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Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University School of Medicine, Stanford, California 94305-5317
Address all correspondence and requests for reprints to: Aaron J. W. Hsueh, Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford, California 94305-5317.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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- 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.
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Materials and Methods |
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Sequence analysis
Based on amino acid sequences of mammalian glycoprotein hormone
receptors, the genomic sequence of the 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
mM L-glutamine. Before transfection, 2 x
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 x 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
x 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 a
-spectrometer. Background binding was determined by adding an
excess amount of the synthetic FLAG peptide at a concentration of 100
µg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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). 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.
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, 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.
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) 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.
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(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).
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, 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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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.
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Note Added in Proof |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionNote Added in ProofReferences |
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Acknowledgments |
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Footnotes |
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Received June 8, 2000.
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