The Nematode Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor (LGR) Protein Homologous to Vertebrate Gonadotropin and Thyrotropin Receptors is Constitutively Activated in Mammalian Cells

Masataka Kudo1, Thomas Chen2, Koji Nakabayashi, Sheau Yu Hsu and Aaron J. W. Hsueh

Division of Reproductive Biology Department of Gynecology and Obstetrics Stanford University School of Medicine 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 interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of an expanding family of homologous leucine-rich repeat-containing, G protein-coupled receptors (LGRs), including the three known glycoprotein hormone receptors; mammalian LGR4 and LGR5; and LGRs in sea anemone, fly, and snail. We isolated nematode LGR cDNA and characterized its gene from the Caenorhabditis elegans genome. This receptor cDNA encodes 929 amino acids consisting of a signal peptide for membrane insertion, an ectodomain with nine leucine-rich repeats, a seven-TM region, and a long C-terminal tail. The nematode LGR has five potential N-linked glycosylation sites in its ectodomain and multiple consensus phosphorylation sites for protein kinase A and C in the cytoplasmic loop and C tail. The nematode receptor gene has 13 exons; its TM region and C tail, unlike mammalian glycoprotein hormone receptors, are encoded by multiple exons. Sequence alignments showed that the TM region of the nematode receptor has 30% identity and 50% similarity to the same region in mammalian glycoprotein hormone receptors. Although human 293T cells expressing the nematode LGR protein do not respond to human glycoprotein hormones, these cells exhibited major increases in basal cAMP production in the absence of ligand stimulation, reaching levels comparable to those in cells expressing a constitutively activated mutant human LH receptor found in patients with familial male-limited precocious puberty. Analysis of cAMP production mediated by chimeric receptors further indicated that the ectodomain and TM region of the nematode LGR and human LH receptor are interchangeable and the TM region of the nematode LGR is responsible for constitutive receptor activation. Thus, the identification and characterization of the nematode receptor provides the basis for understanding the evolutionary relationship of diverse LGRs and for future analysis of mechanisms underlying the activation of glycoprotein hormone receptors and related LGRs.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In vertebrates, gonadotropins (LH and FSH) and the thyroid-stimulating hormone (TSH) are essential for the differentiation and growth of gonads and the thyroid gland, respectively (1). These glycoprotein hormones bind and activate their specific plasma membrane receptors on target cells, leading to increases in Gs coupling and cAMP production. The receptors for LH, FSH, and TSH belong to the large G protein-coupled, seven-transmembrane (TM) protein family but are unique in having a large extracellular (ecto-) domain containing leucine-rich repeats important for interaction with the large glycoprotein ligands (2, 3). Gain-of-function mutants of LH and TSH receptors have been identified in patients with male-limited precocious puberty (4, 5) and nonautoimmune hyperthyroidism (6), respectively, suggesting the constitutive activation of these receptors follows single amino acid alterations.

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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Isolation of Nematode LGR cDNA and Comparison of Deduced Amino Acid Sequences among Different LGRs
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, NIH. A cosmid genomic clone (C50H2) on chromosome V of C. elegans was found to contain the complete sequence of the putative LGR gene. Specific primers with a design based on sequence alignment with known LGRs were then used in RT-PCR using C. elegans total RNA as the template. The isolated full-length nematode LGR cDNA has 2,790 bp of open reading frame.

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 1–8, a TM region by exons 8–11, and the C-terminal tail by exons 11–13 (Fig. 1AGo). 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. 1BGo).



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Figure 1. The Structure of Nematode LGR Gene and cDNA

A, The derivation of the nematode LGR full-length cDNA from 13 exons of the receptor gene. The ectodomain spans exon 1–8 whereas the TM region is encoded by exon 8–11. The C tail corresponds to exon 11–13. B, Intron-exon junctional sequences and the size (nucleotide numbers in parentheses) of individual introns and exons are shown. Bold letters indicate presumptive donor-acceptor sequences. C, Deduced amino acid residues for the full-length nematode receptor cDNA are shown, together with the intron-exon junctions. Conserved cysteine residues (dots), potential N-linked glycosylation sites (inverse triangles), as well as potential phosphorylation sites for protein kinase A (triangle) and protein kinase C (asterisks) are indicated.

 
The deduced nematode LGR protein consists of a 30-amino acid signal peptide that is rich in hydrophobic amino acids followed by an ectodomain of 403 amino acids containing 9 typical-type leucine-rich repeats (Fig. 1CGo). The C-terminal half of this protein shows a 7-TM region of 262 amino acids and a relatively long C-tail of 234 amino acids. Hydropathy analyses of the deduced nematode LGR amino acid sequences identified 7-TM domains connected by predicted intra- and extracellular loops of variable lengths, an arrangement that is characteristic of all G protein-coupled receptors (GPCRs)(Fig. 1CGo). A CCXF motif in the C-flanking region of the ectodomain and a NPCEXXXGY motif preceding the TM I of all mammalian glycoprotein hormone receptors were conserved in the nematode LGR.

The TM region of nematode LGR showed greater than 30% identity and 50% similarity with mammalian glycoprotein hormone receptors (Fig. 1CGo and Fig. 2Go). 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 (546–549 RRLS, 719-7229 RRIT, 792–795 RRAS, 924–927 RRKS, and 925–928 RKST) and C (542–544 SFP, 549–551 SPK, 773–775 TPR, 797–799 SPR, 810–812 TPR, 813–815 SDR, and 874–876 SGR)(Figs. 1CGo and 2Go).




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Figure 2. Comparison of Amino Acid Sequences between the Nematode LGR and Orthologous Receptor Genes from Diverse Species

A, Signal peptide and ectodomain. The ectodomain of LGRs consists of a signal peptide for secretion followed by an N-flanking cysteine-rich region, multiple leucine-rich repeats (LRR), and a C-flanking region. Cysteine residues in the N and C flanking regions of the ectodomain are heavily shaded whereas putative N-linked glycosylation sites are indicated in bold letters. In addition, residues that are conserved in at least three of the six LGRs aligned are lightly shaded. B, C-terminal cysteine-rich region and TM domain. The seven-TM is flanked by a cysteine-rich region at its N terminus. The shaded residues represent a conservation of at least five of the six receptors used for comparison whereas the conserved cysteine residues in the extracellular loop 1 and 2 of GPCRs are marked by asterisks. C, The C-terminal cytoplasmic tail. The nematode LGR has a long C-tail. The potential phosphorylation sites for protein kinase A and C are underlined/bolded and underlined, respectively. Residue numbers are indicated at the right and the stop codons are shown as asterisks. Dashes indicated gaps in sequences that were included for optimal protein alignment.

 
Analysis of amino acid sequences among the nematode receptor, mammalian LGRs, and homologous LGRs found in other invertebrate species (sea anemone, fly, and snail) indicates that the nematode LGR receptor is evolutionarily closer to the mammalian glycoprotein hormone receptors than to the two recently isolated mammalian LGRs, LGR4 and LGR5 (11). As compared with approximately 40% similarity to mammalian glycoprotein hormone receptors, sequence alignments showed that the leucine-rich repeats in the nematode LGR have 30% and 36% similarity to those found in Drosophila Slit and human Toll genes (12, 13), respectively.

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. 3AGo, left panel). As shown in Fig. 3BGo, 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. 3BGo). 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. 3AGo (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. 3BGo). 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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Figure 3. The Nematode LGR Is Constitutively Activated; Comparison between Wild-Type and Mutant Human LH Receptors

A, Left panel: Basal cAMP production was monitored in 293T cells transiently transfected with plasmids (1 µg/well) encoding the cDNAs for nematode (n)LGR, wild- type human LH receptor (LHRWT) or a gain-of-function LH receptor mutant D578Y. Right panel: Cell surface expression of different receptor proteins was monitored using M1 antibodies to detect the FLAG epitope in individual receptors. B, Basal cAMP production by cells transfected with increasing amounts of the empty vector or the expression vector containing nematode LGR or human LH receptor cDNA. Although the extent of its cell surface expression could not be monitored, a nematode LGR without the FLAG epitope (no tag) also showed increases in basal cAMP production when cells were transfected with increasing amounts of the expression plasmid. C, Lack of stimulation of IP turnover in 293T cells expressing nematode LGR. Time-dependent increases in the hydrolysis of phosphatidyl inositols (PIs) were measured for cells transfected with the empty vector or vectors containing different receptor cDNAs. Basal and hCG (1 µg/ml)-stimulated PI hydrolysis by wild-type LH receptor and by the mutant D578Y LH receptor also are included.

 
To test the possibility that known ligands for human glycoprotein hormone receptors might stimulate the orthologous worm LGR, cells expressing the nematode receptor were incubated with each of these ligands at 1 µg/ml. However, the high basal levels of cAMP were not further augmented after treatment with the glycoprotein hormones (% changes, mean ± SD: human CG-treated, 103 ± 8%; FSH-treated, 115 ± 13%; TSH-treated 112 ± 18%; n = 3). In addition, ligand binding analysis using saturating levels of I125-labeled hCG or human FSH showed no specific binding to cells expressing the nematode receptor (data not shown).

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. 3CGo). 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. 4AGo) 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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Figure 4. Ligand Binding and cAMP Production Mediated by Chimeric Receptors with Different Regions of Either Nematode LGR or Human LH Receptor

A, Diagrammatic representation of wild-type and chimeric receptors. Filled circles represent LH receptor sequence whereas the open circles represent nematode receptor sequence. Chimeric receptors were constructed using mutagenesis and gene transfer. Junctional amino acid sequences of the chimeric receptors are listed below together with the residue number for each receptor at the junctional site (represented by a slash): L-N(TM-C): ... PCED 355 (LH receptor)/(nLGR) 426 IVGYPFLR; N-L(TM-C): ... . NPCEN (nLGR) 425/(LH receptor) 356 LMGYDFL ... ; L-N(TM)-L: ... PCED355 (LH receptor)/(nLGR) 426 IVGYPFLR ... VFFT695/(LH receptor) 628 KTFQRD... ; L-N(i3C)-L: LH receptor 565–569 replaced by the corresponding nLGR sequence RALIT. B, Scatchard plot analysis of wild-type and the chimeric receptor L-N(TM-C). C, Basal and hCG-stimulated cAMP production by wild-type LH receptor and the chimeric receptor L-N(TM-C). Basal and hCG (100 ng/ml)-stimulated cAMP production was measured in cells expressing wild-type and the mutant receptor. Results were normalized based on cell surface I125-hCG binding. D, Basal cAMP production by cells transfected with increasing amounts of the expression vector containing wild-type LH receptor (LHRWT) or the chimeric receptor L-N-(TM-C). E, Basal cAMP production by chimeric receptors with different regions from either LH receptor or nematode LGR. The chimeric receptors L-N(TM)-L and L-N(i3C)-L are constitutively activated. Also, introduction of a point mutation (D578Y) based on the human gain-of-function LH receptor in the chimeric receptor, N-L(TM-C), with the TM region from LH receptor leads to receptor activation. Estimated transfection efficiency based on ß-gal activity was comparable for all groups.

 
A chimeric receptor, L-N(TM-C), in which the ectodomain of nematode receptor was replaced with the corresponding ligand-binding domain of LH receptor, was expressed on the cell surface. Based on 125I-hCG binding, this receptor showed lower receptor numbers but exhibited similar binding affinity when compared with the wild type LH receptor (Kd value: 256 ± 67 pM for the chimeric receptor; 199 ± 48 for wild-type LH receptor, mean ± SD n = 3) (Fig. 4BGo). Of interest, this chimeric receptor retained constitutive activity as reflected by increases in basal cAMP levels (Fig. 4CGo). However, treatment with hCG could not further augment cAMP production (Fig. 4CGo). Transfection with increasing amounts of the plasmid containing the chimeric receptor L-N(TM-C) resulted in dose-dependent increases in basal cAMP production whereas the wild-type LH receptor showed negligible increases in basal cAMP production (Fig. 4DGo). In addition, a chimeric receptor, L-N(TM)-L, in which both ectodomain and C-terminal tail of the nematode LGR were replaced with the corresponding region of the LH receptor also showed constitutive activation (Fig. 4EGo). However, another chimeric receptor, N-L(TM-C), in which the ectodomain of LH receptor was replaced with the corresponding ectodomain of the nematode LGR, showed no increases in basal cAMP levels. Increases in basal cAMP levels could also be observed when one of the gain-of-function mutations for the LH receptor was introduced into TM VI (D578Y) of this chimeric receptor (Fig. 4EGo).

Because the C-terminal end of the intracellular loop 3 of the nematode LGR is distinctly different from the three glycoprotein hormone receptors (Fig. 2BGo), we hypothesized that this region might be responsible for its constitutive activation. As shown in Fig. 4EGo, 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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Based on the analysis of high throughput genomic sequences, we have isolated a nematode ortholog of the expanding mammalian LGRs. Analysis of the structure of the encoded polypeptide revealed that the nematode LGR is evolutionary related to the mammalian glycoprotein hormone receptors and two LGRs from Drosophila and sea anemone. The nematode receptor was found to be constitutively activated as reflected by increases in basal cAMP production, but not IP turnover, in transfected mammalian cells. Studies using chimeric receptors further demonstrated the importance of the intracellular loop 3 of the nematode receptor in its constitutive activity.

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-{alpha}, 2 G-ß, and 2 G-{gamma} genes. Among the G-{alpha} genes, one homolog for each of the four mammalian classes of G-{alpha} genes, Gs-{alpha}, Gi/Go-{alpha}, Gq-{alpha}, and G12-{alpha}, has been found (38). Of interest, the Gs-{alpha} 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 {alpha}- 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 {alpha}- 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hormones and Reagents
Purified human CG (CR-129) was supplied by the National Hormone and Pituitary Program (NIDDK, NIH, Bethesda, MD). Human recombinant FSH (Org32489) was provided by Organon (Oss, The Netherlands) whereas recombinant human TSH was from Genzyme Corp. (Thousand Oaks, CA). Anti-FLAG M1 monoclonal antibody and FLAG peptide were purchased from Sigma (St. Louis, MO). 125I-Na and myo-[3H] inositol were purchased from Amersham Pharmacia Biotech (Arlington Heights, IL). Dowex AG1-X8 was from Bio-Rad Laboratories, Inc. (Richmond, CA).

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 manufacturer’s 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/Ham’s 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 70–80% 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 18–24 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 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 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 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, 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 {gamma}-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 {gamma}-spectrometer. Background binding was determined by adding excess amounts of the synthetic FLAG peptide at a concentration of 100 µg/ml.


    ACKNOWLEDGMENTS
 
We thank Dr. Anne Villaneuve (Stanford University, Stanford, CA) for providing C. elegans RNA, Dr. Yuji Kohara of the National Institute of Genetics (Mishima, Japan) for cDNA clones (yk420f1 and yk429f6), Dr. G. Vassart, Universite Libre de Bruxelles (Brussels, Belgium) for providing the human TSH receptor cDNA and the National Pituitary and Hormone Distribution Program for the cAMP antiserum.


    FOOTNOTES
 
Address requests for reprints to: Dr. Aaron Hsueh, Department of Gynecology/Obstetrics, Stanford University School of Medicine, Division of Reproductive Biology, 300 Pasteur Drive, Room A-344, Stanford, California 94305-5317.

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. Back

2 On sabbatical leave from Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville, Tennessee 37996. Back

Received for publication August 16, 1999. Revision received November 11, 1999. Accepted for publication November 15, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Pierce JG, Parsons TF 1981 Glycoprotein hormones: structure and function. Annu Rev Biochem 50:465–495[CrossRef][Medline]
  2. Vassart G, Dumont JE 1992 The thyrotropin receptor and the regulation of thyrocyte function and growth. Endocr Rev 13:596–611[Medline]
  3. Ji TH, Grossmann M, Ji I 1998 G protein-coupled receptors. I. Diversity of receptor-ligand interactions. J Biol Chem 273:17299–17302[Free Full Text]
  4. Shenker A, Laue L, Kosugi S, Merendino JJ, Jr, Minegishi T, Cutler GB, Jr 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365:652–654[CrossRef][Medline]
  5. Laue LL, Wu SM, Kudo M, Bourdony CJ, Cutler GB, Jr, Hsueh AJ, Chan WY 1996 Compound heterozygous mutations of the luteinizing hormone receptor gene in Leydig cell hypoplasia. Mol Endocrinol 10:987–997[Abstract]
  6. Vassart G 1997 New pathophysiological mechanisms for hyperthyroidism. Horm Res 48:47–50[Medline]
  7. Nothacker HP, Grimmelikhuijzen CJ 1993 Molecular cloning of a novel, putative G protein-coupled receptor from sea anemones structurally related to members of the FSH, TSH, LH/CG receptor family from mammals. Biochem Biophys Res Commun 197:1062–1069[CrossRef][Medline]
  8. Hauser F, Nothacker HP, Grimmelikhuijzen CJ 1997 Molecular cloning, genomic organization, and developmental regulation of a novel receptor from Drosophila melanogaster structurally related to members of the thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone/choriogonadotropin receptor family from mammals. J Biol Chem 272:1002–1010[Abstract/Free Full Text]
  9. Tensen CP, Van Kesteren ER, Planta RJ, Cox KJ, Burke JF, van Heerikhuizen H, Vreugdenhil E 1994 A G protein-coupled receptor with low density lipoprotein-binding motifs suggests a role for lipoproteins in G-linked signal transduction. Proc Natl Acad Sci USA 91:4816–4820[Abstract]
  10. Arora KK, Sakai A, Catt KJ 1995 Effects of second intracellular loop mutations on signal transduction and internalization of the gonadotropin-releasing hormone receptor. J Biol Chem 270:22820–22826[Abstract/Free Full Text]
  11. Hsu SY, Liang SG, Hsueh AJ 1998 Characterization of two LGR genes homologous to gonadotropin and thyrotropin receptors with extracellular leucine-rich repeats and a G protein-coupled, seven-transmembrane region. Mol Endocrinol 12:1830–1845[Abstract/Free Full Text]
  12. Rothberg JM, Jacobs JR, Goodman CS, Artavanis-Tsakonas S 1990 Slit: an extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev 4:2169–2187[Abstract]
  13. Rock FL, Hardiman G, Timans JC, Kastelein RA, Bazan JF 1998 A family of human receptors structurally related to Drosophila Toll. Proc Natl Acad Sci USA 95:588–593[Abstract/Free Full Text]
  14. Allgeier A, Laugwitz KL, Van Sande J, Schultz G, Dumont JE 1997 Multiple G-protein coupling of the dog thyrotropin receptor. Mol Cell Endocrinol 127:81–90[CrossRef][Medline]
  15. Hirsch B, Kudo M, Naro F, Conti M, Hsueh AJ 1996 The C-terminal third of the human luteinizing hormone (LH) receptor is important for inositol phosphate release: analysis using chimeric human LH/follicle-stimulating hormone receptors. Mol Endocrinol 10:1127–1137[Abstract]
  16. Kudo M, Osuga Y, Kobilka BK, Hsueh AJW 1996 Transmembrane regions V and VI of the human luteinizing hormone receptor are required for constitutive activation by a mutation in the third intracellular loop. J Biol Chem 271:22470–22478[Abstract/Free Full Text]
  17. Scheer A, Fanelli F, Costa T, De Benedetti PG, Cotecchia S 1996 Constitutively active mutants of the {alpha}1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. EMBO J 15:3566–3578[Abstract]
  18. Brosius J 1999 Many G-protein-coupled receptors are encoded by retrogenes. Trends Genet 15:304–305[CrossRef][Medline]
  19. Gentles AJ, Karlin S 1999 Why are human G-protein-coupled receptors predominantly intronless? Trends Genet 15:47–49[CrossRef][Medline]
  20. Zhang Y, Chou JH, Bradley J, Bargmann CI, Zinn K 1997 The Caenorhabditis elegans seven-transmembrane protein ODR-10 functions as an odorant receptor in mammalian cells. Proc Natl Acad Sci USA 94:12162–12167[Abstract/Free Full Text]
  21. Hamdan FF, Ungrin MD, Abramovitz M, Ribeiro P 1999 Characterization of a novel serotonin receptor from Caenorhabditis elegans: cloning and expression of two splice variants. J Neurochem 72:1372–1383[CrossRef][Medline]
  22. Tiberi M, Caron MG 1994 High agonist-independent activity is a distinguishing feature of the dopamine D1B receptor subtype. J Biol Chem 269:27925–27931[Abstract/Free Full Text]
  23. Barker EL, Westphal RS, Schmidt D, Sanders-Bush E 1994 Constitutively active 5-hydroxytryptamine2C receptors reveal novel inverse agonist activity of receptor ligands. J Biol Chem 269:11687–11690[Abstract/Free Full Text]
  24. Laue L, Wu SM, Kudo M, Hsueh AJW, Cutler GB, Jr, Jelly DH, Diamond FB, Chan WY 1996 Heterogeneity of activating mutations of the human luteinizing hormone receptor in male-limited precocious puberty. Biochem Mol Med 58:192–198[CrossRef][Medline]
  25. O’Dowd BF, Hnatowich M, Regan JW, Leader WM, Caron MG, Lefkowitz RJ 1988 Site-directed mutagenesis of the cytoplasmic domains of the human ß2-adrenergic receptor. Localization of regions involved in G protein- receptor coupling. J Biol Chem 263:15985–15992[Abstract/Free Full Text]
  26. Birnbaumer M 1995 Mutations and diseases of G protein coupled receptors. J Recept Signal Transduct Res 15:131–160[Medline]
  27. Raymond JR 1994 Hereditary and acquired defects in signaling through the hormone-receptor-G protein complex. Am J Physiol 266:F163–174
  28. Lefkowitz RJ 1993 G-protein-coupled receptors. Turned on to ill effect. Nature 365:603–604[CrossRef][Medline]
  29. Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD 1992 Constitutively active mutants of rhodopsin. Neuron 9:719–725[Medline]
  30. Rao VR, Cohen GB, Oprian DD 1994 Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. Nature 367:639–642[CrossRef][Medline]
  31. Robbins LS, Nadeau JH, Johnson KR, Kelly MA, Roselli-Rehfuss L, Baack E, Mountjoy KG, Cone RD 1993 Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72:827–834[Medline]
  32. Schipani E, Kruse K, Juppner H 1995 A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268:98–100[Medline]
  33. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, Seidman JG 1994 Autosomal dominant hypocalcaemia caused by a Ca(2+)-sensing receptor gene mutation. Nat Genet 8:303–307[Medline]
  34. Pollak MR, Brown EM, Chou YH, Hebert SC, Marx SJ, Steinmann B, Levi T, Seidman CE, Seidman JG 1993 Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 75:1297–1303[Medline]
  35. Latronico AC, Anasti J, Arnhold IJ, Mendonca BB, Domenice S, Albano MC, Zachman K, Wajchenberg BL, Tsigos C 1995 A novel mutation of the luteinizing hormone receptor gene causing male gonadotropin-independent precocious puberty. J Clin Endocrinol Metab 80:2490–2494[Abstract]
  36. Parma J, Duprez L, Van Sande J, Cochaux P, Gervy C, Mockel J, Dumont J, Vassart G 1993 Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365:649–651[CrossRef][Medline]
  37. The C elegans Sequencing Consortium 1998 Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 282:2012–2018[Abstract/Free Full Text]
  38. Jansen G, Thijssen KL, Werner P, van der Horst M, Hazendonk E, Plasterk RH 1999 The complete family of genes encoding G proteins of Caenorhabditis elegans. Nat Genet 21:414–419[CrossRef][Medline]
  39. Park JH, Ohshima S, Tani T, Ohshima Y 1997 Structure and expression of the gsa-1 gene encoding a G protein alpha(s) subunit in C. elegans. Gene 194:183–190[CrossRef][Medline]
  40. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ 1997 Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402[Abstract/Free Full Text]
  41. Osuga Y, Kudo M, Kaipia A, Kobilka B, Hsueh AJ 1997 Derivation of functional antagonists using N-terminal extracellular domain of gonadotropin and thyrotropin receptors. Mol Endocrinol 11:1659–1668[Abstract/Free Full Text]
  42. Jia XC, Oikawa M, Bo M, Tanaka T, Ny T, Boime I, Hsueh AJ 1991 Expression of human luteinizing hormone (LH) receptor: interaction with LH and chorionic gonadotropin from human but not equine, rat, and ovine species. Mol Endocrinol 5:759–768[Abstract]
  43. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Medline]
  44. Davoren JB, Hsueh AJ 1985 Vasoactive intestinal peptide: a novel stimulator of steroidogenesis by cultured rat granulosa cells. Biol Reprod 33:37–52[Abstract]
  45. Hall CV, Jacob PE, Ringold GM, Lee F 1983 Expression and regulation of Escherichia coli lacZ gene fusions in mammalian cells. J Mol Appl Genet 2:101–109[Medline]
  46. Su JG, Hsueh AJ 1992 Characterization of mouse inhibin {alpha} gene and its promoter. Biochem Biophys Res Commun 186:293–300[Medline]
  47. Miyachi Y, Vaitukaitis JL, Nieschlag E, Lipsett MB 1972 Enzymatic radioiodination of gonadotropins. J Clin Endocrinol Metab 34:23–28[Medline]