Abstract
The receptors for lutropin (LH), FSH, and TSH belong to the large G protein-coupled receptor (GPCR) superfamily and are unique in having a large N-terminal extracellular (ecto-) domain important for interactions with the large glycoprotein hormone ligands. Recent studies indicated the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals. Based on the sequences of mammalian glycoprotein hormone receptors, we have identified a new LGR in Drosophila melanogaster and named it as fly LGR2 to distinguish it from the previously reported fly LH/FSH/TSH receptor (renamed as fly LGR1). Genomic analysis indicated the presence of 10 exons in fly LGR2 as compared with 16 exons in fly LGR1. The deduced fly LGR2 complementary DNA (cDNA) showed 43 and 64% similarity to the fly LGR1 in the ectodomain and transmembrane region, respectively. Comparison of 12 LGRs from diverse species indicated that these proteins can be divided into three subfamilies and fly LGR1 and LGR2 belong to different subfamilies. Potential signaling mechanisms were tested in human 293T cells overexpressing the fly receptors. Of interest, fly LGR1, but not LGR2, showed constitutive activity as reflected by elevated basal cAMP production in transfected cells. The basal activity of fly LGR1 was further augmented following point mutations of key residues in the intracellular loop 3 or transmembrane VI, similar to those found in patients with familial male precocious puberty. The present study reports the cloning of fly LGR2 and indicates that the G protein-coupling mechanism is conserved in fly LGR1 as compared with the mammalian glycoprotein hormone receptors. The characterization of fly receptors with features similar to mammalian glycoprotein hormone receptors allows a better understanding of the evolution of this unique group of GPCRs and future elucidation of their ligand signaling mechanisms.
THE G PROTEIN-COUPLED receptors (GPCR) represent the largest superfamily of mammalian genes and are essential for almost all physiological functions in the higher organisms (1, 2). These receptors are characterized functionally by their interaction with guanine nucleotide binding proteins and structurally by their seven hydrophobic, α- helical transmembrane domains. Members of this superfamily are functionally diverse and include proteins ranging from the cAMP receptor in slime mold to mammalian neurotransmitter and glycoprotein hormone receptors. TSH and gonadotropin receptors are essential for thyroid and gonadal development, respectively. These glycoprotein hormone receptors belong to the subfamily A, rhodopsin-like GPCRs but diverge structurally from other GPCRs of this subfamily in having large N-terminal extracellular (ecto-) domains that are required for interaction with the large size of the glycoprotein hormones (3, 4) (5, 6). The ectodomains of glyco-protein hormone receptors contain multiple leucine-rich repeats homologous to similar repeats found in small proteoglycans decorin (7), Drosophila slit (8), and ribonuclease inhibitor (9).
The glycoprotein hormone receptors are conserved during evolution. Based on the cDNA sequences of mammalian glycoprotein hormone receptors, homologous receptors were identified in sea anemone (10), fruit fly (11), and snail (12). Recent advances in the genome projects of C. elegans and human have allowed the discovery of one nematode protein as well as several novel mammalian proteins with sequence homology to the mammalian glycoprotein hormone receptors. These leucine-rich repeat-containing, G protein-coupled receptors were named as nematode LGR (13) and mammalian LGR4 and LGR5 (14). The worm LGR contains nine leucine-rich repeats in its ectodomain, whereas both LGR4 and LGR5 contain 17 leucine-rich repeats.
With recent advances in the sequencing of the entire genome for Drosophila melanogaster (15), we have isolated a second fruit fly receptor with homology to mammalian glycoprotein hormone receptors and named it as fly LGR2 to distinguish it from the previously reported fly lutropin (LH)/FSH/TSH receptor (renamed as fly LGR1). The two fly LGR cDNAs were cloned to reveal their structural characteristics, and their evolutionary relationship with LGRs from diverse animals was analyzed. Using transfected mammalian cells, the signaling mechanisms for the two fly LGRs were also investigated. Of interest, fly LGR1, but not fly LGR2, was found to mediate constitutive increases in cAMP production by transfected mammalian cells, suggesting this signaling mechanism was conserved during evolution.
Materials and Methods
Hormones and reagents
Purified hCG (CR-129) and specific antiserum for cAMP were supplied by the National Hormone and Pituitary Program (NIDDK, NIH, Bethesda, MD), human recombinant FSH (Org32489) was provided by Organon (Oss, The Netherlands) and human recombinant TSH was from Genzyme Transgenics Corp. (Cambridge, MA). Anti-FLAG M1 monoclonal antibody and FLAG peptide were purchased from Sigma (St. Louis, MO).
Sequence analysis
Based on amino acid sequences of mammalian glycoprotein hormone receptors, the genomic sequence of the fly LGR2 was identified following searches of the high throughput genomic sequences (HTGS) in the National Center for Biotechnology Information using the tblastn program of the BLAST server (16). A P1 genomic clone (DS00180; D29) on chromosome 2L of D. melanogaster was identified to contain complete sequences of a putative LGR. A potential signal peptide cleavage site was predicted using the SignalP program (http://www.cbs.dtu.dk/services/SignalP/). The leucine-rich repeat motifs in the ectodomain of the LGR2 were identified using the PRINTS library of protein fingerprints (http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html). Alignments among multiple receptor paralogs were performed using the programs OMIGA and CLUSTALW (http://www.hgsc.bcm.tmc.edu/SearchLauncher/) with comparable outcomes. Phylogenetic analysis of the LGRs from fly, mammal, nematode, and snail was performed using the DRAWGRAM program in Biology Workbench (http://workbench.sdsc.edu/CGI/BW.cgi).
Cloning of full-length fly LGR2 cDNA
Full-length fly LGR2 cDNA was obtained by RT-PCR using adult D. melanogaster messenger RNA (CLONTECH Laboratories, Inc., Palo Alto, CA). Two micrograms of messenger RNA were reverse-transcribed by using 25 U of avian myoblastosis virus reverse transcriptase (AMV RNase) with oligo (dT) primer, 0.5 mm dNTP, and 20 U of RNase inhibitor. Specific primers were designed based on predicted sequences in the open reading frame of fly LGR2. All PCR amplifications were performed under highly stringent conditions (annealing temperature >67 C) using Advantage cDNA polymerase (CLONTECH Laboratories, Inc.) or Pfu DNA polymerase (Stratagene, La Jolla, CA) to minimize mismatching and infidelity during PCR amplification. PCR products were fractionated using agarose electrophoresis and specific bands showing hybridization with radiolabeled cDNA probes were subcloned into the pUC18 vector (Invitrogen Corp., Carlsbad, CA). The PCR products were phenol/chloroform-extracted, precipitated with ethanol, phosphorylated with T4 polynucleotide kinase, and blunt-ended with the Klenow enzyme. They were then subcloned into the SmaI site in the pUC18 vector. At least two independent PCR clones were sequenced to verify the authenticity of the coding sequences. The resulting fly LGR2 sequence was assembled into contigs using the Blast2 sequences server (http://www.ncbi.nlm.nih.gov/gorf/bl2.html) and ClustalW 1.7 at BCM Search Launcher (http://dot.imgen.bcm.tmc.edu:9331/multialign).
Full-length cDNA for fly LGR1 was also obtained by RT-PCR using adult D. melanogaster messenger RNA and specific primers which were designed based on published sequences of fly LH/FSH/TSH receptor (11).
Construction of expression plasmids for wild-type and mutant fly LGR1 and LGR2
To facilitate the cell surface expression of the fly receptors in mammalian cells, their signal peptides were replaced with the PRL signal peptide for secretion with or without tagging with the FLAG M1 epitope as previously described (13). Amino acid alignment of the tagged construct is: PRL signal peptide, FLAG M1 epitope (DYKDDDDVD), followed by the receptor sequences for fly LGR1 (VYAT… . ) or fly LGR2 (YFCN… . ).
To study signal transduction by the fly LGRs, gain-of-function mutants of the fly receptors were also generated based on the mutations found in the LH receptor gene of patients with familial male precocious puberty (17). PCR-based mutagenesis was performed using overlapping primers as described previously (18). To substitute Gly for Glu at residue 687 of the fly LGR1 receptor, PCR was performed with VENT DNA polymerase (New England Biolabs, Inc., Beverly, MA) following manufacturer’s instructions. The PCR fragment containing the mutated sequence was used to replace the corresponding region in the wild-type receptor construct. In the same manner, three other mutant cDNAs for fly LGR1 (N701Y) and fly LGR2 (D744G, and D758Y) were constructed. Wild-type and mutant cDNAs were subcloned into the expression vector pcDNA3.1 Zeo (Invitrogen Corp.) and the plasmids were purified using the Qiafilter plasmid Maxi kit (QIAGEN, Inc., Valencia, CA). Fidelity of PCR was confirmed by DNA sequencing on both strands of the final constructs before use in expression studies.
Transfection of cells and analysis of signal transduction
Human 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 mml-glutamine. Before transfection, 2 × 105 cells were seeded on each well of 12-well tissue culture plates (Corning, Inc., Corning, NY). When cells were 70–80% confluent, transient transfection was performed using 1 μg of plasmid for each well by the calcium phosphate precipitation method (19). After 18 h of incubation with the calcium phosphate-DNA precipitates, each well was washed once with Dulbecco’s PBS (d-PBS), replaced with DMEM/F12 supplemented with 1 mg/ml of BSA and 0.25 mm 3-isobutyl-1-methyl xanthine (IBMX, Sigma), and incubated at 37 C for 16 h with or without hormones. Transfection using increasing amounts of plasmid in 12-well culture plates was also performed as described above to test the effects of increasing receptor expression on basal cAMP production. Each well was transfected separately with different amounts of plasmids. At 48 h 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 (20). All experiments were repeated at least three times using cells from independent transfection.
Determination of epitope-tagged receptors on the cell surface
293T cells (2 × 106) seeded in 10-cm dishes (Nalge Nunc International, Naperville, IL) were transfected as described above using 10 μg of plasmid. After 18 h incubation with the calcium phosphate-DNA precipitates, media were replaced with DMEM/F12 containing 10% FBS. At 48 h after transfection, cells were washed twice with d-PBS before harvesting from culture dishes for cell surface expression analysis. Transfected cells were resuspended and 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 following centrifugation at 14,000× g for 15 sec. The 125I-labeled second antibody (antimouse IgG from sheep: ∼400,000 cpm) was added to the resuspended cell pellet and incubated for 1 h at room temperature. Cells were then washed twice with 1 ml of assay buffer by repeated centrifugation before determination of radioactivities in the pellets using aγ -spectrometer. Background binding was determined by adding an excess amount of the synthetic FLAG peptide at a concentration of 100μ g/ml.
Results
Isolation of fly LGR2 cDNA and analysis of intron-exon arrangement of the fly LGR2 gene
Based on amino acid sequences of mammalian glycoprotein hormone receptors, the genomic sequence of the fly LGR2 was identified following searches of the high throughput genomic sequences in the GenBank. A P1 genomic clone on chromosome 2L of D. melanogaster was found to contain the complete sequence of the putative LGR2 gene. Specific primers were designed based on sequence alignment of predicted fly LGR2 open reading frame consistent with known LGR cDNAs. These primers were used in RT-PCR using adult D. melanogaster total RNA as the template. The isolated full-length fly LGR2 cDNA has 3,153 bp of open reading frame. Comparison of this cDNA with the corresponding genomic sequence revealed that the fly LGR2 gene has at least 10 exons and expands over 7 kb in length (Fig. 1A). The deduced 1,050 amino acid sequence of this receptor consists of an ectodomain encoded by exons 1 to 10 as well as a transmembrane region and the C-terminal tail encoded by exon 10. Six of the nine introns are smaller than 100 bp in size with a 1,747-bp intron between exons 3 and 4 (Fig. 1B). Comparison of exon arrangement among the two fly LGRs, nematode LGR, and the prototypic human FSH receptor (Fig. 1C) indicated that fly LGR2 is similar to human FSH receptor in having the transmembrane region and C-terminal tail encoded by a single exon.
Structure of the fly LGR2 gene and the corresponding cDNA sequence. A, Derivation of the fly LGR2 full-length cDNA from the genomic sequence. The ectodomain spans exons 1 to 10, whereas the transmembrane (TM) region and C-terminal tail are encoded by exon 10. B, Intron-exon junctional sequences. The size (nucleotide numbers in parentheses) of individual introns and exons are shown. C, Comparison of the derivation of cDNAs from different exons of the genes for human FSH receptor (FSHR), nematode (n) LGR as well as fly LGR1 and fly LGR2. The junctions between the ectodomain, the transmembrane region (TM), and the C-terminal tail are shown as dashed lines. Numbers denote the portions of cDNA corresponding to individual exons. D, Deduced amino acid residues for the full-length fly LGR2 cDNA. The intron-exon junctions are indicated by arrows. In addition, potential N-linked glycosylation sites (inverse triangles), as well as potential phosphorylation sites for protein kinase A (triangles) and protein kinase C (asterisk) are indicated. The stop codon is also denoted by an asterisk.
The deduced fly LGR2 protein consists of a 26 amino acid signal peptide, which is rich in hydrophobic amino acids followed by an ectodomain of 517 amino acids that contains five potential N-linked glycosylation sites (Fig. 1D, inverted triangles). In the intracellular loop 2, consensus protein kinase A (656–659 KRLS) and C (659–661 SLK) sites were found (Fig. 1D). In addition, the unusually long C-terminal tail has two consensus phosphorylation sites for protein kinase A (987–990 RRHS and 996–999 RKTS) (Fig. 1D, triangles).
The deduced fly LGR2 cDNA showed 43 and 64% similarity to the fly LGR1 in the ectodomain and transmembrane region, respectively. Nine typical-type leucine-rich repeats are present in the ectodomain of fly LGR2, showing high homology to similar repeats in other LGRs (Fig. 2A, LRRs). A CCAF motif in the C-flanking region of leucine-rich repeats (Fig. 2A), and a FXPCXD motif preceding the transmembrane I of human glycoprotein hormone receptors (Fig. 2B) were conserved in fly LGR2. The C-terminal half of this protein shows a transmembrane region of 270 amino acids (Fig. 2B) and a long C-terminal tail of 237 amino acids (Fig. 2C). Hydropathy analyses of the deduced fly LGR2 amino acid sequences identified seven-transmembrane domains connected by predicted intra and extracellular loops of variable lengths, an arrangement that is characteristic of all GPCRs. The transmembrane region of fly LGR2 showed greater than 40% identity and 65% similarity with mammalian glycoprotein hormone receptors (Fig. 2B). Although distinctive GPCR motifs were identified, including the highly conserved cysteine residues for disulfide bond formation in the extracellular loops 1 and 2 (Fig. 2B), the unique DRY (ERW in glycoprotein hormone receptors) motif found at the junction between transmembrane III and intracellular loop 2 of many GPCRs (21), has diverged to an ERN sequence in the fly LGR2.
Comparison of deduced amino acid sequences among fly LGR1, fly LGR2, nematode LGR, and human glycoprotein hormone receptors. A, Signal peptide and ectodomain. The ectodomain of different LGRs consists of a signal peptide for secretion followed by an N-flanking cysteine-rich region, multiple leucine-rich repeats (LRRs), and a C-flanking region. Cysteine residues in the N- and C-flanking regions of the ectodomain are in dark shading. In addition, residues that are conserved in at least three out of the six LGRs aligned are in light shading. B, Junctional cysteine-rich region and transmembrane (TM) domain. The seven-transmembrane region is flanked by a junctional cysteine-rich region at its N terminus. The shaded residues represent the conservation of at least four out of the six receptors used for comparison, whereas the conserved cysteine residues in the extracellular loop (EL)-1 and -2 of GPCRs are marked by asterisks. The conserved E687 (fly LGR1) and D744 (fly LGR2) in the intracellular loop (IL)-3 are also indicated by an inverted triangle, together with the key residues N701 (fly LGR1) and D758 (fly LGR2) in the TM VI. C, The C-terminal cytoplasmic tail. The fly LGR2 has a long C-tail similar to that found in nematode LGR. The stop codons are shown as asterisks. Residue numbers are indicated at right, and dashes represent gaps in sequences that were included for optimal protein alignment.
Comparison between deduced amino acid sequences for fly LGR2 and different LGRs from diverse species
In addition to five mammalian LGRs and homologous LGRs found in the invertebrate species (sea anemone, nematode, snail, and fly), we have recently identified two new human LGRs and named them as LGR6 and LGR7 (22). Analysis of full-length amino acid sequences of all 12 known LGRs (Fig. 3) indicated that the fly LGR1 is evolutionarily closer to the mammalian glycoprotein hormone receptors and the LGRs from nematode and sea anemone, whereas the fly LGR2 is more related to mammalian LGR4, LGR5, and LGR6. In addition, the human LGR7 can be categorized into a third subgroup together with snail LGR. These results are consistent with the hypothesis that three subgroups of LGRs evolved in metazoans; and fly LGR1 and fly LGR2 represent two branches of the evolutionary tree.
Phylogenetic relatedness of LGRs from diverse species. Based on full-length sequence comparison of 12 LGR proteins from diverse species, the evolutionary relationship was analyzed for fly LGR1, fly LGR2, LGRs from sea anemone (Ae: Anthopleura elegantissima), nematode (n), and snail together with human glycoprotein hormone receptors and mammalian LGR4–7. The LGR proteins from diverse species can be divided into three subgroups with the fly LGR1 clustered with the human glycoprotein hormone receptors and LGRs from sea anemone and nematode. In contrast, fly LGR2 belongs to the second subgroup together with mammalian LGR4–6. Subgroup 3 contains human LGR7 and the snail LGR.
Fly LGR1, but not fly LGR2, is constitutively activated and mediates ligand-independent cAMP biosynthesis by transfected mammalian cells
To test the signaling mechanisms for the two fly LGRs, we constructed expression vectors containing full-length cDNAs for these receptors. Mammalian 293T cells were transfected with expression plasmids containing cDNA encoding fly LGR1, fly LGR2, wild-type human LH receptor, or a mutant LH receptor. As shown in Fig. 4A (top panel), transfection of 293T cells with increasing amounts of the fly LGR1 expression plasmid led to dose-dependent increases in basal cAMP production in a ligand-independent fashion. The observed basal activity of fly LGR1 receptor is about 50% to that found in cells expressing a constitutively activated mutant human LH receptor (D564G LHR), whereas minimal increases in basal cAMP production were found in cells transfected with the empty vector and cells expressing fly LGR2 or the wild-type LH receptor (LHRWT). Although basal cAMP production was minimal in cells expressing fly LGR2 and the wild-type human LH receptor, basal cAMP production produced by these cells was still higher than cells transfected with the empty vector (Fig. 4A, lower panel).
The fly LGR1, but not LGR2, protein is constitutively activated in transfected mammalian cells. A, Basal cAMP production was monitored in 293T cells transiently transfected with increasing amounts of plasmids encoding cDNAs for either fly LGR1, fly LGR2, wild-type human LH receptor (LHR WT) or an LH receptor mutant D564G. Some cells were also transfected with the empty vector (vector). Basal cAMP production by cells in individual cultures was monitored. Data for selected groups were replotted in the bottom panel to compare low levels of cAMP production. B, Cell surface expression of fly LGRs as well as wild-type and mutant human LH receptors. Levels of cell surface proteins for transfected cells were monitored using M1 antibodies to detect the FLAG epitope in individual receptors. Mean ± se (n = 3).
Because these receptors were tagged at the N-terminal ends with a FLAG epitope, their expression levels on the cell surface were also determined using the M1 antibody. As shown in Fig. 4B, the levels of tagged proteins expressed on the cell surface were comparable for all receptors tested; however, negligible FLAG epitope expression was found in cells transfected with the empty vector.
Further activation of fly LGR1 following point mutation of key residues in intracellular loop 3 and transmembrane VI
We further tested the possibility that alternation of key residues in fly LGRs could enhance basal cAMP production mediated by these proteins. We constructed mutant fly receptors with a design based on gain-of-function mutations found in patients with male-limited precocious puberty and nonimmune hyperthyroidism (17, 23, 24). Two key aspartic acid residues in the intracellular loop 3 and transmembrane VI regions of human LH and TSH receptors known to be related to receptor activation are conserved in fly LGR1 and fly LGR2 (Fig. 2B). We constructed mutants for the two fly LGRs by altering these conserved residues in intracellular loop 3 and transmem- brane VI. These mutants were named as fly LGR1 E687G and fly LGR2 D744G (intracellular loop 3 mutants) plus fly LGR1 N701Y and fly LGR2 D758Y (transmembrane VI mutants). As shown in Fig. 5A, basal cAMP production mediated by fly LGR1 was further augmented more than 2-fold following incorporation of a point mutation in either region of the receptor. Although minimal basal cAMP production was mediated by the wild-type fly LGR2, incorporation of the intracellular loop 3 mutation (D744G), but not the transmembrane VI mutation (D578Y), led to further increases in basal cAMP production (Fig. 5A, inset). As shown in Fig. 5B, analysis of cell surface expression of these receptors based on their epitope tags indicated that comparable proteins were expressed by transfected cells.
Augmentation of basal cAMP production by fly LGRs following introduction of single point mutations in the intracellular loop 3 or transmembrane VI. A, Basal cAMP production was monitored in 293T cells transiently transfected with different plasmids encoding the cDNAs for wild-type and mutant fly LGRs. Data for selected groups were replotted in the inset to compare low levels of cAMP production. B, Surface expression of wild-type fly LGR1 or LGR2 and their mutants. Cell surface protein levels were monitored using M1 antibodies to detect the FLAG epitope in individual receptors.
Discussion
Recent advances in the sequencing of the genome of D. melanogaster allowed the present cloning of a new fly LGR2 and facilitated the characterization of the signaling mechanism for both fly LGR1 and LGR2. Sequence analysis of 12 LGRs from diverse animal species indicated the existence of three subgroups of LGRs with fly LGR1 and fly LGR2 as belonging to two separate subgroups. Overexpression of fly LGR1, but not fly LGR2, in 293T cells led to major increases in ligand-independent basal cAMP production, suggesting constitutive activation of this fly protein in mammalian cells and the conservation of the second messenger system for this subgroup of LGRs during evolution.
Recent completion of the nematode and fly genome projects revealed that there are 19,000 and 13,000 genes in C. elegans and D. melanogaster, respectively. It is interesting to note that there is only one LGR in nematode, whereas our preliminary data indicated that, in addition to the two fly LGRs reported here, there are likely two more LGRs in the fly genome. In the human genome, seven genes in the LGR family have been reported, including the three glycoprotein hormone receptors, LGR4, LGR5/FEX/HG38 (14, 25, 26), LGR6, and LGR7. Based on sequence comparison of all 12 known LGRs from diverse species, the expanding LGR genes can be divided into three subgroups: 1) three glycoprotein hormone receptors together with fly LGR1 and LGRs from sea anemone and nematode; 2) mammalian LGR4, LGR5, and LGR6 together with fly LGR2; and 3) human LGR7 together with the snail LGR. Although fly LGR1 and LGR2 are closely related based on sequence comparison, these two fly receptors belong to two separate subgroups. Of interest, the fly LGR1, similar to mammalian glycoprotein hormone receptors, is capable of coupling to the Gs protein. In contrast, both wild-type fly LGR2 and its mutants, similar to mammalian wild-type LGR4 and LGR5 as well as their mutants (14), did not confer major increases in basal cAMP production.
Analysis of the genomic and cDNA structures of the newly identified fly LGR2 indicated that this fly gene is similar to mammalian glycoprotein hormone receptor genes (27, 28) in having 10–11 exons and the entire transmembrane region of the protein is encoded by a single exon. Because the fly LGR1 gene (11) and the nematode LGR gene (13) both contain multiple exons encoding their transmembrane region, it is likely that the ancestral LGR could have multiple exons for the transmembrane region and the intron-exon arrangement of fly LGR2 and mammalian glycoprotein hormone receptors are the result of intron loss during evolution. Analysis of multiple olfactory receptor genes also indicated that fly and worm genes are interrupted by introns but their human counterparts are intronless (29).
Mutant LH and TSH receptors, found in patients with familial precocious puberty and nonimmune hyperthyroidism, respectively, are characterized by ligand-independent constitutive activity (17, 23, 24). However, the wild-type glycoprotein hormone receptors in human show negligible constitutive activation in the unliganded state, in direct contrast to the high constitutive activity associated with unliganded fly LGR1. Of interest, the nematode LGR also exhibited constitutive activity when transfected into mammalian cells (13). The allosteric ternary complex model suggests that the GPCRs usually exist in the inactive state in the absence of ligands, whereas ligand binding converts the receptor into an active state capable of coupling to the G proteins. This isomerization of receptor proteins involves conformational changes, which may occur spontaneously or may be induced by agonists or appropriate mutations that abrogate the normal constraining function of the receptor, allowing it to relax into the active conformation (30). Based on the observed constitutive activity found in LGRs from the two lower species, one could speculate that the ancestral LGRs are constitutively active, whereas the receptors in higher species develop ligand-induced activation accompanied by a loss of basal activity during evolution. Concomitant decreases in basal constitutive activity and increases in ligand-induced cAMP lead to an increase in the signal to noise ratios characteristic of an advanced ligand-signaling mechanism.
Studies on mutant LH and TSH receptors indicated that at least two regions of the human glycoprotein hormone receptors are important for their activation. Two prominent LH receptor point mutations have been analyzed in greater detail. The Asp 578 side chain in the transmembrane VI serves as a properly positioned hydrogen bond acceptor that is important for stabilizing the inactive state of the LH receptor. A bulky aromatic side chain at this position, rather than the negative charge, destabilizes the inactive receptor conformation (31). In addition, the activation mechanism for the point mutant in Asp 564 in the intracellular loop 3 of the human LH receptor has also been studied. It was found that a negative charge at position 564 might be important for maintaining the inactive LH receptor conformation. Replacement of the negatively charged aspartic acid at position 564 by a neutral amino acid (glycine or asparagine) or by a positively charged lysine led to agonist-independent cAMP formation, whereas introduction of a negatively charged glutamic acid led to a silent mutation. Thus, an anionic amino acid at this position may be required to constrain the receptor by interacting with a cationic residue (32, 33). Based on the conservation of these two residues in the two fly genes, we constructed putative gain-of-function mutants of the fly receptors and observed further augmentation of the basal activity of fly LGR1 following point mutation at either transmembrane VI or intracellular loop 3. These findings suggest that the molecular mechanism underlying the constraint of fly LGR1, like the nematode LGR (13), is similar to that for the human LH receptor.
Although no major increase in basal cAMP production was observed in cells overexpressing fly LGR2, one could still observe low basal cAMP production mediated by this receptor when compared with cells transfected with the empty vector (Fig. 4A). In addition, the D744G fly LGR2 mutant also showed a higher basal cAMP production than the wild-type receptor (Fig. 5A). These data suggested that fly LGR2 is more constrained than fly LGR1, similar to the differing constraints found for human LH and FSH receptors (18). Further work is needed to elucidate the signaling mechanism for fly LGR2.
The ligands for the two fly LGRs are presently unknown and treatment of cells expressing wild-type fly LGR1 or fly LGR2 with human gonadotropins led to negligible increases in cAMP production (data not shown). The availability of the cDNAs for fly LGR1 and fly LGR2 and the understanding of the potential signaling pathway for fly LGR1 allow future testing of putative fly glycoprotein hormones in the activation of these receptor proteins. Future studies on the structural-functional relationship of LGRs from fly and other species could also provide understanding of the co-evolution of ligand receptor pairs for this important family of GPCRs.
Note Added in Proof
A recent paper described the molecular cloning, genomic organization, developmental regulation, and a knock-out mutant of fly LGR-2. (Eriksen, K. K., F. Hauser, M. Schiott, K. M. Pedersen, L. Sondergaard, and C. J. Grimmelikhuijzen, Genome Res 2000 10:924–938).
Acknowledgments
We thank the National Pituitary and Hormone Distribution Program for the cAMP antiserum and Ms. Caren Spencer for editorial assistance.
