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Division of Reproductive Biology (S.Y.H., M.K., T.C. K.N.,
A.B., A.J.W.H.) Department of Gynecology and Obstetrics
Stanford University School of Medicine Stanford, California
94305-5317
Scientific Development Group (P.J. v.d.S.,
M.v.D.) N.V. Organon Oss, The Netherlands
5340
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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- and
specific ß- subunits, LH, FSH, and TSH are secreted by the
anterior pituitary whereas the human chorionic gonadotropin (hCG) is
secreted by placenta cells. These heterodimers bind specific plasma
membrane receptors on target cells and signal mainly through the
cAMP-dependent pathway. The receptors for these glycoprotein hormones
belong to the large G protein- coupled, seven-transmembrane protein
superfamily but are unique in having a large N-terminal extracellular
(ecto-) domain important for interaction with the large glycoprotein
hormone ligands (5, 6). Hallmarks of this subgroup of G protein-coupled
receptors (GPCRs) are the leucine-rich repeats in the ectodomain that
have been postulated to form a horseshoe-shaped interaction motif for
ligand binding (7, 8, 9). Recently, putative receptors homologous to the
mammalian glycoprotein hormone receptors were found in sea anemone (10, 11), nematode (12), pond snail Lymnaea stagnalis (13), and
Drosophila (14), suggesting that this subgroup of GPCR
evolved early during evolution and that these invertebrate
receptors represent ancient homologs of mammalian glycoprotein hormone
receptors. Based on the conserved sequences of mammalian glycoprotein hormone receptors and invertebrate homologs, we and others have recently isolated two novel mammalian leucine-rich repeat-containing, G protein-coupled receptors (LGRs) based on a homologous sequence search of the expressed sequence tags (ESTs) database (15, 16, 17). Because phylogenetic analysis showed that sea anemone LGR shares a closer relatedness to mammalian glycoprotein hormone receptors than to an LGR isolated from pond snail Lymnaea stagnalis (13, 15), one can predict that there are additional LGRs in mammalian genomes. Indeed, a recent search of the EST and genomic databases and subsequent characterization revealed that there are at least two additional mammalian LGRs. These two genes were named as LGR6 and LGR7 based on the chronological order of discovery. Analysis of primary sequences and domain arrangement in these LGRs showed that LGR6 is closely related to LGR4 and LGR5; whereas LGR7 and snail LGR are likely derived from a common ancestor. Together with the three known glycoprotein hormone receptors, these studies define the existence of three subgroups of LGRs in mammals. Based on the conserved mechanisms identified in constitutively activated LH and TSH receptors (18, 19, 20), studies of putative gain-of-function point mutants of LGR7 showed that this orphan receptor could mediate signaling through the protein kinase A-dependent pathway. Thus, site-directed mutagenesis of key residues in the functional domains of seven-transmembrane receptors provided a novel approach to reveal the signal transduction pathway of selective orphan GPCRs and to facilitate future identification of their cognate ligands.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Analysis of
multiple cDNA clones showed that the deduced methionine translation
start site is preceded with an in-frame stop codon at position -177.
Using a similar approach, two splicing variants of LGR7 were obtained
which differ in the N terminus of the coding region. The long form of
LGR7, LGR7(1), is 3759 nucleotides long and encodes an ORF of 757 amino
acids with a calculated molecular mass of 87 kDa; whereas the short
form of LGR7, LGR7(2), encodes an ORF of 723 amino acids with a
corresponding mass of 82.9 kDa (Fig. 2).
The LGR7(2) variant is 34 amino acids shorter than the LGR7(1), and
these two variants could be derived from alternative splicing events at
the N terminus of the coding region.
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). Among the 13 leucine-rich repeats of
LGR6, the first seven repeats align perfectly with leucine-rich repeats
1–3 and leucine-rich repeats 8–11 of LGR4 and LGR5, but the remaining
leucine-rich repeats showed disparity in these receptors. In addition,
distinctive sequence motifs, including a PYAYQCC motif and a GXFKPCE
motif in the hinge region of the ectodomain, and the highly conserved
cysteine residues for disulfide bond formation in extracellular loops 1
and 2, were completely conserved in LGR4, LGR5, and LGR6 (Fig. 1, A and
B). A motif search using the ScanProsite program showed that both LGR6
and LGR7 contain N-glycosylation sites at the ectodomain and that LGR7
carries a protein kinase C phosphorylation site at the intracellular
C-terminal sequences [STR, residues 750–752 of LGR7(1)]. Unlike LGR4
and LGR5 (15), both LGR6 and LGR7 lack protein kinase A phosphorylation
sites in their C termini.
Among all known LGRs, LGR7 shares the highest identity with snail LGR
(33%) (13) and less with the three mammalian glycoprotein hormone
receptors (24%). As shown in Fig. 2, LGR7 and snail (Ls) LGR shared
similar primary sequences and common domain arrangement as shown by the
presence of the N-terminal low density lipoprotein (LDL)
receptor cysteine-rich motif followed by leucine-rich repeats and the
seven transmembrane region. However, the predicted tertiary structure
of the LGR7 ectodomain differed from that of snail LGR; the ectodomain
of snail LGR is bulkier and contains approximately 760 amino acids
instead of the 410 amino acids found in LGR7(1) (Fig. 2A). In addition
to the 10 leucine-rich repeats at the C terminus of the ectodomain,
snail LGR contains 12 LDL-receptor cysteine-rich motifs at the N
terminus. In contrast, both LGR7 variants have only one such motif
[residue 40–62 of LGR7(1), CLPQLLHCNGVDDCGNQADEDNC] preceding the
conserved leucine-rich repeat domain. In addition, these two receptors
are distinct in their hinge region of the ectodomain. In LGR7 and snail
LGR, the hinge regions are approximately 30 amino acids long as
compared with 72–123 amino acids found in other LGRs. The unique
PYAYQCC and GXFKPCE motifs found in this region of other LGRs are
absent in these two receptors. Thus, the overall structural features of
LGR7 are similar to that of snail LGR.
Mammalian LGRs Have Diverged into Three Distinct Subtypes
Phylogenetic analysis using the neighbor-joining and parsimony
methods showed that the 11 known LGRs from vertebrates and
invertebrates can be divided into three distinct subgroups (Fig. 3A). The first subgroup contains the
mammalian gonadotropin and TSH receptors, and LGRs from sea anemone,
Caenorhabditis elegans, and Drosophila. The
second branch contains only vertebrate receptors including LGR4, LGR5,
and LGR6, whereas snail LGR and LGR7 belong to the third subgroup. To
gain insight into the evolution of LGRs, phylogenetic analysis was
performed together with diverse GPCRs with a polypeptide or
neurotransmitter ligand using full-length receptor sequences. As shown
in Fig. 3B, LGRs share a branch with diverse GPCRs known to have a
peptide ligand, including a subgroup of angiotensin receptor-like GPCRs
[angiotensin receptor, platelet-activating factor (PAF)
receptor, and formyl-methionyl-leucyl-phenylalanine (FMLP)
receptor] and another subgroup of bombesin receptor-like GPCRs
(bombesin, gastrin, thrombin, and neuropeptide Y receptors). In
contrast, the relatedness to other family 1 GPCRs (21), such as
receptors for various kinins, amine derivatives, and somatostatin
(SST), is more remote.
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). In addition, a minor
LGR6 transcript of >5.5 kb was also detected in the oviduct and heart,
whereas a smaller transcript was found in the spleen. Likewise, LGR7
transcripts were also found in multiple tissues. As shown in Fig. 4B, a
major LGR7 transcript of 5.5 kb was detected in all tissues tested with
the exception of spleen. Furthermore, multiple smaller
transcripts (2 or 3 kb) were detected in oviduct, uterus, colon,
and brain.
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, A and B,
LGR6 and LGR7 genes were localized to banded DNA in chromosome 1q32 and
4q32, respectively.
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As shown in Fig. 6, transfection of 293T
cells with increasing concentrations of the expression plasmid encoding
the mutant LGR7 receptors [LGR7(1) D637Y or LGR7(2) D603Y], similar
to those transfected with the plasmid encoding the D578Y
gain-of-function mutant LH receptor, resulted in dose-dependent
increases of basal cAMP production by transfected cells. In contrast,
cAMP levels in cells transfected with different amounts of wild-type LH
receptor or LGR7 did not show an increase in basal cAMP production. To
allow quantitative comparison of basal cAMP production by different
receptors, cAMP levels in transfected cells were normalized based on
the level of cell surface expression of an N-terminally tagged FLAG
epitope in different LGRs (Fig. 6, shown as percentage changes
vs. wild-type LH receptor). Although the D637Y mutation
caused significant increases of basal cAMP levels in both LGR7(1) and
LGR7(2) constructs, cells expressing mutant LGR7(1) consistently showed
greater levels of cAMP increase as compared with those expressing the
mutant LGR7(2) construct. To evaluate the specificity of the activating
LGR7 mutation for the Gs protein, the effect of this mutation on a
different signal transduction pathway, phosphatidyl inositol (PI)
turnover, was measured. As shown in Table 1, the Gs-activating mutants, LGR7(1)
D637Y and LGR7(2) D603Y, did not stimulate inositol phosphate (IP)
turnover by transfected 293T cells whereas hCG treatment significantly
increased the IP content in cells expressing either wild-type or
constitutively active mutant LH receptors.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Recent expansion of the nucleic acid sequence database for diverse organisms have provided opportunities to identify novel mammalian gene paralogs through pairwise sequence comparison (30). Although the physiological roles of most new genes are not known, alignment of their primary or secondary structures have allowed their preliminary grouping. Studies on the entire nematode genome indicated that the GPCR protein superfamily represents one of the most abundant signaling molecules that allows the cell to communicate with its environment (31, 32), and the GPCR family proteins could account for more than 1% of human genes. Among the known mammalian GPCRs, the glycoprotein hormone receptors represent a unique group of family 1 GPCRs with a large ectodomain for interaction with their ligands. Based on modeling with a prototypic leucine-rich repeat- containing polypeptide, ribonuclease inhibitor (8, 9), it was envisioned that the multiple leucine-rich repeats in the ectodomain of mammalian glycoprotein hormone receptors are arranged in a horseshoe-shaped binding motif and the inwardly ß-sheet structures interact with the specific ligand (7, 8, 9, 33). Unlike the glycoprotein hormone receptors with nine leucine-rich repeats, newly isolated mammalian LGRs contain varying numbers of repeats, indicating that the binding domain in these receptors could have the same configuration, but with distinct curvature and size. The glycoprotein hormones have been widely used in the treatment of diverse diseases; the current finding of new LGRs and the possibility of finding additional hormones as ligands for these orphan LGRs could reveal novel endocrine regulatory mechanisms.
The physiological and pathophysiological actions of glycoprotein hormone receptors are mediated mainly through interaction with the Gs protein. Recent characterization of constitutively activated LH and TSH receptors, based on patients with specific etiology (18, 19, 20, 25), has allowed analyses of structural requirements for signaling by these receptors. Taking advantage of these observations, mutant LGR7 constructs were made to explore the putative signaling pathways of LGR7. Similar to LH and TSH receptors, mutation of a single structure-determining amino acid in the transmembrane VI region of LGR7 caused constitutive signaling, as reflected by increases in basal cAMP levels in transfected cells. It has been proposed that mutations causing constitutive activation alter the receptor conformation from an inactive to an active state, mimicking the ligand stimulation of these receptors (34, 35, 36). Thus, the increase of basal cAMP production by mutant LGR7 receptors could be the result of conformational changes as found for constitutively active LH and TSH receptors. The present findings suggest that wild-type LGR7 may signal through the cAMP-protein kinase A pathway, a mechanism similar to the related glycoprotein hormone receptors. Although the Gs activating mutants LGR7(1) D637Y and LGR7(2) D603Y do not stimulate IP turnover by transfected 293T cells, it is important to note that the coupling of LGR7 to other G proteins cannot be excluded. Other examples of signaling by orphan GPCRs include a wild-type orphan ACCA (adenylate cyclase constitutive activator) receptor found to constitutively activate adenylate cyclase in transfected cells (37).
Recently, endogenous ligands for several orphan receptors have been isolated by monitoring the stimulation of downstream signaling transduction (38, 39, 40, 41, 42, 43, 44). With no knowledge of the G protein-signaling mechanism, several of these studies were made possible by coexpressing the orphan receptor with chimeric G proteins or G proteins that are promiscuous in receptor coupling. The present study demonstrates that mutagenesis of key residues in the transmembrane domain of GPCR is a novel approach to characterize signaling pathways for orphan GPCRs. While the present study demonstrated that mutagenesis of conserved residues is a useful approach for the characterization of signaling by the orphan LGR7, previous studies indicated that an FSH receptor mutant equivalent to D578Y in the LH receptor does not lead to receptor activation (27), suggesting this approach is only applicable to selective orphan GPCRs.
Because chimeric receptors with the ectodomain and transmembrane region from different glycoprotein hormone receptors have been shown to be functional (5, 27), we have attempted to characterize the potential coupling of LGR6 to Gs or Gq pathways by fusing the transmembrane region of LGR6 with the ectodomain of the LH receptor in a chimeric construct. Although the chimeric receptor could be expressed on the cell surface, no activation of the chimeric receptor by the LH receptor ligand was observed. Previous studies on similar chimeric receptors containing the ectodomain domain of the LH receptor and the transmembrane domain of LGR4 or LGR5 also suggest that such chimeric receptors do not react to ligand stimulation (15). Thus, either the ectodomain of the LH receptor and the transmembrane region of these orphan receptors are incompatible, or they could signal through other unknown mechanisms. Indeed, a similar construct of the LH receptor ectodomain plus the LGR7 transmembrane domain failed to allow expression of the chimeric receptor on the cell surface of transfected cells.
The newly identified LGR7 shows close sequence homology with the only known snail LGR (13). Interestingly, the putative ligand-binding domains of these two receptors appear to have diverged. The most obvious difference is the number of LDL receptor cysteine-rich motifs in the N terminus of these two receptors. In the ectodomain of snail receptor, there are 12 LDL receptor cysteine-rich motifs, each encoding three conserved cysteine residues (13, 45) important for disulfide bond formation and ligand binding (46). Unlike snail LGR, LGR7 contains only one typical LDL receptor cysteine-rich motif in its N terminus. Assuming the motifs in these two related receptors comprise part of their ligand-binding domain, the respective ligands for these two receptors could have diverged during evolution. Future studies on the LGR7 gene could reveal additional LGR7 variants that are closer to snail LGR, and uncover the evolutionary relationship of these two receptors.
Analysis of LGR6 sequences showed that LGR6 belongs to a subgroup of LGRs which includes LGR4 and LGR5, suggesting that these receptors may share similar ligand binding and signal transduction characteristics. However, the ectodomain of LGR6 is unique and contains only 13 leucine-rich repeats instead of the 17 repeats found in LGR4 and LGR5. It is unclear whether the cloned LGR6 cDNA is the only transcript encoded by the LGR6 gene. Observed differences in the number of leucine-rich repeats in the ectodomain of these receptors may be the result of alternative splicing. Recent studies on GPCRs have shown that within a given subfamily functional diversity is most often conferred by the existence of multiple receptor subtypes, each encoded by a distinct gene. Additional diversity results from alternative splicing of a given gene to form receptor variants (47). Indeed, our unpublished results showed that the LGR4 gene encodes multiple splicing variants, including one with only 14 leucine-rich repeats. Likewise, glycoprotein hormone receptor genes also encode multiple splicing variants with distinct functional characteristics (48), and two splicing variants of LGR7 were isolated here. Alternative splicing of these receptors, especially in the ectodomain, could result in alternative binding characteristics or specificity.
Sequence alignment showed that the three subgroups of LGRs could be distinguished merely by the amino acid sequences in their hinge regions between leucine-rich repeats and the transmembrane domain. In the glycoprotein hormone receptors, this region is flanked by the conserved YPSHCC and DXFNPCED motifs whereas LGR4, LGR5, and LGR6 contain YAYQCC and GXFKPCEX sequences in the corresponding regions, respectively. In contrast, these two motifs are absent in LGR7. Interestingly, recent studies on the TSH receptor have shown that point mutation of the serine residue in the conserved YPSHCC motif resulted in constitutive activation of the TSH receptor, leading to severe congenital hyperthyroidism in patients (49, 50, 51). Because LGR4, LGR5, and LGR6 share similar structural determinants in the hinge region, investigations on this region of the orphan receptors could provide insights toward the activation mechanisms of different LGRs.
Analysis using 11 known LGRs from diverse organisms indicated that LGRs from sea anemone, nematode, and Drosophila grouped under a single branch with mammalian glycoprotein hormone receptors while the newly isolated LGR4, LGR5, LGR6, and LGR7 diverged early during evolution. Assuming the evolutionary pressures on these receptors are constant, the sequence divergence in mammalian LGRs suggests that the ancestral gene giving rise to modern LGRs could have evolved before the emergence of cnidarians for cell-cell communication. Also of interest, analysis of the completely sequenced C. elegans genome showed that this nematode contains only one GPCR with LGR characteristics (12), suggesting a possible gene loss during the evolution of modern nematodes and that different LGRs in present day organisms evolved to serve adaptive functions in different phylogenies.
Findings of multiple LGRs allow a better comparison of the relatedness of the LGR subfamily with other GPCRs in the superfamily. Previous studies have shown that known GPCRs can be divided into six major families with distinct evolutionary origins, and the majority of GPCRs with a peptide or neurotransmitter ligand belong to family 1 (21). Phylogenetic analysis of LGRs with other GPCRs in family 1 indicated that LGRs belong to a distinct branch and share the closest relatedness with a subgroup of GPCRs including receptors for bombesin, gastrin, thrombin, neuropeptide Y, angiotensin, PAF, and FMLP. This classification would allow a better understanding of the ligand signaling mechanisms for these receptors through comparison of their structure-function relationship.
In conclusion, we have cloned two novel mammalian LGRs (LGR6 and LGR7) and identified the putative signal transduction pathway for LGR7. The constitutive activation of cAMP production by the mutant LGR7 suggests that this orphan receptor could signal through the cAMP-dependent pathway. This study represents the first demonstration of the elucidation of the signaling mechanism for an orphan GPCR based on single-point mutations to allow constitutive activation of the protein. The present study also defines the existence of three subclasses of leucine-rich repeat-containing GPCRs in the human genome and possibly other metazoans. Identification and functional characterization of these novel LGRs allow elucidation of the evolutionary relationship of this subfamily of GPCRs with leucine-rich repeats. It also facilitates future studies on the physiology of this expanding subgroup of GPCRs and the identification of their cognate ligands.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Computational Analysis
cDNA sequences related to invertebrate LGRs and mammalian
glycoprotein hormone receptors were identified from the EST and genomic
survey sequences (GSS) database at the National Center for
Biotechnology Information and an Incyte EST database using the
BLAST and Gapped BLAST server with the BLOSUM62 comparison matrix (52).
Initially, four independent human DNA entries (three ESTs and one GSS
AQ053279) were found to encode sequences homologous, but not identical,
to the known mammalian LGRs. Based on these sequences, RACE experiments
were used to clone the full-length cDNA sequences. After RACE, the four
original DNA entries were found to encode different portions of two
novel LGRs. The alignment of primary sequences for genes in the LGR
family was carried out by the Blocks WWW server
(http://blocks.fhcrc.org/blocks/blockmkr). The Block Maker program also
calculated the branching order and phylogenetic relatedness of aligned
sequences by the Cobbler and Gibbs algorithms. To compare the
phylogenetic relationship of LGRs with diverse peptide and
neurotransmitter GPCRs, neighbor-joining
(http://www.biophys.kyoto-u.ac.jp/maketree2.html) and parsimony methods
(Phylogeny Inference Package) were used. In all studies, the
full-length amino acid sequences of different GPCRs were used for
phylogenetic analyses to provide the maximum possible information
within families and subfamilies.
Additionally, the analyses of primary and secondary structures of these novel LGRs were conducted using the BCM search launcher (http://gc.bcm.tmc.edu:8088/search-launcher/launcher.html), Biology Workbench (http://gc.bcm.tmc.edu:8088/search-launcher/launcher.html), Blocks WWW server (http://www.blocks.fhcrc.org/blocks/), the eMotif maker server (http://dna.stanford.edu/emotif/), or the ExPASy Molecular Biology Server (http://expasy.hcuge.ch/).
Identification and Isolation of the Full-Length cDNAs for LGR6
and LGR7
For the isolation of full-length cDNA fragments, specific
primers with a design based on EST sequences were used to prepare cDNA
pools enriched with the candidate cDNAs derived from human ovary and
testis mRNA. Two micrograms of mRNA were reverse transcribed by using
25 U of avian myoblastosis virus reverse transcriptase with
oligo(dT) primer, 0.5 mM deoxynucleoside
triphosphate (dNTP), and 20 U of RNAse inhibitor. After second
strand synthesis with T4 DNA polymerase, the cDNA pool was tailed at
both ends with adaptor sequences to allow PCR amplification using
specific primers. The tailed cDNA products were then employed as a
template for 5'- and 3'-RACE with adaptor- and gene-specific
primers. All PCR amplifications were performed under highly stringent
conditions (annealing temperature >67 C) using Advantage DNA
polymerase (CLONTECH Laboratories, Inc., Palo Alto,
CA) or Pfu DNA polymerase (Stratagene, La Jolla, CA) to
minimize mismatching and infidelity during PCR amplification. PCR
products were fractionated using agarose electrophoresis, and specific
bands showing hybridization with radiolabeled cDNA probes were
subcloned into the pUC18 vector (Invitrogen, San Diego,
CA) to further identify candidate clones. The PCR products were
phenol/chloroform-extracted, precipitated with ethanol, phosphorylated
with T4 polynucleotide kinase, and blunt-ended with the Klenow enzyme.
The PCR products were then subcloned into the SmaI site in
the pUC18 vector. At least two independent PCR clones were sequenced to
verify the authenticity of the coding sequences of each cDNA. The
resulting sequences were assembled into contigs using the Blast2
sequences server (http://www.ncbi.nlm.nih.gov/gorf/bl2.html) and
ClustalW 1.7 at the BCM Search Launcher
(http://dot.imgen.bcm.tmc.edu:9331/multi-align). After the initial
round of RACE, it was determined that the multiple homologous sequences
belong to two independent LGR genes, LGR6 and LGR7. The full-length
sequences were obtained and confirmed after three rounds of RACE.
Northern Blot Analysis
For mRNA analysis, membranes containing poly(A)+ RNA from
various rat tissues were hybridized with specific cRNA probes. The rat
mRNAs were extracted from 27-day-old rats using Trizol solution
(Life Technologies, Inc., Gaithersburg, MD) followed by
the Oligotex mRNA purification columns (QIAGEN Inc.
Chatsworth, CA) to select for poly(A)+ RNA. The cRNA probes were
synthesized using a Riboprobe Combination System (Promega Corp., Madison, WI). For hybridization using cRNA probes,
membranes were prehybridized for 1 h at 60 C in the ExpressHyb
solution (CLONTECH Laboratories, Inc.). This was followed
by hybridization under the same conditions for 2 h but with 1
x 106 cpm/ml of
32P-labeled LGR6 or LGR7 cRNA probes. After
hybridization, the membranes were washed twice in 0.2 x sodium
chloride/sodium citrate (SSC), 0.5% SDS at 60 C, followed by
two washes under high stringency conditions (0.1 x SSC, 1% SDS
at 75 C) before exposure to RX film (Eastman Kodak Co.,
Rochester, NY) with intensifying screens (Amersham Pharmacia Biotech, Buckinghamshire, UK). To monitor the loading of mRNA
samples from different tissues, membranes were stripped and
rehybridized with a 32P-labled ß-actin cDNA
probe. The cDNA probe was generated by random priming (Life Technologies, Inc.). For hybridization using cDNA probes,
membranes were washed to a stringency of 0.1 x SSC, 1% SDS at 60
C.
Expression of LGR6 and LGR7 in Mammalian Cells
Wild-type and mutant LGR6 and LGR7 cDNAs were constructed by
sequential PCR amplification and standard restriction digest and
ligation procedures. To allow efficient targeting of receptors to the
cell surface and immunodetection in vitro, a lead cDNA
sequence containing a PRL signal peptide for cell surface expression
(MNIKGSPWKGSLLLLL-VSNLLLCQSVAP) and a FLAG (DYKDDDDK)
epitope were added to the N terminus of the mature region of LGRs in
all expression constructs (53). To construct chimeric LH/LGR6 and
LH/LGR7 receptors, junctional amino acid sequences were designed to be
CAPEPPDAFN/PCEYLFESWGIRL and CAPEPPDAFN/SCEDLMSNHVLRVS, respectively.
For expression in eukaryotic cells, the receptor cDNAs were subcloned
into the eukaryotic cell expression vector pcDNA3.1 Zeo
(Invitrogen, San Diego, CA), and the plasmids were
purified using the Maxi plasmid preparation kit
(QIAGEN, Inc.). Each construct was sequenced on both
strands using vector-derived primers and gene-specific primers before
use in transfection experiments for the analyses of signal transduction
and/or receptor binding.
Mammalian 293T cells derived from human embryonic kidney fibroblast were maintained in DMEM/Ham’s F-12 (Life Technologies, Inc.) supplemented with 10% FBS, 100 µg/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The cells were transfected with expression plasmids using the calcium phosphate precipitation method (54). After 18–24 h of incubation with the calcium phosphate-DNA precipitates, media were replaced with DMEM/F12 containing 10% FBS. Forty-eight hours after transfection, cells were washed twice with Dulbecco’s PBS (D-PBS), harvested from culture dishes, and centrifuged at 400 x g for 5 min. Cell pellets were then resuspended in DMEM/F12 supplemented with 1 mg/ml of BSA. Cells (2 x 105/ml) were placed on 12-well tissue culture plates (Corning, Inc. Corning, NY) and preincubated at 37 C for 30 min in the presence of 0.25 mM 3-isobutyl-1-methyl xanthine (IBMX; Sigma) to prevent hydrolysis of cAMP before hormonal treatment for 16 h. To study basal cAMP production mediated by increasing numbers of receptors, each well was transfected separately with different amounts of the expression plasmid. To detect signaling by the wild-type and mutated LGRs, levels of cAMP production by transfected cells were measured by specific RIA using [125I] cAMP (Amersham Pharmacia Biotech) (27). Cells transfected with the empty plasmid (mock) were routinely used as negative controls. At the end of incubation, cells and medium in each well were frozen and thawed once before heating at 95 C for 3 min to inactivate phosphodiesterase activity. Total cAMP in each well was measured in triplicate. For IP measurement, transfected cells were labeled for 24 h with myo-[3H]-inositol at 4 µCi/ml in inositol-free DMEM supplemented with 5% FBS. After washing three times with D-PBS, 2 x 105cells were preincubated for 30 min in D-PBS containing 20 mM LiCl, and treated with or without hormones at 37 C for 1 h. Total IPs were extracted and separated as previously described (29). All experiments were repeated three times using cells from independent transfections. To monitor transfection efficiency, 0.5 µg of RSV-ß-gal plasmid was routinely included in the transfection mixture, and the ß-galactosidase activity in the cell lysate was measured as previously described (55). Statistical analysis was performed using Student’s t test.
Radioligand Binding Assays
For ligand binding analysis of the wild-type and mutant
receptors, human CG (CR-129) was iodinated by the lactoperoxidase
method (56) and characterized by a radioligand receptor assay using
human LH receptors stably expressed in 293T cells. Specific activity
and maximal binding of the labeled hCG were 100,000–150,000 cpm/ng and
40–50%, respectively. To estimate ligand binding to the cell surface,
transfected cells were washed twice with D-PBS and collected in D-PBS
before centrifugation at 400 x g for 5 min. Pellets
were resuspended in D-PBS containing 1 mg/ml BSA (binding assay
buffer). Resuspended cells (2 x 105/tube)
were incubated with increasing doses (or a saturating dose) of labeled
hCG at room temperature for 22 h in the presence or absence of
unlabeled hCG (Pregnyl, 100 IU/tube; Organon, West Orange,
NJ). At the end of incubation, cells were centrifuged and washed
twice with the binding assay buffer. Radioactivity in the pellets was
determined with a 
-spectrometer (53).
Determination of FLAG Epitope-Tagged Receptors on the Cell
Surface
Transfected cells were washed twice with D-PBS, and resuspended
cells (2 x 106/tube) were incubated with
FLAG M1 antibody (50 µg/ml) in Tris-buffered saline (pH 7.4)
containing 5 mg/ml BSA and 2 mM CaCl2
(assay buffer) for 4 h at room temperature in siliconized
centrifuge tubes. Cells were then washed twice with 1 ml of assay
buffer after centrifugation at 14,000 x g for 15 sec.
The 125I-labeled second antibody (antimouse IgG
from sheep: 
400,000 cpm/tube) was added to the resuspended cell
pellet and incubated for 1 h at room temperature. Cells were again
washed twice with 1 ml of assay buffer by repeated centrifugation
before determination of radioactivity in cell pellets. Background
binding was determined by adding excess amounts of the synthetic FLAG
peptide at a concentration of 100 µg/ml.
Genomic Analysis and Chromosomal Localization of LGR6 and
LGR7
To isolate genomic clones for LGR6, a human bacterial artificial
chromosome (BAC) genomic DNA library was screened using the
transmembrane region of LGR6 cDNA as a probe. The LGR7 genomic clone
was identified by a sequence search of the GSS database and obtained
from Genome Systems. These genomic fragments were then
confirmed by Southern blot hybridization. For the identification of the
chromosomal localization of LGR6 and LGR7 genes, genomic fragments
(>50 kb) were used as probes for FISH of human metaphase chromosomes.
Denatured chromosomes from synchronous cultures of human lymphocytes
were hybridized with biotinylated probes for signal localization.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This study was supported by NIH Grant HD-31566. S.Y.H. was supported by NIH Training Grant T32 DK-07217. The GenBank accession numbers for LGR6 and LGR7 are AF190501 and AF190500, respectively.
1 On sabbatical leave from the Department of Biochemistry and Cellular
and Molecular Biology, The University of Tennessee, Knoxville,
Tennessee 37996. 
Received for publication November 9, 1999. Revision received May 4, 2000. Accepted for publication May 9, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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