Characterization of Two LGR Genes Homologous to Gonadotropin and Thyrotropin Receptors with Extracellular Leucine-Rich Repeats and a G Protein-Coupled, Seven-Transmembrane Region

Sheau Yu Hsu1, Shan-Guang Liang1 and Aaron J. W. Hsueh

Division of Reproductive Biology Department of Gynecology and Obstetrics Stanford University Medical School Stanford, California 94305-5317


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane (TM) protein family and are unique in having a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interaction with the glycoprotein ligands. We have identified two new leucine-rich repeat-containing, G protein-coupled receptors and named them as LGR4 and LGR5, respectively. The ectodomains of both receptors contain 17 leucine-rich repeats together with N- and C-terminal flanking cysteine-rich sequences, compared with 9 repeats found in known glycoprotein hormone receptors. The leucine-rich repeats in LGR4 and LGR5 are arrays of 24 amino acids showing similarity to repeats found in the acid labile subunit of the insulin-like growth factor (IGF)/IGF binding protein complexes as well as slit, decorin, and Toll proteins. The TM region and the junction between ectodomain and TM 1 are highly conserved in LGR4, LGR5, and seven other LGRs from sea anemone, fly, nematode, mollusk, and mammal, suggesting their common evolutionary origin. In contrast to the restricted tissue expression of gonadotropin and TSH receptors in gonads and thyroid, respectively, LGR4 is expressed in diverse tissues including ovary, testis, adrenal, placenta, thymus, spinal cord, and thyroid, whereas LGR5 is found in muscle, placenta, spinal cord, and brain. Hybridization analysis of genomic DNA indicated that LGR4 and LGR5 genes are conserved in mammals. Comparison of overall amino acid sequences indicated that LGR4 and LGR5 are closely related to each other but diverge, during evolution, from the homologous receptor found in snail and the mammalian glycoprotein hormone receptors. The identification and characterization of new members of the LGR subfamily of receptor genes not only allow future isolation of their ligands and understanding of their physiological roles but also reveal the evolutionary relationship of G protein-coupled receptors with leucine-rich repeats.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Proteins in the large seven-transmembrane (TM), G protein-coupled receptor (GPCR) superfamily are functionally diverse and include receptors ranging from the cAMP receptor in slime mold to mammalian neurotransmitter and glycoprotein hormone receptors (1, 2, 3, 4, 5). Agonist occupancy of these plasma membrane proteins leads to the activation of different G proteins, which in turn modulate the activity of different effector enzymes and ion channels (6, 7). Gonadotropins (LH, FSH, CG) and TSH are essential for the growth and differentiation of the gonads and thyroid gland, respectively. These glycoprotein hormones bind specific membrane-bound GPCRs on target cells to activate the Gs-cAMP-protein kinase A pathway (8, 9, 10, 11). The glycoprotein hormone receptors represent a subgroup of GPCRs that have a large N-terminal extracellular (ecto-) domain containing leucine-rich repeats important for interaction with glycoprotein hormones from adenohypophysis and placenta, which leads to cAMP production in target cells.

Based on the conserved sequences of putative glycoprotein hormone receptors in Drosophila and sea anemone (12, 13), the expression sequence tags (EST) in the GenBank were searched, and fragments of two new mammalian receptors in this subfamily of leucine-rich repeat-containing, G-protein-coupled receptor (LGR) were identified.2 We report here the molecular cloning of these putative mammalian receptors with a protein architecture that is similar to the known glycoprotein hormone receptors and their invertebrate homologs in both ectodomains and TM segments. In addition to the three known receptors, the ectodomains of LGR4 and LGR5 show high homology with the acid labile subunit (ALS) (14, 15, 16), slit (17), decorin (18), and Toll proteins (19) containing leucine-rich repeats, suggesting a common evolutionary origin. In contrast to the restricted tissue expression pattern of known gonadotropin and TSH receptors, these new receptors were found in multiple tissues. Identification of this expanding family of LGRs has implications for future studies to identify putative ligands for these orphan receptors and for the understanding of the evolutionary origin of proteins in this expanding subfamily of leucine-rich repeat-containing seven-TM receptors.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Conserved Architecture of Ectodomain, TM Region, and C-Terminal Tail of LGR4 and LGR5
Human sequences related to the sea anemone and Drosophila glycoprotein hormone receptors (12, 13) were identified from the EST database based on their similarities to receptors found in the lower species and nonidentity to the three known human glycoprotein hormone receptors. The full-length cDNAs for novel receptors were isolated using RT-PCR and repeated screening of sublibraries from rat ovary or human placenta enriched with each receptor cDNA. Positive clones with long inserts were sequenced and aligned to identify the open reading frames (ORFs) of individual receptors. The prototypic LGR consists of an ectodomain with leucine-rich repeats and a C-terminal half with seven-TM domains similar to other GPCRs. Because three known glycoprotein hormone receptors have the same leucine-rich repeat-containing ectodomain and G protein-coupled TM region, the new mammalian receptors were named LGR4 and LGR5, respectively.

LGR4 cDNA from rat ovary consists of 3,504 bp with a predicted ORF of 951 amino acids, whereas LGR5 from human placenta has 4,208 bp with a 907-amino acid ORF (Fig. 1Go). 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. 1CGo, Table 1Go, and Ref. 18). Six and four consensus N-linked glycosylation sites (Fig. 1CGo, underlined N; and Table 1Go) were found in the ectodomains of LGR4 and LGR5, respectively, and two of these sites were conserved between LGR4 and LGR5.





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Figure 1. Comparison of Deduced Amino Acid Sequence of LGR4 and LGR5 cDNAs and Those Encoding FSH, LH, and TSH Receptors and Other Leucine-Rich Repeat-Containing Proteins

Sequence alignment for different regions of LGR4, LGR5, and three human glycoprotein hormone receptors were performed. Heavily shaded residues are identical in at least four of the five receptor proteins shown, whereas the lightly shaded residues show identity in LGR4 and LGR5. Amino acid numbers are shown on the right. Asparagine residues in the potential N-linked glycosylation sites are underlined and shown in bold. Gaps introduced for optimal alignment are indicated as dashes. A, Signal peptide. B, N-flanking cysteine-rich sequence. C, Leucine-rich repeats. Seventeen leucine-rich repeat sequences in LGR4 and LGR5 are indicated by arrows at the top of the sequences. Because there are only nine repeat sequences in the three glycoprotein hormone receptors, they were arbitrarily aligned with the first nine repeats in LGR4 and LGR5. D, C-flanking cysteine-rich sequence. E, TM region. Seven putative TM (TM) domains are indicated at the top of the sequence. Intracellular loops (IL) and outside loops (OL) are also indicated. F, C-terminal tail. Conserved protein kinase A phosphorylation sites are underlined. The GenBank accession numbers for LGR4 and LGR5 are AF061443 and AF061444, respectively. G, Comparison of leucine-rich repeats found in LGRs and diverse other proteins with typical type leucine-rich repeats. Consensus leucine-rich repeats in LGR4 and LGR5 were compared with those found in three human glycoprotein hormone receptors (LHR/FSHR/TSHR) and LGRs from lower species. In addition, the leucine-rich repeats of several other secretory proteins (ALS of IGF/IGF binding protein complexes, slit, and decorin) and single-TM domain receptors (Toll and Tartan) (36 62 ) with homologous repeats are shown. The number of repeats found in each protein is shown in parenthesis, whereas consensus amino acid residues are shaded. Uppercase letters indicate more than half of the leucine-rich repeats found in a given protein are conserved, whereas lowercase letters denote less than 50% consensus. a, Aliphatic residues, c, charged residues; r, rat; h, human; Ae, Anthopleura elegantissima (sea anemone); Ce, Caenorhabditis elegans (nematode); Dm, Drosophila melanogaster (fly); Ls: Lymnaea stagnalis (snail).

 

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Table 1. Structural Features of LGR4 and LGR5 in Comparison with Human Gonadotropin and TSH Receptors

 
Although there are 17 leucine-rich repeats in LGR4 and LGR5 as compared with 9 repeats in the glycoprotein hormone receptors, alignment of the N-terminal 9 repeats of the five mammalian LGRs showed that the third potential N-glycosylation site in LGR4 (Asn 199) and the second potential N-glycosylation site in LGR5 (Asn 208) align perfectly with the N-glycosylation site found in the sixth repeat of gonadotropin and TSH receptors. In addition, clusters of cysteines (cysteine-rich sequences) are present in the N-terminal region and the junction between the ectodomain and TM 1. Because the four N-flanking cysteine residues are conserved in all of the mammalian LGRs and other leucine-rich repeat proteins (Fig. 1BGo), 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. 1DGo). 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 {alpha}-helices (SOUSI server, www.tuat.ac.jp/cgi/~mitaku/& NAKAI server, http://psort.nibb.ac.jp/cgi-bin; Fig. 1EGo). 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. 1FGo), 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. 1FGo, 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. 1Go, 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. 1GGo). 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. 1GGo). 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. 1GGo). 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. 2AGo, 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. 2BGo). A transcript of 4.3 kb, together with a minor transcript of 2.4 kb for LGR5 mRNA, was found to be highest in the skeletal muscle. This transcript is also present in placenta, spinal cord, brain, adrenal, colon, stomach, and bone marrow.



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Figure 2. Expression Pattern of LGR4 and LGR5 mRNA Transcripts in Different Tissues

For Northern blot analysis, 2 µg of poly (A)+-selected RNA from different human tissues were probed with a 32P-labeled LGR4 or LGR5 cDNA probe at 60 C. After washing under stringent conditions, the blots were exposed to x-ray films with an intensifying screen at -80 C for 7 days. Subsequent hybridization with a ß-actin cDNA probe was performed to estimate nucleic acid loading (8 h exposure; data not shown). A, LGR4 Northern blot. B, LGR5 Northern blot. Specific LGR transcripts are indicated by arrows. Pa, Pancreas; Ki, kidney; Mu, skeletal muscle; Li, liver; Lu, lung; Pl, placenta; Br, brain, He, heart; Co, colon; In, small intestine; Ov, ovary; Te, testis; Pr, prostate; Th, thymus; Sp, spleen; Bm, bone marrow; Ad, adrenal; Tr, trachea; ly, lymph node; Si, spinal cord; Ty, thyroid; St, stomach.

 
Lack of Gs Stimulation Mediated by Chimeric Receptors Comprising the Ectodomain of LH Receptor and the TM Region of LGR4 or LGR5
Because chimeric receptors among the known glycoprotein hormone receptors have been successfully used to study signal transduction by these proteins (29), cDNAs for chimeric receptors were generated by fusing the ectodomain of human LH receptor with the TM region and C-terminal tail of either LGR4 or LGR5 [named as L(EC)LGR4(TM) and L(EC)LGR5(TM), respectively]. Cells transfected with the plasmid encoding L(EC)LGR4(TM) showed moderate binding to labeled human (h)CG with a dissociation constant (Kd) value similar to that of the wild-type LH receptor whereas cells transfected with the plasmid encoding L(EC)LGR5(TM) showed lower binding but with high affinity. The Bmax (ng hCG bound/105 cells) and Kd (pM) values for different receptors are: LH receptor, 4.6±3.3 and 195±99; L(EC)LGR4(TM), 2.5±0.8 and 183±114 and L(EC)LGR5(TM), 0.46±0.28 and 549±206, respectively. Despite detectable hCG binding, treatment with increasing doses of hCG did not increase cAMP production by either one of the chimeric receptors. At 10 µg/ml hCG, a 62-fold increase of cAMP production was mediated by the wild type LH receptor but no stimulation of cAMP was found in cells expressing either chimeric receptors (P < 0.05).

Isolation of LGR4 and LGR5 Genes, Their Conservation in Vertebrates, and Chromosomal Localization in Humans
Using LGR4 and LGR5 cDNA fragments as probes, a bacterial artificial chromosome-based human genomic DNA library was screened and several genomic clones for LGR4 and LGR5 were isolated. To assess the conservation of the LGR4 and LGR5 genes in diverse vertebrates, Southern blot hybridization of genomic DNA from different species was performed. Under medium stringency washing conditions, the rat LGR4 cDNA and human LGR5 cDNAs hybridized with genomic DNA from all mammalian species tested, suggesting that both LGR4 and LGR5 genes are conserved during mammalian evolution (Fig. 3AGo).



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Figure 3. Conservation of LGR4 and LGR5 Genes in Diverse Vertebrate Species and Their Chromosomal Localization in Human Cells

A, Southern blot hybridization of genomic DNA isolated from different vertebrate species was performed using LGR4 and LGR5 cDNA probes. Four micrograms of genomic DNA was digested with the EcoRI restriction enzyme and probed with LGR4 or LGR5 cDNA. After hybridization at 60 C, the membrane was washed under medium stringency conditions (0.5% SDS, 0.2 x SSC at 60C) before exposure. B, Using DNA fragments of bacterial artificial chromosome containing human LGR4 and LGR5 genes as probes, chromosomal localization of LGR4 and LGR5 was detected using the FISH method to chromosome 5q34–35.1 and 12q15, respectively. Denatured chromosomes from synchronous cultures of human lymphocytes were hybridized with biotinylated probes for localization. Assignment of the FISH mapping data (left) was achieved by superimposing signals with 4,6-diamdino-2-phenylindole-banded chromosome (center). Analyses are summarized in the form of human chromosome ideograms (right). Upper panel, LGR4; lower panel, LGR5.

 
Genomic fragments (>100 kb) of LGR4 and LGR5 were used as probes in fluorescence in situ hybridization (FISH) analysis to identify the chromosomal localization of LGR 4 and LGR5 genes. As shown in Fig. 3BGo, 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. 4AGo, 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. 4AGo). Consensus secondary structure analysis of these receptors further indicated that, in addition to the seven {alpha}-helical membrane-spanning domains, one unique ß-strand structure could be found in the outside loop 2. Of interest, this region has been shown to be important for the modulation of hormone binding of LH receptor (33); conservation of the secondary structure in this region suggests the outside loop 2 may have a similar role in LGR4 and LGR5.




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Figure 4. Comparison of Protein Architectures of Mammalian LGRs and Their Relationship to Similar Receptors Found in Sea Anemone, Nematode, Fly, and Snail

A, Sequence comparison of the highly conserved TM regions of LGRs from diverse species [sea anemone: Ae, Anthopleura elegantissima; nematode: Ce, Caenorhabditis elegans; fly: Dm, Drosophila melanogaster; snail: Ls, Lymnaea stagnalis; rat (r) and human (h)]. Based on BLOCK Maker analysis, four highly conserved ungapped blocks were identified in the TM and flanking sequences of nine LGRs from diverse species. Boxed areas are sequence blocks with multiply aligned ungapped segments corresponding to the most highly conserved regions of proteins. Secondary structure predictions ({alpha}-helices: curved arrows; ß- strand: straight arrow) below the sequences were derived based on the PHD algorithm and DSC (60 61 ). Chemically similar residues are lightly shaded whereas conserved charged residues are heavily shaded. Consensus residues represent identity among at least five of the nine receptors, B, Phylogenetic relatedness of LGRs from diverse species. Based on sequence comparison of the entire receptor proteins, LGRs can be divided into three subgroups: one containing the snail LGR, one containing mammalian LGR4 and LGR5, and a third one containing human gonadotropin and TSH receptors together with LGRs from fly, nematode, and sea anemone.

 
Further analysis of the phylogenetic relatedness of nine LGRs from diverse species, based on either the full-length receptor sequence (Fig. 4BGo) 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. 4BGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The mammalian LGR family of proteins comprises at least five gene products: LH and FSH receptors essential for gonadal development; TSH receptor for thyroid differentiation; and the two new orphan LGR4 and LGR5 without known ligands (Table 1Go). 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 {alpha}-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 {alpha}-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. 3BGo). 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. 5Go) (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 {alpha}-helical motifs of largely hydrophobic residues. An ancestral gene with leucine-rich repeats could evolve into genes with different functions through gene rearrangement. Drosophila slit represents a fusion of leucine-rich repeat domains with an epidermal growth factor domain, whereas genes of the Toll family are derived from the fusion of leucine-rich repeats to the interleukin-1 receptor-like motif (19, 36). The LGR family of proteins represents the fusion of the leucine-rich repeats with an ancestral GPCR. Although closely related to different LGRs, the GRL101 gene found in the central nervous system of snail is unique and may represent a fusion of low-density lipoprotein-binding motifs and leucine-rich repeats together with the seven-TM region (31).



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Figure 5. Hypothetical Model on the Evolution of Diverse Genes Containing Leucine-Rich Repeats

Gene duplication and recombination probably account for the evolution of LGRs and related leucine-rich repeat-containing proteins. The motifs containing leucine-rich sequences or TM domains are the basic units to build modules of leucine-rich repeats and seven-TM helices through gene duplication. Fusion of ancestral leucine-rich repeat modules with epidermal growth factor (EGF) domain or interleukin (IL) 1 receptor (R)-like domain led to the formation of secreted slit protein and Toll receptors (R), respectively. In contrast, fusion of leucine-rich repeats with seven-TM modules led to the evolution of LGRs, whereas the fusion of leucine-rich repeats with seven-TM domains plus a low density lipoprotein (LDL)-binding region led to the formation of the GRL101 receptor found in snail.

 
It is clear that the LGR genes encode a conserved subgroup of seven TM receptors of ancient origin. Based on the evolutionary relationship of LGRs in diverse species and the homologous amino acid sequences found in nine vertebrate and invertebrate LGRs, future searches could identify additional members of this subfamily of GPCRs in the mammalian genome. Because LGR4 and LGR5 appear to diverge from known gonadotropin and TSH receptors early during evolution before the formation of the pituitary gland and exhibit high similarity to LGRs found in lower vertebrates, they could subserve physiological functions associated with the primitive LGRs found in Cnidarians, one of the most primitive animals with a sensory system in the animal kingdom. A wide tissue distribution pattern of the mRNAs for these proteins further suggested that their physiological roles might be different from mammalian gonadotropin and TSH receptors known to play tissue-specific functions. The availability of cDNAs for LGR4 and LGR5 allows for future identification of their specific ligands by employing an anchored receptor approach found to be useful for the solubilization of the ligand-binding domains of glycoprotein hormone receptors (56) and for the elucidation of their physiological functions.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Computational Analysis
Human sequences related to the sea anemone and Drosophila glycoprotein hormone receptors (12, 13) were identified from the EST database (dbEST) at the National Center for Biotechnology Information (NIH, Bethesda, MD) by using the BLAST server with the BLOSUM62 protein comparison matrix (57). The alignment of LGR ecto- and TM domain sequences was carried out by CLUSTALW (58); this program also calculated the branching order of aligned sequences by the neighbor-joining algorithm (10,000 bootstrap replications provided confidence values for the tree groupings). Conserved alignment patterns were drawn by the CONSENSUS program (Internet URL http://www. bork.embl-heidelberg.de/Alignment/consensus.html). The PRINTS library of protein fingerprints (http://www.biochem. ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html) (18, 59) identified the myriad leucine-rich repeats present in the ectodomains of LGRs with a compound "Leurichrpt" motif that flexibly matches N- and C-terminal features of divergent leucine-rich repeats. The BLOCK Maker website (http://blocks.fhcrc.org) was used to align and generate the highly conserved ungapped blocks of the TM regions of diverse LGRs from vertebrates and invertebrates using both full-length receptor and TM segment sequences. The use of two different methods, Motif and Gibbs samplings, confirms for close relatedness. The blocks predicted from this alignment were then used to construct the neighbor-joining tree for the examination of possible subfamily relationships. Two algorithms whose three-state accuracy is greater than 72%, the neural network program PHD (60) and the statistical prediction method DSC (61; http://bonsai.lif.icnet.uk/dsc/manual.html), were used to derive a consensus secondary structure for the TM domain of different LGRs.

Cloning of Full-Length LGR4 and LGR5 cDNAs
Human ESTs showing high homology to two nonoverlapping regions of the gonadotropin receptors were identified. Clones AA312798 and AA298810 were found to encode TM 4 to TM 5 of the putative receptor (LGR4), whereas AA460529 and AA424098 encode TM 2 to TM 3 of another putative receptor (LGR5). Using these ESTs to further search the GenBank EST division database, overlapping EST sequences were aligned to obtain the longest ORF for each initial clone. Relevant EST clones were obtained from the I.M.A.G.E. consortium (info@image.llnl.gov) via Genome System, Inc. (St. Louis, MO).

Based on the longest human ORF, specific primers were designed for PCR amplification of LGR4 and LGR5 cDNA fragments from rat ovary and human placenta, respectively. After hybridization with labeled EST clones and confirmation of DNA sequences by dideoxy DNA sequencing, specific receptor fragments isolated were used to design primers to prepare sub-cDNA libraries enriched with specific receptor cDNAs. For 5' extension, reverse transcription was performed using rat ovarian and human placenta mRNA preparations and receptor-specific primers. After second-strand synthesis, the enriched cDNA pool was tailed at 5'-ends with specific adaptor sequences to allow further PCR amplification. For 3'-extension, rat ovarian or human placenta mRNAs were reversed transcribed using oligo-dT, followed by second-strand synthesis using receptor-specific primers and adaptor tailing. These minilibraries were further used as templates for PCR amplification of upstream or downstream cDNAs specific for each receptor using internal primers. PCR products with a strong hybridization signal to each receptor cDNA fragment were subcloned into the pUC18 or pcDNA3 vectors. After screening of these sublibraries based on colony hybridization using specific receptor probes, clones with 5'- or 3'-sequences of the putative receptors were identified and isolated for DNA sequencing. As needed, the procedure was repeated up to three times to generate cDNAs encoding the complete ORF of each putative receptor for sequence analysis and for the expression of receptor proteins in eukaryotic cells. The entire coding sequences of each gene were also amplified with specific primers flanking the entire ORF in independent experiments. At least three independent PCR clones were sequenced to verify the authenticity of coding sequences.

Tissue Expression of LGR4 and LGR5 mRNAs
Human multiple tissue blots, containing ~2 µg of poly(A)+ RNA per lane, were purchased from CLONTECH (Palo Alto, CA; catalog number 7759, 7760, and 7767). Northern blot analyses were performed using tissue blots after hybridization of labeled receptor cDNA probes. Membranes were prehybridized for 1 h at 60 C in the ExpressHyb solution (CLONTECH). This was followed by hybridization under the same condition for 2 h but with 1 x 106 cpm/ml of 32P-labeled LGR4, LGR5, or ß-actin cDNA probe. After hybridization, the membranes were washed twice in 2 x saline-sodium citrate (SSC), 0.5% SDS at room temperature, followed by two washes in 0.2 x SSC, 0.5% SDS at 60 C before exposure to Kodak RX films (Eastman Kodak, Rochester, NY).

Construction of Chimeric Receptor cDNAs and Analysis of Signal Transduction and Ligand Binding
PCR-based mutagenesis was performed using overlapping primers to construct cDNAs for chimeric LH/LGR4 and LH/LGR5 receptors as described previously (29). L(EC)LGR4(TM) and L(EC)LGR5(TM) represent chimeric receptors with the ectodomain of human LH receptor and the TM and C-terminal tail of LGR4 or LGR5 with the junctional sequences of PEPDA-FKPCEYLLGS and PEPDA-FKPCEHLLDG, respectively. All cDNAs were subcloned into the expression vector pcDNA3 (Invitrogen, San Diego, CA). Both the fidelity of PCR-amplified regions and the junctional sequences were confirmed by DNA sequencing on both strands. 293 cells derived from human embryonic kidney fibroblast were maintained in DMEM/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.


    ACKNOWLEDGMENTS
 
We thank C. Spencer for editorial assistance.


    FOOTNOTES
 
Address requests for reprints to: Dr. A. J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology/Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, California 94305-5317. E-mail: aaron.hsueh{at}stanford.edu

This work was supported by NIH Grant HD-23273.

1 The first two authors contributed equally to this work. S.L. is on leave from the Department of Obstetrics and Gynecology, Teikyo University, Tokyo, Japan. Back

2 GenBank accession numbers for LGR4 and LGR5 are AF061443 and AF061444, respectively. Back

Received for publication July 21, 1998. Revision received September 9, 1998. Accepted for publication September 11, 1998.


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