Figure 1. 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. Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591. Endocrinology. 2000;141(11):4081-4090. doi:10.1210/endo.141.11.7744 Endocrinology | Copyright © 2000 by the Endocrine Society

Figure 1. 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. Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591. Endocrinology. 2000;141(11):4081-4090. doi:10.1210/endo.141.11.7744 Endocrinology | Copyright © 2000 by the Endocrine Society

Figure 2. 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. Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591. Endocrinology. 2000;141(11):4081-4090. doi:10.1210/endo.141.11.7744 Endocrinology | Copyright © 2000 by the Endocrine Society

Figure 2. 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. Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591. Endocrinology. 2000;141(11):4081-4090. doi:10.1210/endo.141.11.7744 Endocrinology | Copyright © 2000 by the Endocrine Society

Figure 3. Phylogenetic relatedness of LGRs from diverse species Figure 3. 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. Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591. Endocrinology. 2000;141(11):4081-4090. doi:10.1210/endo.141.11.7744 Endocrinology | Copyright © 2000 by the Endocrine Society

Figure 4. 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). Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591. Endocrinology. 2000;141(11):4081-4090. doi:10.1210/endo.141.11.7744 Endocrinology | Copyright © 2000 by the Endocrine Society

Figure 5. 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. Characterization of Two Fly LGR (Leucine-Rich Repeat-Containing, G Protein-Coupled Receptor) Proteins Homologous to Vertebrate Glycoprotein Hormone Receptors: Constitutive Activation of Wild-Type Fly LGR1 But Not LGR2 in Transfected Mammalian Cells**This study was supported by NIH Grant HD-23273. The GenBank submission number for fly LGR2 is AF274591. Endocrinology. 2000;141(11):4081-4090. doi:10.1210/endo.141.11.7744 Endocrinology | Copyright © 2000 by the Endocrine Society