receptor is also found in spinal cord, thyroid, stomach, trachea,
heart, pancreas, kidney, prostate, and spleen. In contrast, the
expression pattern of LGR5 mRNA is more restricted (Fig. 2B
). A
transcript of 4.3 kb, together with a minor transcript of 2.4 kb for
LGR5 mRNA, was found to be highest in the skeletal muscle. This
transcript is also present in placenta, spinal cord, brain, adrenal,
colon, stomach, and bone marrow.

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Figure 2. Expression Pattern of LGR4 and LGR5 mRNA
Transcripts in Different Tissues
For Northern blot analysis, 2 µg of poly (A)+-selected
RNA from different human tissues were probed with a
32P-labeled LGR4 or LGR5 cDNA probe at 60 C. After washing
under stringent conditions, the blots were exposed to x-ray films with
an intensifying screen at -80 C for 7 days. Subsequent hybridization
with a ß-actin cDNA probe was performed to estimate nucleic acid
loading (8 h exposure; data not shown). A, LGR4 Northern blot. B, LGR5
Northern blot. Specific LGR transcripts are indicated by
arrows. Pa, Pancreas; Ki, kidney; Mu, skeletal muscle;
Li, liver; Lu, lung; Pl, placenta; Br, brain, He, heart; Co, colon; In,
small intestine; Ov, ovary; Te, testis; Pr, prostate; Th, thymus; Sp,
spleen; Bm, bone marrow; Ad, adrenal; Tr, trachea; ly, lymph node; Si,
spinal cord; Ty, thyroid; St, stomach.
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Lack of Gs Stimulation Mediated by Chimeric Receptors Comprising
the Ectodomain of LH Receptor and the TM Region of LGR4 or LGR5
Because chimeric receptors among the known glycoprotein hormone
receptors have been successfully used to study signal transduction by
these proteins (29), cDNAs for chimeric receptors were generated by
fusing the ectodomain of human LH receptor with the TM region and
C-terminal tail of either LGR4 or LGR5 [named as L(EC)LGR4(TM) and
L(EC)LGR5(TM), respectively]. Cells transfected with the plasmid
encoding L(EC)LGR4(TM) showed moderate binding to labeled human (h)CG
with a dissociation constant (Kd) value similar to that of
the wild-type LH receptor whereas cells transfected with the plasmid
encoding L(EC)LGR5(TM) showed lower binding but with high affinity. The
Bmax (ng hCG bound/105 cells) and
Kd (pM) values for different receptors are: LH receptor,
4.6±3.3 and 195±99; L(EC)LGR4(TM), 2.5±0.8 and 183±114 and
L(EC)LGR5(TM), 0.46±0.28 and 549±206, respectively. Despite
detectable hCG binding, treatment with increasing doses of hCG did not
increase cAMP production by either one of the chimeric receptors. At 10
µg/ml hCG, a 62-fold increase of cAMP production was mediated by the
wild type LH receptor but no stimulation of cAMP was found in cells
expressing either chimeric receptors (P < 0.05).
Isolation of LGR4 and LGR5 Genes, Their Conservation in
Vertebrates, and Chromosomal Localization in Humans
Using LGR4 and LGR5 cDNA fragments as probes, a bacterial
artificial chromosome-based human genomic DNA library was screened and
several genomic clones for LGR4 and LGR5 were isolated. To assess the
conservation of the LGR4 and LGR5 genes in diverse vertebrates,
Southern blot hybridization of genomic DNA from different species was
performed. Under medium stringency washing conditions, the rat LGR4
cDNA and human LGR5 cDNAs hybridized with genomic DNA from all
mammalian species tested, suggesting that both LGR4 and LGR5 genes are
conserved during mammalian evolution (Fig. 3A
).

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Figure 3. Conservation of LGR4 and LGR5 Genes in Diverse
Vertebrate Species and Their Chromosomal Localization in Human Cells
A, Southern blot hybridization of genomic DNA isolated from different
vertebrate species was performed using LGR4 and LGR5 cDNA probes. Four micrograms of genomic DNA was
digested with the EcoRI restriction enzyme and probed
with LGR4 or LGR5 cDNA. After hybridization at 60 C, the membrane was
washed under medium stringency conditions (0.5% SDS, 0.2 x SSC
at 60C) before exposure. B, Using DNA fragments of bacterial artificial
chromosome containing human LGR4 and LGR5 genes as probes, chromosomal
localization of LGR4 and LGR5 was detected using the FISH method to
chromosome 5q3435.1 and 12q15, respectively. Denatured chromosomes
from synchronous cultures of human lymphocytes were hybridized with
biotinylated probes for localization. Assignment of the FISH mapping
data (left) was achieved by superimposing signals with
4,6-diamdino-2-phenylindole-banded chromosome (center).
Analyses are summarized in the form of human chromosome ideograms
(right). Upper panel, LGR4; lower
panel, LGR5.
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Genomic fragments (>100 kb) of LGR4 and LGR5 were used as probes in
fluorescence in situ hybridization (FISH) analysis to
identify the chromosomal localization of LGR 4 and LGR5 genes. As shown
in Fig. 3B
, LGR4 and LGR5 genes were localized to banded DNA in
chromosomal 5q3435.1 and 12q15, respectively.
Conservation of TM and Flanking Regions in Nine LGRs from Diverse
Species and the Phylogenetic Relationship of These Receptors
In addition to the three glycoprotein hormone receptors and the
two new LGRs discussed here, four similar receptors have been found in
lower species. Sequence analysis of mammalian LGRs and homologous
receptors from sea anemone (13), fly (12), nematode (30), and snail
(31) indicated that the TM region and the junction between ectodomain
and TM 1, shown to be important for signal transduction of the known
glycoprotein hormone receptors, can be aligned based on BLOCK search
(32). In Fig. 4A
, BLOCK Maker analysis
showed that the TM region and sequences 5' to TM 1 are highly conserved
in all nine receptors and four ungapped blocks can be identified. In
addition, the chemical property of residues in this region is highly
similar (lightly hatched in Fig. 4A
). Consensus secondary
structure analysis of these receptors further indicated that, in
addition to the seven
-helical membrane-spanning domains, one unique
ß-strand structure could be found in the outside loop 2. Of interest,
this region has been shown to be important for the modulation of
hormone binding of LH receptor (33); conservation of the secondary
structure in this region suggests the outside loop 2 may have a
similar role in LGR4 and LGR5.


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Figure 4. Comparison of Protein Architectures of Mammalian
LGRs and Their Relationship to Similar Receptors Found in Sea Anemone,
Nematode, Fly, and Snail
A, Sequence comparison of the highly conserved TM regions of LGRs
from diverse species [sea anemone: Ae, Anthopleura
elegantissima; nematode: Ce, Caenorhabditis
elegans; fly: Dm, Drosophila melanogaster;
snail: Ls, Lymnaea stagnalis; rat (r) and human
(h)]. Based on BLOCK Maker analysis, four highly conserved
ungapped blocks were identified in the TM and flanking sequences of
nine LGRs from diverse species. Boxed areas are sequence
blocks with multiply aligned ungapped segments corresponding to
the most highly conserved regions of proteins.
Secondary structure predictions ( -helices: curved
arrows; ß- strand: straight arrow)
below the sequences were derived based on the PHD
algorithm and DSC (60 61 ). Chemically similar residues are
lightly shaded whereas conserved charged residues are
heavily shaded. Consensus residues represent identity
among at least five of the nine receptors, B, Phylogenetic relatedness
of LGRs from diverse species. Based on sequence comparison of the
entire receptor proteins, LGRs can be divided into three subgroups: one
containing the snail LGR, one containing mammalian LGR4 and LGR5, and a
third one containing human gonadotropin and TSH receptors together with
LGRs from fly, nematode, and sea anemone.
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Further analysis of the phylogenetic relatedness of nine LGRs from
diverse species, based on either the full-length
receptor sequence (Fig. 4B
) or the TM regions (data not shown),
suggested that LGR4 and LGR5 diverged early during evolution from the
known glycoprotein hormone receptors and from a homologous LGR found in
the central nervous system of snail (Lymnaea stagnalis; Fig. 4B
). In contrast, the three mammalian glycoprotein hormone receptors
are more related to the receptor identified in Drosophila,
and all four of these receptors can be categorized into the same branch
of the evolutionary tree together with LGRs found in sea anemone and
nematode.
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DISCUSSION
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The mammalian LGR family of proteins comprises at
least five gene products: LH and FSH receptors essential for gonadal
development; TSH receptor for thyroid differentiation; and the two new
orphan LGR4 and LGR5 without known ligands (Table 1
). In contrast to
known glycoprotein hormone receptors with 9 leucine-rich repeats in
their ectodomain, LGR4 and LGR5 have 17 leucine-rich repeats. Although
the TM helices are highly conserved among the five mammalian LGRs, the
inside loops of LGR4 and LGR5 are diverged from the glycoprotein
hormone receptors. The two new receptors, as FSH, LH, and TSH
receptors, are likely to be glycoproteins. In contrast to LH and FSH
receptors found in the same region of human chromosome 2p21 (34), LGR4
and LGR5 were localized to distinct human chromosomes. Although the
physiological roles of LGR4 and LGR5 are presently unclear, these
putative receptors appear to have a wider tissue distribution than
gonadotropin and TSH receptors. The availability of LGR4 and LGR5 cDNAs
allows future identification of ligands for these orphan receptors and
elucidation of their physiological function.
Diverse proteins containing leucine-rich repeats have been identified
in prokaryotes, plants, yeast, and many metazoans (18). Leucine-rich
repeats represent amphipathic sequences with leucine as the predominant
hydrophobic residue and are important for protein-protein interaction
(35). The packing of similar repeats allows the formation of a specific
hydrogen bond network between neighboring repeats to form a unique
secondary structure (21). The leucine-rich repeats in LGR4 and LGR5
belong to the typical type repeats with a conserved asparagine in the
middle (18). Conserved cysteine residues flanking leucine-rich repeats
are also present in LGR4 and LGR5. Except for Toll-like receptors and a
related 18-wheeler receptor (19, 36, 37) containing only a C-terminal
cysteine-rich domain, other leucine-rich repeat proteins, like LGRs,
have conserved cysteines at both N- and C-flanking regions. These
cysteine residues are likely to form disulfide bridges to maintain the
overall folding of repeat modules regardless of the number of repeat
(21).
Several models for leucine-rich repeats in the ectodomains of mammalian
glycoprotein hormone receptors have been postulated (38, 39). These
models are based on the crystal structure of the porcine
ribonuclease-ribonuclease inhibitor complex in which the repeats of 28
or 29 residues each have an inwardly directed ß-sheet (at the concave
surface) that might interact with specific ligands and an outwardly
directed
-helix (at the convex surface of the horseshoe). The
consensus repeat sequences found in LGR4 and LGR5 are most similar to
the leucine-rich repeats found in the ALS in the IGF/IGF binding
protein complexes important for maintaining the serum IGF reserve (40).
They are also similar to the Drosophila slit secreted by
glia cells in developing neurons (17) and the Drosophila and
mammalian Toll-like receptors important for dorsal-ventral polarization
during embryogenesis and the innate immune responses in adults
(19, 36). In addition, a small dermatan sulfate proteoglycan
decorin has homologous repeats; this proteoglycan interacts with
extracellular matrix and may serve as a reservoir of transforming
growth factor-ß (TGFß) (41). All repeats are believed to be
involved in protein-protein interactions: RNase inhibitor binds RNase;
ALS interacts with IGF-binding protein 3; slit binds laminin; Toll
receptor binds Spatzel (42); proteoglycan decorin binds TGFß and
collagen (41, 43); and biglycan binds laminin and fibronectin. For FSH,
LH, and TSH receptors, the repeat-containing ectodomains are
responsible for binding of cystine-knot fold glycoprotein hormones
(38). Based on structural homology with other LGRs, leucine-rich
repeats in the ectodomains of LGR4 and LGR5 might also bind specific
ligands. Although the putative ligands could be related to known
glycoprotein hormones, they could also be related to
Drosophila Spatzel protein based on the similarity between
leucine-rich repeats found in LGR4, LGR5, and the Toll receptors. Of
interest, both Spatzel and 8a related ligand Trunk have a conserved
cysteine-knot tertiary structure similar to FSH, LH, and TSH (44).
Because leucine-rich repeats in the two novel LGRs are also similar to
that found in the ALS of the IGF/IGF binding protein complexes and the
proteoglycan decorin, they might also interact with proteins related to
IGF-binding protein 3 or TGFß, ligands for ALS and decorin,
respectively.
Both LGR4 and LGR5 contain multiple consensus N-linked glycosylation
sites in their ectodomain. In all mammalian LGRs, an N-glycosylation
site in leucine-rich repeat 6 was conserved. In addition, the Ala-Phe
residues 5' to this site were also found in LGR4 and LGR5 with the
exception of an amino acid insertion. Interestingly, mutation of the
conserved Ala to Val in the FSH receptor gene leads to ovarian
dysgenesis (45) and spermatogenic failure (46). The conservation of
this motif among different LGRs underlines its functional
importance.
Alignment of four blocks of homology domains in the TM and flanking
regions of nine LGRs from diverse species indicated that multiple
-helices are important for membrane orientation and functional
integrity. The first homologous block not only contains TM helix 1 but
also extends into
15 residues in the junction of the ectodomain and
the TM region. This conserved region represents a cysteine-rich,
chemokine-like structure likely important for correct orientation of
the ectodomain to the TM region (47). Based on mutagenesis and chimeric
receptor studies, this junction is important for signal transduction
and folding of the LH receptor (24, 47, 48). Also, several residues
(LGR4 residues: 783K, 791P, and 801Y) at the border and inside the TM
helix 7, identified as essential for signal transduction of LH and TSH
receptors based on extensive site-directed mutagenesis (8, 47), are
highly conserved in all nine LGRs. Sequence alignment further indicated
that several key features that distinguish the known glycoprotein
hormone receptors from other GPCRs are conserved in the two new LGRs,
including the lack of a conserved proline in TM 5, an extra proline in
TM 7, and substitution of aromatic residues in TM 5 and 6 of
nonglycoprotein hormone receptors with polar residues in LGRs (49, 50, 51).
However, a third proline residue in TM 7 was found only in LGR4 and
LGR5. These data suggest that the seven TM bundles of LGR4 and LGR5
could have similar but distinct spatial orientation as compared with
the known glycoprotein hormone receptors. Thus, structural comparison
of the expanding group of LGRs could predict the functional importance
of critical residues for proper topology of these proteins.
During the preparation of this manuscript, an orphan GPCR (HG38) was
reported showing sequence identity to LGR5 except for two amino acids
in the ectodomain (52). Using radiation hybrid mapping, HG38 was
localized to human chromosome 12q2223 instead of 12q15 as was found
based on the FISH method (Fig. 3B
). Although the former method gives
greater resolution, further characterization using physical mapping
could provide the precise location of LGR5/HG38.
Using a chimeric receptor approach, the signal transduction property of
LGR4 and LGR5 was tested. Cells expressing chimeric receptors showed
high-affinity hCG binding, but no cAMP stimulation by hCG was
detected. Although these findings suggest the two new receptors might
not be coupled to the Gs protein, one cannot rule out the possibility
that the ectodomain of LH receptor might not be compatible with the
exoloops and TM helices of LGR4 and LGR5 for signal transduction. Of
interest, a conserved Glu-Arg-Trp triplet motif found in the junction
between TM 3 and inside loop 2, postulated to be involved in the
interaction between receptors and G proteins (53), is present in the
LGR4 and LGR5 but shows substitution in the last residue. In addition,
unique SH2 and SH3 interacting sequences, believed to be important for
protein-protein interaction in the mitogen kinase cascade (28), were
found in the C-terminal tail of LGR5 but not in glycoprotein hormone
receptors. The exact ligand-signaling mechanisms for the new LGRs
remain to be elucidated.
LGRs most likely represent the evolution of composite proteins or
chimeras derived from the duplication of different functional motifs to
form protein modules followed by gene rearrangement or exon shuffling
(Fig. 5
) (54, 55). The basic modules for
leucine-rich repeats are stretches of 24 amino acids, whereas the
seven-TM region is composed of membrane-spanning
-helical motifs of
largely hydrophobic residues. An ancestral gene with leucine-rich
repeats could evolve into genes with different functions through gene
rearrangement. Drosophila slit represents a fusion of
leucine-rich repeat domains with an epidermal growth factor
domain, whereas genes of the Toll family are derived from the fusion of
leucine-rich repeats to the interleukin-1 receptor-like motif (19, 36).
The LGR family of proteins represents the fusion of the leucine-rich
repeats with an ancestral GPCR. Although closely related to different
LGRs, the GRL101 gene found in the central nervous system of snail is
unique and may represent a fusion of low-density
lipoprotein-binding motifs and leucine-rich repeats together
with the seven-TM region (31).

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|
Figure 5. Hypothetical Model on the Evolution of Diverse
Genes Containing Leucine-Rich Repeats
Gene duplication and recombination probably account for the evolution
of LGRs and related leucine-rich repeat-containing proteins. The motifs
containing leucine-rich sequences or TM domains are the basic units to
build modules of leucine-rich repeats and seven-TM helices through gene
duplication. Fusion of ancestral leucine-rich repeat modules with
epidermal growth factor (EGF) domain or interleukin (IL) 1
receptor (R)-like domain led to the formation of secreted slit protein
and Toll receptors (R), respectively. In contrast, fusion of
leucine-rich repeats with seven-TM modules led to the evolution of
LGRs, whereas the fusion of leucine-rich repeats with seven-TM domains
plus a low density lipoprotein (LDL)-binding region led to the
formation of the GRL101 receptor found in snail.
|
|
It is clear that the LGR genes encode a conserved subgroup of seven TM
receptors of ancient origin. Based on the evolutionary relationship of
LGRs in diverse species and the homologous amino acid sequences found
in nine vertebrate and invertebrate LGRs, future searches could
identify additional members of this subfamily of GPCRs in the mammalian
genome. Because LGR4 and LGR5 appear to diverge from known gonadotropin
and TSH receptors early during evolution before the formation of the
pituitary gland and exhibit high similarity to LGRs found in lower
vertebrates, they could subserve physiological functions associated
with the primitive LGRs found in Cnidarians, one of the most primitive
animals with a sensory system in the animal kingdom. A wide tissue
distribution pattern of the mRNAs for these proteins further suggested
that their physiological roles might be different from mammalian
gonadotropin and TSH receptors known to play tissue-specific functions.
The availability of cDNAs for LGR4 and LGR5 allows for future
identification of their specific ligands by employing an anchored
receptor approach found to be useful for the solubilization of the
ligand-binding domains of glycoprotein hormone receptors (56) and for
the elucidation of their physiological functions.
 |
MATERIALS AND METHODS
|
---|
Computational Analysis
Human sequences related to the sea anemone and
Drosophila glycoprotein hormone receptors (12, 13) were
identified from the EST database (dbEST) at the National Center for
Biotechnology Information (NIH, Bethesda, MD) by using the BLAST
server with the BLOSUM62 protein comparison matrix (57). The
alignment of LGR ecto- and TM domain sequences was carried out by
CLUSTALW (58); this program also calculated the branching order of
aligned sequences by the neighbor-joining algorithm (10,000 bootstrap
replications provided confidence values for the tree groupings).
Conserved alignment patterns were drawn by the CONSENSUS program
(Internet URL http://www.
bork.embl-heidelberg.de/Alignment/consensus.html). The PRINTS
library of protein fingerprints (http://www.biochem.
ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html) (18, 59) identified
the myriad leucine-rich repeats present in the ectodomains of
LGRs with a compound "Leurichrpt" motif that flexibly matches N-
and C-terminal features of divergent leucine-rich repeats. The BLOCK
Maker website (http://blocks.fhcrc.org) was used to align and
generate the highly conserved ungapped blocks of the TM regions of
diverse LGRs from vertebrates and invertebrates using both full-length
receptor and TM segment sequences. The use of two different methods,
Motif and Gibbs samplings, confirms for close relatedness. The blocks
predicted from this alignment were then used to construct the
neighbor-joining tree for the examination of possible subfamily
relationships. Two algorithms whose three-state accuracy is greater
than 72%, the neural network program PHD (60) and the statistical
prediction method DSC (61; http://bonsai.lif.icnet.uk/dsc/manual.html),
were used to derive a consensus secondary structure for the TM
domain of different LGRs.
Cloning of Full-Length LGR4 and LGR5 cDNAs
Human ESTs showing high homology to two nonoverlapping
regions of the gonadotropin receptors were identified. Clones AA312798
and AA298810 were found to encode TM 4 to TM 5 of the putative receptor
(LGR4), whereas AA460529 and AA424098 encode TM 2 to TM 3 of another
putative receptor (LGR5). Using these ESTs to further search the
GenBank EST division database, overlapping EST sequences were aligned
to obtain the longest ORF for each initial clone. Relevant EST clones
were obtained from the I.M.A.G.E. consortium (info@image.llnl.gov) via
Genome System, Inc. (St. Louis, MO).
Based on the longest human ORF, specific primers were designed for PCR
amplification of LGR4 and LGR5 cDNA fragments from rat ovary and human
placenta, respectively. After hybridization with labeled EST clones and
confirmation of DNA sequences by dideoxy DNA sequencing, specific
receptor fragments isolated were used to design primers to prepare
sub-cDNA libraries enriched with specific receptor cDNAs. For 5'
extension, reverse transcription was performed using rat ovarian and
human placenta mRNA preparations and receptor-specific primers. After
second-strand synthesis, the enriched cDNA pool was tailed at 5'-ends
with specific adaptor sequences to allow further PCR amplification. For
3'-extension, rat ovarian or human placenta mRNAs were reversed
transcribed using oligo-dT, followed by second-strand synthesis using
receptor-specific primers and adaptor tailing. These minilibraries were
further used as templates for PCR amplification of upstream or
downstream cDNAs specific for each receptor using internal primers. PCR
products with a strong hybridization signal to each receptor cDNA
fragment were subcloned into the pUC18 or pcDNA3 vectors. After
screening of these sublibraries based on colony hybridization using
specific receptor probes, clones with 5'- or 3'-sequences of the
putative receptors were identified and isolated for DNA sequencing. As
needed, the procedure was repeated up to three times to generate cDNAs
encoding the complete ORF of each putative receptor for sequence
analysis and for the expression of receptor proteins in eukaryotic
cells. The entire coding sequences of each gene were also amplified
with specific primers flanking the entire ORF in independent
experiments. At least three independent PCR clones were sequenced to
verify the authenticity of coding sequences.
Tissue Expression of LGR4 and LGR5 mRNAs
Human multiple tissue blots, containing
2 µg of poly(A)+
RNA per lane, were purchased from CLONTECH (Palo Alto, CA; catalog
number 7759, 7760, and 7767). Northern blot analyses were performed
using tissue blots after hybridization of labeled receptor cDNA probes.
Membranes were prehybridized for 1 h at 60 C in the ExpressHyb
solution (CLONTECH). This was followed by hybridization under the same
condition for 2 h but with 1 x 106 cpm/ml of
32P-labeled LGR4, LGR5, or ß-actin cDNA probe. After
hybridization, the membranes were washed twice in 2 x
saline-sodium citrate (SSC), 0.5% SDS at room temperature,
followed by two washes in 0.2 x SSC, 0.5% SDS at 60 C before
exposure to Kodak RX films (Eastman Kodak, Rochester, NY).
Construction of Chimeric Receptor cDNAs and Analysis of Signal
Transduction and Ligand Binding
PCR-based mutagenesis was performed using overlapping primers to
construct cDNAs for chimeric LH/LGR4 and LH/LGR5 receptors as described
previously (29). L(EC)LGR4(TM) and L(EC)LGR5(TM) represent chimeric
receptors with the ectodomain of human LH receptor and the TM and
C-terminal tail of LGR4 or LGR5 with the junctional sequences of
PEPDA-FKPCEYLLGS and PEPDA-FKPCEHLLDG, respectively. All cDNAs were
subcloned into the expression vector pcDNA3 (Invitrogen, San Diego,
CA). Both the fidelity of PCR-amplified regions and the junctional
sequences were confirmed by DNA sequencing on both strands. 293 cells
derived from human embryonic kidney fibroblast were maintained in
DMEM/Hams F-12 (Life Technologies, Inc., Gaithersburg, MD)
supplemented with 10% FBS, 100 µg/ml penicillin, 100 µg/ml
streptomycin, and 2 mM L-glutamine. The cells
were transfected with receptor cDNAs as described (29) by the calcium
phosphate precipitation method. Cells transfected with the empty
plasmid (mock) served as negative controls. Cells were placed on
24-well tissue culture plates (Corning, Corning, NY) and preincubated
at 37 C for 30 min in the presence of 0.25 mM
3-isobutyl-1-methyl xanthine (Sigma Chemical Co., St. Louis, MO) before
treatment with or without hCG for 5 h. At the end of incubation,
cells and medium in each well were frozen and thawed once and then
heated to 95 C for 3 min to inactivate phosphodiesterase activity.
Total cAMP was measured in triplicates by specific RIA. All experiments
were repeated at least three times using cells from independent
transfection. Statistical analysis was performed using Students
t test.
For ligand binding analysis of the chimeric receptors, hCG was
iodinated by the lactoperoxidase method and characterized by
radioligand receptor assay using human LH receptors stably expressed in
293 cells (29). Specific activity and maximal binding of the labeled
hCG were 100,000150,000 cpm/ng and 4050%, respectively. To
estimate ligand binding on the cell surface, cells were washed twice
with PBS and collected in PBS before centrifugation at 400 x
g for 5 min. Pellets were resuspended in PBS containing
0.1% BSA, and 200,000 cells/300 µl were incubated with a nearly
saturating amount of labeled hCG at room temperature for 1822 h in
the presence or the absence of hCG. At the end of incubation, cells
were centrifuged and washed twice with PBS. Radioactivities in the
pellets were determined in a ß-counter.
Genomic Analysis and Chromosomal Localization of LGR4 and
LGR5
For studies on the conservation of LGR4 and LGR5 genes, the Zoo
blots (CLONTECH) containing genomic DNA from different vertebrates were
hybridized with 32P-labeled rat LGR4 or human LGR5 cDNA
probe under moderate stringency conditions.
To isolate genomic clones for LGR4 and LGR5, several genomic DNA
fragments were isolated from a human bacterial artificial chromosome
(BAC) genomic DNA library (Genome Systems, Inc.) using the near
full-length LGR4 or LGR5 cDNA probes. The genomic fragments were then
confirmed by Southern blot hybridization. For the identification of the
chromosomal localization of LGR4 and LGR5 genes, genomic fragments
(>100 Kb) of LGR4 and LGR5 were used as probes for FISH to human
metaphase chromosomes (SeeDNA Biotech, Inc., Toronto, Ontario, Canada).
Denatured chromosomes from synchronous cultures of human lymphocytes
were hybridized with biotinylated probes for signal localization.
 |
ACKNOWLEDGMENTS
|
---|
We thank C. Spencer for editorial assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. A. J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology/Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu
This work was supported by NIH Grant HD-23273.
1 The first two authors contributed equally to this work. S.L. is on
leave from the Department of Obstetrics and Gynecology, Teikyo
University, Tokyo, Japan. 
2 GenBank accession numbers for LGR4 and LGR5 are
AF061443 and AF061444, respectively. 
Received for publication July 21, 1998.
Revision received September 9, 1998.
Accepted for publication September 11, 1998.
 |
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