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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 (18–23%
identity; 33–35% 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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), 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
(25–27% identity; 48–52% 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
878–881 and 888–891, 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 22–24 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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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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, LGR4 and LGR5 genes were localized to banded DNA in
chromosomal 5q34–35.1 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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) 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. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). 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 12q22–23 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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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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/Ham’s 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 Student’s
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 100,000–150,000 cpm/ng and 40–50%, 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 18–22 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 S. Mazerbourg, D. M. Bouley, S. Sudo, C. A. Klein, J. V. Zhang, K. Kawamura, L. V. Goodrich, H. Rayburn, M. Tessier-Lavigne, and A. J. W. Hsueh Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor 4 Null Mice Exhibit Intrauterine Growth Retardation Associated with Embryonic and Perinatal Lethality Mol. Endocrinol., September 1, 2004; 18(9): 2241 - 2254. [Abstract] [Full Text] [PDF]
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L. Montanelli, J. J. J. Van Durme, G. Smits, M. Bonomi, P. Rodien, E. J. Devor, K. Moffat-Wilson, L. Pardo, G. Vassart, and S. Costagliola Modulation of Ligand Selectivity Associated with Activation of the Transmembrane Region of the Human Follitropin Receptor Mol. Endocrinol., August 1, 2004; 18(8): 2061 - 2073. [Abstract] [Full Text] [PDF]
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P. M. Apaja, K. T. Harju, J. T. Aatsinki, U. E. Petaja-Repo, and H. J. Rajaniemi Identification and Structural Characterization of the Neuronal Luteinizing Hormone Receptor Associated with Sensory Systems J. Biol. Chem., January 16, 2004; 279(3): 1899 - 1906. [Abstract] [Full Text] [PDF]
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M. A. M. Krajnc-Franken, A. J. M. van Disseldorp, J. E. Koenders, S. Mosselman, M. van Duin, and J. A. Gossen Impaired Nipple Development and Parturition in LGR7 Knockout Mice Mol. Cell. Biol., January 15, 2004; 24(2): 687 - 696. [Abstract] [Full Text] [PDF]
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S. Sudo, J. Kumagai, S. Nishi, S. Layfield, T. Ferraro, R. A. D. Bathgate, and A. J. W. Hsueh H3 Relaxin Is a Specific Ligand for LGR7 and Activates the Receptor by Interacting with Both the Ectodomain and the Exoloop 2 J. Biol. Chem., February 28, 2003; 278(10): 7855 - 7862. [Abstract] [Full Text] [PDF]
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 M. Conti Specificity of the Cyclic Adenosine 3',5'-Monophosphate Signal in Granulosa Cell Function Biol Reprod, December 1, 2002; 67(6): 1653 - 1661. [Abstract] [Full Text] [PDF]
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 V. Garcia-Campayo, T. R. Kumar, and I. Boime Thyrotropin, Follitropin, and Chorionic Gonadotropin Expressed as a Single Multifunctional Unit Reveal Remarkable Permissiveness in Receptor-Ligand Interactions Endocrinology, October 1, 2002; 143(10): 3773 - 3778. [Abstract] [Full Text] [PDF]
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Y.-X. Tao, D. Mizrachi, and D. L. Segaloff Chimeras of the Rat and Human FSH Receptors (FSHRs) Identify Residues that Permit or Suppress Transmembrane 6 Mutation-Induced Constitutive Activation of the FSHR via Rearrangements of Hydrophobic Interactions Between Helices 6 and 7 Mol. Endocrinol., August 1, 2002; 16(8): 1881 - 1892. [Abstract] [Full Text] [PDF]
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S. Y. Hsu, K. Nakabayashi, and A. Bhalla Evolution of Glycoprotein Hormone Subunit Genes in Bilateral Metazoa: Identification of Two Novel Human Glycoprotein Hormone Subunit Family Genes, GPA2 and GPB5 Mol. Endocrinol., July 1, 2002; 16(7): 1538 - 1551. [Abstract] [Full Text] [PDF]
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C. P. Leo, S. Y. Hsu, and A. J. W. Hsueh Hormonal Genomics Endocr. Rev., June 1, 2002; 23(3): 369 - 381. [Abstract] [Full Text] [PDF]
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 J. W. M. Martens, S. Lumbroso, M. Verhoef-Post, V. Georget, A. Richter-Unruh, M. Szarras-Czapnik, T. E. Romer, H. G. Brunner, A. P. N. Themmen, and Ch. Sultan Mutant Luteinizing Hormone Receptors in a Compound Heterozygous Patient with Complete Leydig Cell Hypoplasia: Abnormal Processing Causes Signaling Deficiency J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2506 - 2513. [Abstract] [Full Text] [PDF]
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M. Ascoli, F. Fanelli, and D. L. Segaloff The Lutropin/Choriogonadotropin Receptor, A 2002 Perspective Endocr. Rev., April 1, 2002; 23(2): 141 - 174. [Abstract] [Full Text] [PDF]
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 S. Y. Hsu, K. Nakabayashi, S. Nishi, J. Kumagai, M. Kudo, O. D. Sherwood, and A. J. W. Hsueh Activation of Orphan Receptors by the Hormone Relaxin Science, January 25, 2002; 295(5555): 671 - 674. [Abstract] [Full Text] [PDF]
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 L. L. Heckert and M. D. Griswold The Expression of the Follicle-stimulating Hormone Receptor in Spermatogenesis Recent Prog. Horm. Res., January 1, 2002; 57(1): 129 - 148. [Abstract] [Full Text] [PDF]
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H. Shinozaki, F. Fanelli, X. Liu, J. Jaquette, K. Nakamura, and D. L. Segaloff Pleiotropic Effects of Substitutions of a Highly Conserved Leucine in Transmembrane Helix III of the Human Lutropin/Choriogonadotropin Receptor with Respect to Constitutive Activation and Hormone Responsiveness Mol. Endocrinol., June 1, 2001; 15(6): 972 - 984. [Abstract] [Full Text] [PDF]
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 K. M. Wasson and A. J. W. Hsueh Ovarian Gene Database Reproductive Sciences, January 1, 2001; 8(1_suppl): S37 - S39. [Abstract] [PDF]
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S. Nishi, S. Y. Hsu, K. Zell, and A. J. W. Hsueh Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells Endocrinology, November 1, 2000; 141(11): 4081 - 4090. [Abstract] [Full Text] [PDF]
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A. P. N. Themmen and I. T. Huhtaniemi Mutations of Gonadotropins and Gonadotropin Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal Function Endocr. Rev., October 1, 2000; 21(5): 551 - 583. [Abstract] [Full Text]
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S. Y. Hsu, M. Kudo, T. Chen, K. Nakabayashi, A. Bhalla, P. J. van der Spek, M. van Duin, and A. J. W. Hsueh The Three Subfamilies of Leucine-Rich Repeat-Containing G Protein-Coupled Receptors (LGR): Identification of LGR6 and LGR7 and the Signaling Mechanism for LGR7 Mol. Endocrinol., August 1, 2000; 14(8): 1257 - 1271. [Abstract] [Full Text]
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 T. Brody and A. Cravchik Drosophila melanogaster G Protein-coupled Receptors J. Cell Biol., July 24, 2000; 150(2): F83 - F88. [Abstract] [Full Text] [PDF]
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 K. K. Eriksen, F. Hauser, M. Schiøtt, K.-M. Pedersen, L. Søndergaard, and C. J.P. Grimmelikhuijzen Molecular Cloning, Genomic Organization, Developmental Regulation, and a Knock-out Mutant of a Novel Leu-rich Repeats-containing G Protein-coupled Receptor (DLGR-2) from Drosophila melanogaster Genome Res., July 1, 2000; 10(7): 924 - 938. [Abstract] [Full Text]
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S. Y. Hsu and A. J. W. Hsueh Discovering New Hormones, Receptors, and Signaling Mediators in the Genomic Era Mol. Endocrinol., May 1, 2000; 14(5): 594 - 604. [Full Text]
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M. Kudo, T. Chen, K. Nakabayashi, S. Yu Hsu, and A. J. W. Hsueh The Nematode Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor (LGR) Protein Homologous to Vertebrate Gonadotropin and Thyrotropin Receptors is Constitutively Activated in Mammalian Cells Mol. Endocrinol., February 1, 2000; 14(2): 272 - 284. [Abstract] [Full Text]
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H. Kishi and M. Ascoli Multiple Distant Amino Acid Residues Present in the Serpentine Region of the Follitropin Receptor Modulate the Rate of Agonist-induced Internalization J. Biol. Chem., September 29, 2000; 275(40): 31030 - 31037. [Abstract] [Full Text] [PDF]
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K. Nakabayashi, M. Kudo, B. Kobilka, and A. J. W. Hsueh Activation of the Luteinizing Hormone Receptor Following Substitution of Ser-277 with Selective Hydrophobic Residues in the Ectodomain Hinge Region J. Biol. Chem., September 22, 2000; 275(39): 30264 - 30271. [Abstract] [Full Text] [PDF]
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C. Govaerts, A. Lefort, S. Costagliola, S. J. Wodak, J. A. Ballesteros, J. Van Sande, L. Pardo, and G. Vassart A Conserved Asn in Transmembrane Helix 7 Is an On/Off Switch in the Activation of the Thyrotropin Receptor J. Biol. Chem., June 15, 2001; 276(25): 22991 - 22999. [Abstract] [Full Text] [PDF]
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S. Nishi, K. Nakabayashi, B. Kobilka, and A. J. W. Hsueh The Ectodomain of the Luteinizing Hormone Receptor Interacts with Exoloop 2 to Constrain the Transmembrane Region. STUDIES USING CHIMERIC HUMAN AND FLY RECEPTORS J. Biol. Chem., February 1, 2002; 277(6): 3958 - 3964. [Abstract] [Full Text] [PDF]
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