Division of Reproductive Biology Department of Gynecology and Obstetrics Stanford University School of Medicine Stanford, California 94305-5317
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ABSTRACT |
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INTRODUCTION |
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Recently, putative receptors homologous to mammalian glycoprotein hormone receptors were found in sea anemone (7), fly (8), and snail (9), indicating that this subfamily of G protein-coupled receptors has an ancient origin. Based on the sequence homology of mammalian glycoprotein hormone receptors, we identified an LGR (leucine-rich repeat-containing, G protein-coupled receptor) ortholog gene in the C. elegans genome and isolated its cDNA. Transfection studies indicated that the nematode receptor is constitutively activated in a ligand-independent manner. The use of chimeric receptor constructs further suggested that different regions of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode receptor is important for its constitutive activity.
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RESULTS |
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Comparison of cDNA sequences with the genomic sequences in the GenBank
showed that the open reading frame of the nematode LGR contains 929
amino acids with a calculated molecular mass of 104 kDa instead of 889
amino acids, as originally predicted by the Genefinder program (cosmid
C50H2.1, GenBank accession no. 1402971). The differences resulted from
an incorrect prediction of the 43 amino acids at the N terminus and an
incorrect exon-intron junction assignment. The methionine start site
predicted by the present cDNA sequence should be accurate because the
new methionine translation initiation codon is preceded by a stop codon
and is followed by a string of hydrophobic amino acid characteristic of
the signal peptide for membrane insertion or secretion. Comparison of
this cDNA with the corresponding genomic sequence revealed that the
nematode gene has at least 13 exons and expands over 4.7 kb in
length. The deduced 929 amino acid sequence of this receptor consists
of an ectodomain encoded by exons 18, a TM region by exons 811, and
the C-terminal tail by exons 1113 (Fig. 1A).
Twelve of the 13 introns are smaller than 200 bp in size with a
1,055-bp intron between exon 1 and 2. In the exon-intron junctions,
three types of donor-acceptor patterns could be found (Fig. 1B
).
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The TM region of nematode LGR showed greater than 30% identity and
50% similarity with mammalian glycoprotein hormone receptors (Fig. 1C
and Fig. 2
).
Although distinctive GPCR motifs were identified, including the highly
conserved cysteine residues for disulfide bond formation in the
extracellular loops 1 and 2, the unique DRY (ERW in glycoprotein
hormone receptors) motif found at the junction between TM III and
intracellular loop 2 of many GPCRs (10) has diverged to an EMS
sequence in the nematode receptor. In its ectodomain, the nematode
receptor has five potential N-linked glycosylation sites whereas the
unusually long C-terminal tail has several consensus phosphorylation
sites for protein kinase A (546549 RRLS, 719-7229 RRIT, 792795
RRAS, 924927 RRKS, and 925928 RKST) and C (542544 SFP, 549551
SPK, 773775 TPR, 797799 SPR, 810812 TPR, 813815 SDR, and
874876 SGR)(Figs. 1C
and 2
).
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Nematode LGR Is Constitutively Activated in cAMP but Not Inositol
Phosphate (IP) Signaling in Transfected Mammalian
Cells
To test the signaling mechanisms for the nematode LGR, mammalian
293T cells were transfected with an expression plasmid containing the
nematode receptor cDNA. Cells expressing the nematode LGR showed
increases in basal cAMP production in a ligand-independent fashion
(Fig. 3A, left panel). As
shown in Fig. 3B
, transfection of 293T cells with increasing amounts of
the nematode LGR expression plasmid (nLGR) led to dose-dependent
increases in cAMP production. The observed basal activity of the
nematode receptor is comparable to that found in cells expressing a
constitutively activated mutant human LH receptor (D578Y LHR), whereas
minimal increases in basal cAMP production were found in cells
expressing the wild-type LH receptor (LHRWT) and cells transfected with
the empty vector (Fig. 3B
). Because these receptors were tagged at the
N-terminal ends with a FLAG epitope, the levels of expression on the
cell surface were also determined using the M1 antibody. As shown in
Fig. 3A
(right panel), the levels of proteins expressed on
the cell surface were high for all receptors tested; however,
negligible FLAG epitope expression was found in cells transfected with
the empty vector. Although the use of the FLAG epitope allowed
comparison of the cell surface expression of different receptors, a
nematode LGR construct without the epitope tag also showed constitutive
activation (Fig. 3B
). Because the cell surface expression of the
nontagged nLGR is unknown, it is difficult to compare the degree of
constitutive activation between this and the tagged nLGR. In contrast
to the major increases (>100-fold) in basal cAMP production mediated
by nLGR as compared with the wild-type LH receptor, the wild-type human
TSH receptor (nontagged) only showed 2.4-fold increases in basal cAMP
production (n = 3).
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Because human TSH and LH receptors have been shown to mediate
ligand-stimulated increases in IP turnover (14, 15), phosphatidyl
inositol hydrolysis was also determined in the transfected cells.
However, the synthesis of total IPs after the addition of LiCl was
similar among cells expressing wild-type LH receptor and the nematode
LGR with approximately 1.4-fold increases during the 45-min incubation
(Fig. 3C). In contrast, production of IPs by a wild-type LH receptor
expressing cells after hCG treatment showed a 2.8-fold increase in PI
hydrolysis. Likewise, cells expressing a mutant LH receptor D578Y
showed a 3.8-fold increase in IP turnover only after hCG treatment.
These data suggest the nematode receptor does not activate the
phospholipase C pathway.
The Intracellular Loop 3 Region of Nematode LGR Is Responsible for
Its Constitutive Activation: Analysis Using Chimeric Receptors
Earlier studies indicated that different domains of the
three mammalian glycoprotein hormone receptors are interchangeable (10, 16). Based on the similar secondary structure of the nematode LGR and
human LH receptors, several chimeric receptors were constructed (Fig. 4A) to investigate the compatibility of
different domains of these two receptors and the region of nematode LGR
important for constitutive activation.
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Because the C-terminal end of the intracellular loop 3 of the
nematode LGR is distinctly different from the three glycoprotein
hormone receptors (Fig. 2B), we hypothesized that this region might be
responsible for its constitutive activation. As shown in Fig. 4E
, a
chimeric receptor with five amino acids in the C-terminal end of
intracellular loop 3 of the LH receptor (TKIAK) replaced by the
nematode LGR (RALIT) showed increases in basal cAMP production. These
results indicated that different domains of the nematode LGR and human
LH receptor are interchangeable. Furthermore, the findings demonstrated
that sequences in the C-terminal end of intracellular loop 3 of the
nematode LGR are likely responsible for its constitutive activity.
One of the hallmarks of GPCR is at the junction of the TM III and the second intracellular loop, showing DRY/ERW triplets. Instead of this consensus sequence, EMS was found in the nematode LGR. In adrenergic receptors, this positively charged arginine (R) is thought to be important to keep the receptor inactive (17). Therefore, we performed mutagenesis to change EMS to ERS, but the mutated receptor did not differ from wild-type receptor in the magnitude of constitutive activation (data not shown).
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DISCUSSION |
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In contrast to the mammalian glycoprotein hormone receptors, the TM region and the C-tail of nematode and fly LGRs are encoded by multiple exons. In the mammalian glycoprotein hormone receptors, this region is encoded by a single exon whereas the same region is split into 4 and 6 exons in fly (8) and nematode LGR genes, respectively. While the number of exons encoding the TM region and the C tail of nematode LGR is greater than that of fly LGR, the number of exons for the ectodomain of nematode LGR is less than that found in the fly LGR gene. These findings suggest the ancestral gene for LGRs could be encoded by even larger numbers of exons as compared with those found in modern organisms; and intron loss during evolution leads to the presence of fewer exons in LGRs of higher organisms. Like many other GPCRs (18, 19), it is possible that gene duplication, diversification, movement in the genome, and intron loss account for the evolution of the mammalian glycoprotein hormone receptors.
Analysis of the nematode genomic sequences showed that nematode LGR is unique among all nematode GPCRs. Since the genome of C. elegans has been completely sequenced, the nematode LGR characterized here is likely to be the only nematode GPCR carrying the hallmark of mammalian LGRs, an ectodomain with multiple leucine-rich repeats followed by a 7-TM domain. This finding is interesting because phylogenetic analysis of all known LGRs indicates that the homology between the nematode LGR and the mammalian glycoprotein protein receptors is closer than the homology between the glycoprotein hormone receptors and two newly isolated mammalian LGR4 and LGR5. The nematode LGR not only is similar to LH, FSH, and TSH receptors in having 9 leucine-rich repeats as compared with 17 repeats found in the LGR4 and LGR5, but also shows a closer sequence homology in the TM region (11). The existence of divergent LGRs in mammals predicted that LGRs diverged early during evolution.
The mammalian glycoprotein hormone receptors have distinctive structural features and mainly couple through the cAMP-dependent pathway for signal transduction. Because the nematode receptor shares structural characteristics with mammalian glycoprotein hormone receptors, the nematode LGR is likely to share similar signaling properties with their mammalian counterparts. Surprisingly, when the nematode LGR cDNA was expressed in mammalian cells, the expressed receptors showed constitutive increases in basal cAMP production. Although it is possible that the observed constitutive activity of the nematode LGR is related to a mismatch between the worm receptor and mammalian membrane environment or G proteins, analysis of G protein-coupled serotonin 5-HT2 and odorant ODR-10 receptors from C. elegans indicated that ligands are needed to activate these nematode proteins expressed in mammalian cells (20, 21). Several wild-type G protein-coupled receptors including dopamine D1B (22), 5-hydroxytryptamine 2C (23) and TSH (2) receptors exhibit constitutive activity when overexpressed but the nematode LGR showed much higher constitutive activity than the TSH receptor in 293T cells. The number of cell surface nematode LGRs detected by antibody binding to the FLAG epitope was similar to or lower than that of wild-type LH receptors expressed in the same cells. Because negligible basal cAMP production was observed for the wild-type LH receptor and related LGR4 and LGR5 (11), the observed constitutive activity of the nematode receptor is not due to higher receptor numbers but likely the result of its intrinsic activity as found for mutant LH receptors (4, 24).
In addition to the observed constitutive activity of several wild-type receptors, multiple mutant receptors in the large GPCR superfamily have been found. However, the precise mechanisms underlying receptor activation are not entirely clear. The concept of constitutive or ligand-independent activation of GPCRs was originally discovered in the ß2-adrenergic receptor after introduction of point mutations in the intracellular loop 3 (25). Recently, multiple naturally occurring point mutations have been found in different disease states (26, 27, 28). These include mutations of rhodopsin (retinitis pigmentosa) (29, 30), MSH receptor (coat color in mice) (31), PTH-PTHrP receptor (Jansen-type metaphyseal chondrodysplasia) (32), and Ca2+-sensing receptor (familial hypercalciuric hypercalcemia and neonatal severe hyperparathyroidism) (33, 34). For LH and TSH receptors, activating mutations are also present in patients with familial male-limited precocious puberty and nonautoimmune hyperthyroidism (4, 6), respectively.
To understand the mechanisms for the constitutive activation of the nematode receptor, several chimeric and mutant receptors were tested for basal cAMP production in transfected cells. Although a chimeric receptor with the ectodomain from LH receptor and the TM region and C-tail from the nematode LGR showed reduced cell surface expression, the protein still exhibited constitutive activity. Furthermore, a chimeric receptor, L-N(i3C)-L, in which only the C-terminal portion of intracellular loop 3 is from the nematode receptor, also showed constitutive activation. These results suggest that the different regions of the nematode LGR and human LH receptor are compatible to maintain the overall receptor structure for cell surface expression whereas constitutive activation of the nematode LGR is due to an active conformation of its TM bundles. Because the five amino acids in intracellular loop 3 of the LH receptor replaced by the nematode sequence are highly conserved among the glycoprotein hormone receptors (T-K/R-I-A-K), it is likely that these residues are involved in the constrained state of these proteins. Of interest, point mutations of the conserved alanine to valine of the LH receptor were found in patients with familial precocious puberty (35) whereas replacement of the corresponding alanine to isoleucine in the TSH receptor is associated with nonimmune hyperthyroidism (36). Future studies are needed to elucidate the exact residue(s) important for the active conformation of these receptors.
The G protein-coupled receptor superfamily, consisting of 170
rhodopsin-like receptors, 650 seven-TM chemoreceptors, and other
similar proteins, represents the largest nematode gene family and
accounts for more than 5% of the entire C. elegans genome
(37). Recent studies further indicated that the C. elegans
has 20 G-, 2 G-ß, and 2 G-
genes. Among the G-
genes, one
homolog for each of the four mammalian classes of G-
genes, Gs-
,
Gi/Go-
, Gq-
, and G12-
, has been found (38). Of interest, the
Gs-
is highly conserved, with 66% identity in amino acid sequence,
between human and nematode (39), consistent with the observed coupling
between the nematode LGR and the orthologous mammalian Gs protein found
in the human 293T cells.
It has been proposed that mutations causing constitutive activation
alter the receptor conformation from an inactive state to an active
state, mimicking the ligand stimulation of the receptor (28). Although
the physiological role of the nematode LGR in C. elegans is
unknown, it is likely that this LGR can mediate cAMP signaling. Based
on the homology of the ligand-binding ectodomains between nematode LGR
and mammalian glycoprotein hormone receptors, the possibility was
tested that extremely high concentrations of the known glycoprotein
hormones (heterodimers of - and ß-subunit) can stimulate or bind
the nematode LGR. However, none of the known mammalian ligands
interacted with the nematode receptor. Although the similarity of the
nematode LGR to mammalian glycoprotein hormone receptors suggests that
the worm receptor could have a glycoprotein hormone-like ligand, no
nematode homolog of the mammalian
- and ß-subunit genes has been
reported and the nematode LGR may represent an ancestral protein with
constitutive activity. Future studies on the ligand signaling
mechanisms for LGRs from other lower species could help elucidate the
evolution of LGRs and their ligands.
In conclusion, we cloned and functionally expressed a nematode LGR homologous to mammalian glycoprotein hormone receptors. The nematode receptor showed constitutive activity when overexpressed in mammalian cells. While other LGRs have also been identified in lower organisms (sea anemone and fly), the nematode LGR is the first found to signal through a pathway similar to that of mammalian glycoprotein hormone receptors. Identification and functional characterization of the nematode LGR allows elucidation of the evolutionary relationship of this subfamily of GPCR with leucine-rich repeats as well as facilitation of future studies on the constitutive activation, structural-functional relationship, and physiology of this expanding subgroup of receptors.
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MATERIALS AND METHODS |
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Sequence Analysis
Based on amino acid sequences of mammalian glycoprotein hormone
receptors, the genomic sequence of the nematode LGR was identified
after searches of the high throughput genomic sequences in NCBI using
the tblastn program ofthe BLAST server (40). A cosmid genomic
clone (C50H2)on chromosome V of C. elegans was identified
to contain complete sequences of a putative LGR. A potential signal
peptide cleavage site was predicted using the SignalPprogram
(http://www.cbs.dtu.dk/services/SignalP/).
Theleucine-rich repeat motifs in the ectodomain of the LGR
were identified using the PRINTS library of protein
fingerprints(http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html).
Various protein sequence alignments, between either the nematode LGR
and individual glycoprotein hormone receptors or among multiple
receptor paralogs, were performed using the programs OMIGA and CLUSTALW
(http://www.hgsc.bcm.tmc.edu/SearchLauncher/) with comparable
outcomes.
Cloning of Nematode LGR cDNA and Construction of Expression
Plasmids for Wild-Type and Chimeric Receptors
Full-length cDNA was obtained by RT-PCR using C.
elegans total RNA (provided by Dr. Anne Villaneuve, Stanford
University) and specific primers that were predicted based on sequence
alignment with known LGRs. Two additional cDNA clones (yk420f1 and
yk429f6) kindly provided by Dr. Yuji Kohara of the National Institute
of Genetics (Mishima, Japan) were also used as templates to obtain
full-length receptor cDNA and to verify cDNA sequences. Both strands of
cDNAs from different PCRs were sequenced and found to be identical to
published genomic sequences. To facilitate the cell surface expression
of the nematode receptor in mammalian cells, its signal peptide was
replaced with the PRL signal peptide for secretion with or without
tagging with the FLAG M1 epitope as previously described (41). Amino
acid alignment of the tagged construct is: MDSKSS ... (PRL signal
peptide) ... QGVVS/DYKDDDD (FLAG M1 epitope)/VD/QNAL ...
(receptor sequence).
To study signal transduction by the nematode LGR, chimeric receptors containing fragments of nematode LGR and human LH receptor cDNA (42) were constructed using PCR-based mutagenesis (16). Furthermore, gain-of-function mutants of the human LH receptor were also generated to serve as positive controls. PCR was performed with Vent DNA polymerase (New England Biolabs, Inc., Beverly, MA) in accordance with manufacturers instructions. All cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen, San Diego, CA), and the plasmids were purified using the Maxi plasmid preparation kit (QIAGEN, Chatsworth, CA). Fidelity of PCR was confirmed by sequencing on both strands of the final constructs before use in expression studies.
Transfection of Cells and Analysis of Signal
Transduction
HEK 293T cells derived from human embryonic kidney fibroblast
were maintained in DMEM/Hams F-12 (DMEM/F12) supplemented with 10%
FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2
mM L-glutamine. Before transfection, 2 x
106 cells were seeded in 10-cm dishes (Nunc,
Naperville, IL). When cells were 7080% confluent, transient
transfection was performed using 10 µg of plasmid by the calcium
phosphate precipitation method (43) after replacement of culture media
with DMEM supplemented with 10% FBS, 100 µg/ml penicillin, 100
µg/ml streptomycin, and 2 mM L-glutamine.
After 1824 h incubation with the calcium phosphate-DNA precipitates,
media were replaced with DMEM/F12 with 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 24-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) before
treatment with or without hormones for 16 h. Transfection using
increasing amounts of plasmid in 12-well culture plates was also
performed as described above. Each well was transfected separately with
different amounts of plasmids. Forty-eight hours after transfection,
each well was washed once with D-PBS, replaced with DMEM/F12
supplemented with 1 mg/ml of BSA and 0.25 mM
IBMX, and incubated for 16 h.
Total cAMP in each well was measured in triplicate by specific RIA as previously described (44). 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 in a 10-cm dish. After washing cells three times with D-PBS, 2 x 105 cells 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 (15). All experiments were repeated at least three times using cells from independent transfection. To monitor transfection efficiency, 0.5 µg of RSV-ß-gal plasmid (45) was routinely included in the transfection mixture and ß-galactosidase activity in cell lysate was measured as previously described (46).
Ligand Binding Analysis
Human CG (CR-129) was iodinated by the lactoperoxidase method
(47) and characterized by radioligand receptor assay using recombinant
human LH receptors expressed in 293T cells. Specific activity and
maximal binding of the labeled hCG were 200,000 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, Organon, 100 IU/tube). At the end
of incubation, cells were centrifuged and washed twice with binding
assay buffer. Radioactivities in the pellets were determined in a
-spectrometer. Data from saturation binding studies were used to
derive equilibrium constant (Kd) values based on
Scatchard plot analysis.
Determination of Epitope-Tagged Receptor 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 (anti-mouse IgG
from sheep: 400,000 cpm) 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 radioactivities in the pellets using a
-spectrometer. Background binding was determined by adding excess
amounts of the synthetic FLAG peptide at a concentration of 100
µg/ml.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by NIH Grant HD-23273. SYH was supported by NIH training grant T32 DK-07217.
1 On sabbatical from Department of Obstetrics and Gynecology, Hokkaido
University School of Medicine, Sapporo, Japan.
2 On sabbatical leave from Department of Biochemistry and Cellular and
Molecular Biology, University of Tennessee, Knoxville, Tennessee
37996.
Received for publication August 16, 1999. Revision received November 11, 1999. Accepted for publication November 15, 1999.
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REFERENCES |
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