Molecular Cloning, Genomic Organization, Developmental Regulation, and a Knock-out Mutant of a Novel Leu-rich Repeats-containing G Protein-coupled Receptor (DLGR-2) from Drosophila melanogaster

Abstract

After screening the Berkeley Drosophila Genome Project database with sequences from a recently characterized Leu-rich repeats-containing G protein-coupled receptor (LGR) fromDrosophila (DLGR-1), we identified a second gene for a different LGR (DLGR-2) and cloned its cDNA. DLGR-2 is 1360 amino acid residues long and shows a striking structural homology with members of the glycoprotein hormone [thyroid-stimulating hormone (TSH); follicle-stimulating hormone (FSH); luteinizing hormone/choriogonadotropin (LH/CG)] receptor family from mammals and with two additional, recently identified mammalian orphan LGRs (LGR-4 and LGR-5). This homology includes the seven transmembrane region (e.g., 49% amino acid identity with the human TSH receptor) and the very large extracellular amino terminus. This amino terminus contains 18 Leu-rich repeats—in contrast with the 3 mammalian glycoprotein hormone receptors and DLGR-1 that contain 9 Leu-rich repeats, but resembling the mammalian LGR-4 and LGR-5 that each have 17 Leu-rich repeats in their amino termini. The DLGR-2 gene is >18.6 kb pairs long and contains 15 exons and 14 introns. Four intron positions coincide with the intron positions of the three mammalian glycoprotein hormone receptors and have the same intron phasing, showing that DLGR-2 is evolutionarily related to these mammalian receptors. The DLGR-2 gene is located at position 34E-F on the left arm of the second chromosome and is expressed in embryos and pupae but not in larvae and adult flies. Homozygous knock-out mutants, where the DLGR-2 gene is interrupted by a P element insertion, die around the time of hatching. This finding, together with the expression data, strongly suggests that DLGR-2 is exclusively involved in development.[The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL database with accession no. AF142343.]

The Ectodomain of the Luteinizing Hormone Receptor Interacts with Exoloop 2 to Constrain the Transmembrane Region

“…The observed ligand signaling of different chimeric receptor mutants consisting of human LH receptor and fly LGR2 also suggested that fly LGR2 likely belongs to the same subgroup of LGRs together with glycoprotein hormone receptors and fly LGR1 (8). Despite the lack of knowledge on the endogenous ligand(s) for the fly LGR2, understanding of the signaling pathway for this orphan receptor could facilitate future identification of its ligands using bioinformatic and biochemical approaches (36).…”

“…Recent studies indicate the evolution of a large family of the leucine-rich repeat-containing, G protein-coupled receptors (LGRs) with at least seven members in mammals, including the well studied glycoprotein hormone receptors (3,4) and LGR4 -7 (5,6). In addition, homologous LGRs were found in fly (LGR1 and LGR2) (7,8), nematode (nLGR) (9), sea anemone (10), and snail (11). These genes can be divided into three subgroups, each with unique structural characteristics (6).…”

“…1 The abbreviations used are: LH, lutropin; LGR: leucine-rich repeatcontaining G protein-coupled receptor; FSH: follitropin; TSH, thyrotropin; GPCR, G protein-coupled receptor; TM, transmembrane; D-PBS, Dulbecco's phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium. ously described (22) using cDNA encoding human LH receptors (23), human FSH receptors (22), or fly LGR2 (8). PCR was performed with Vent DNA polymerase (New England Biolabs, Inc., Beverly, MA) in accordance with the manufacturer's instructions.…”

A Novel Approach for Directing Transgene Expression in Drosophila: T2A-Gal4 In-Frame Fusion

“…Characterizing RK and the cells that express it has been impeded in the fly by the complexity of the rk gene locus. Two splice variants, rk-RA and rk-RF, with different 59 start sites, are predicted from the rk genomic sequence, and other transcripts have been identified (Eriksen et al 2000;Nishi et al 2000). The longest variant, rk-RA, has highly conserved, putative regulatory elements throughout its first, large intron and multiple possible translation start sites, the first of which is preceded by six out-of-frame ATG triplets in the putative 59 UTR (Eriksen et al 2000).…”

“…Two splice variants, rk-RA and rk-RF, with different 59 start sites, are predicted from the rk genomic sequence, and other transcripts have been identified (Eriksen et al 2000;Nishi et al 2000). The longest variant, rk-RA, has highly conserved, putative regulatory elements throughout its first, large intron and multiple possible translation start sites, the first of which is preceded by six out-of-frame ATG triplets in the putative 59 UTR (Eriksen et al 2000). Direct knock-in of the Gal4 transgene into the predicted start site by homologous recombination resulted in no detectable expression of Gal4 (F. Diao and B. H. White, unpublished observations).…”