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) from Drosophila (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.

Figure 1

Schematic representation of the DLGR-2 cDNA and genomic clones and the organization of the DLGR-2 gene. (A) Positions of the PCR clones. (B) Schematic drawing of the composite cDNA (top) and the organization of the receptor gene (bottom). The exons are given as bars and numbered 1–15. We named the introns after the preceeding exons(e.g., intron 1 follows exon 1). The narrow and broad bars represent noncoding and coding regions, respectively. The DNA region coding for the transmembrane domain is black and that coding for the Leu-rich repeats are gray. (C) The positions of the genomic P1 clones, DS00180 and DS01514, from the Berkeley Drosophila Genome Project.

Figure 2

cDNA and deduced amino acid sequence of DLGR-2. This figure is compiled from the sequences of the overlapping cDNA clones 5′C-2, 5B, and 3′4-J (Fig. 1A). Nucleotides are numbered from 5′ to 3′ end, and the amino acid residues are numbered starting with the first ATG in the open reading frame. Introns are indicated by arrows and numbered 1–14. The seven membrane-spanning domains are boxed and labeled TM I—VII. The proposed signal sequence is shaded. Spades indicate potential amino-glycosylation sites. The inframe stop codon in the 5′ region, upstream of the assigned start codon is underlined twice; the translation termination codon is indicated by an asterisk (*). Putative polyadenylylation sites at the 3′ end are underlined.

Figure 2

cDNA and deduced amino acid sequence of DLGR-2. This figure is compiled from the sequences of the overlapping cDNA clones 5′C-2, 5B, and 3′4-J (Fig. 1A). Nucleotides are numbered from 5′ to 3′ end, and the amino acid residues are numbered starting with the first ATG in the open reading frame. Introns are indicated by arrows and numbered 1–14. The seven membrane-spanning domains are boxed and labeled TM I—VII. The proposed signal sequence is shaded. Spades indicate potential amino-glycosylation sites. The inframe stop codon in the 5′ region, upstream of the assigned start codon is underlined twice; the translation termination codon is indicated by an asterisk (*). Putative polyadenylylation sites at the 3′ end are underlined.

Figure 3

Amino acid sequence comparison between DLGR-2, LGR-5, LGR-4, DLGR-1, the human TSH receptor (TSHR), and the LGR from the sea anemone Anthopleura elegantissima (ALGR). Broken lines represent spaces introduced to optimize alignment. The Gly-rich repeats of ALGR (Nothacker and Grimmelikhuijzen 1993) were omitted to facilitate alignment. Amino acid residues that are identical between DLGR-2 and at least one of the other receptors are boxed. Known intron–exon transitions in the genes coding for the receptors are shaded at the corresponding amino acid residues. The positons of the aliphatic and aromatic residues characteristic for the Leu-rich repeats in DGLR-2 are marked by asterisks (*) or solid circles (●). The solid circles also mark intron–exon transitions in the DGLR-2 gene that occur at the same positions and have the same intron phasing as in the DLGR-1, TSHR, and ALGR genes. The seven membrane-spanning domains are indicated by I–VII. The open circles (○) mark conserved cystein residues, flanking the region containing the Leu-rich repeats. The amino acid residue positions are given at right. The amino acid sequences of DLGR-1 and the mammalian and sea anemone receptors as well as the intron–exon positions in their genes are from Misrahi et al. (1990); Gross et al. (1991); Nothacker and Grimmelikhuijzen (1993); Hauser et al. (1997); Hsu et al. (1998); and Vibede et al. (1998).

Figure 3

Amino acid sequence comparison between DLGR-2, LGR-5, LGR-4, DLGR-1, the human TSH receptor (TSHR), and the LGR from the sea anemone Anthopleura elegantissima (ALGR). Broken lines represent spaces introduced to optimize alignment. The Gly-rich repeats of ALGR (Nothacker and Grimmelikhuijzen 1993) were omitted to facilitate alignment. Amino acid residues that are identical between DLGR-2 and at least one of the other receptors are boxed. Known intron–exon transitions in the genes coding for the receptors are shaded at the corresponding amino acid residues. The positons of the aliphatic and aromatic residues characteristic for the Leu-rich repeats in DGLR-2 are marked by asterisks (*) or solid circles (●). The solid circles also mark intron–exon transitions in the DGLR-2 gene that occur at the same positions and have the same intron phasing as in the DLGR-1, TSHR, and ALGR genes. The seven membrane-spanning domains are indicated by I–VII. The open circles (○) mark conserved cystein residues, flanking the region containing the Leu-rich repeats. The amino acid residue positions are given at right. The amino acid sequences of DLGR-1 and the mammalian and sea anemone receptors as well as the intron–exon positions in their genes are from Misrahi et al. (1990); Gross et al. (1991); Nothacker and Grimmelikhuijzen (1993); Hauser et al. (1997); Hsu et al. (1998); and Vibede et al. (1998).

Figure 4

Leu (Ile/Val/Ala/Phe)-rich repeats in the amino-terminal region of DLGR-2 and the rat LH receptor. (A) Consecutive segments (L1–L18) within the amino terminus of DLGR-2 were aligned and small gaps (–) were introduced to show that many of the aliphatic and aromatic residues in one segment occur at a similar position in the other segments. These residues are boxed. In addition, Asn residues at positions typical for Leu-rich repeats (Kobe and Deisenhofer 1994, 1995) are also marked. The shaded aliphatic residues correspond to intron–exon transitions in the receptor gene and are given at the start of the repeating segments. Most of the repeating segments, therefore, are coded for by distinct exons. Only complete segments, lying within the cluster of Cys residues bordering the Leu-rich repeats of Leu-rich repeats-containing proteins (Kobe and Deisenhofer 1994, 1995), are taken into account. However, 0.8 repeat flanking L1 and 0.3 repeat flanking L18 might contribute to an additional repeat (altogether 19 Leu-rich repeats) in DLGR-2. (B) Similar alignment of the rat LH receptor. Data from Bhowmick et al. (1996).

Figure 5

Partial nucleotide sequence of the genomic DNA around intron 1. The numbers in this figure refer to the nucleotide positions of the cDNA of Fig. 2. Uppercase and lowercase letters represent the nucleotides in exons 1 and 2 and in intron 1, respectively. The three gt splicing donor sites and the two ag acceptor sites are underlined and printed in boldface type. The three donor and two acceptor sites give six possible mRNAs, of which four have been identified. The four identified mRNAs have the combinations d1/a1 (donor 1/acceptor 1), d1/a2, d2/a2, and d3/a2. Fig. 2 corresponds to d3/a2.

Figure 6

Southern blot analysis. Genomic DNA from D. melanogaster Canton S. was digested with one of five restrictions enzymes (BamHI, EcoRV, SacI, SalI, and XbaI). After electrophoresis and blotting, the genomic fragments were hybridized with a cDNA fragment coding for the Leu-rich repeats 10–18 and the transmembrane domain of DLGR-2. The size of the markers (left) is in kb. All lanes show a single hybridization band with exception of the lane containing the XbaI fragment.

Figure 7

Northern blot analysis of the expression of the DLGR-2 gene at several developmental stages. Marker size (left) is given in kb. (A) Poly(A)⁺ RNA from each developmental stage was hybridized with a cDNA fragment coding for the seven-transmembrane domain of DLGR-2. This Northern blot shows that mRNA is only present in embryos and pupae, not in larvae and adult (mixed male and female) flies. (B) Hybridization of the same blot as in A with a cDNA probe coding for RP49. The RP49 gene is regarded to be expressed in all developmental stages (O'Connell and Rosbash 1984; Kerrebrock et al. 1995).

Figure 8

Results of crossing 15 different deficiency stains (carrying a well-defined deletion in an area of chromosome 2 close to or including the DLGR-2 gene, see Table 4) with mutant P919 that carries a P element insertion in the DLGR-2 gene. (A) Schematic description of the crossings. (P) The DLGR-2 gene, carrying the P element insertion; (Cy^-) curly wing mutation; (D) deletion mutation. The Cy^- mutation is dominant and homozygous lethal. Therefore, if the offspring only consists of curly wing flies (P/Cy^- or Cy^-/D), the P/D combination is nonviable and the deletion can not be rescued by the P element-inserted DLGR-2 region. On the other hand, if the offspring contains flies with normal flat wings (P/D), the deletion mutant can be rescued. (B) Map of the deletion strains used in the rescue experiments (horizontal lines). The abscissa shows the region 34A-35F of Drosophila chromosome 2L. The stock numbers of the mutants are given at right (ordinate), together with the information about whether the deletion mutant can be rescued (+) or not (−) in a cross with mutant fly P919. For stock numbers 45490 and 42850 only the left ends of the deletions are shown, as these deletions are very large. The vertical shaded bar indicates the borders of the chormosomal region determined by the inability of mutant P919 to rescue the deletion mutants. This region is 34E5-F1, which is exactly the region where the DLGR-2 gene has been located. This is an independent and strong indication that the P element that we have earlier shown to be inserted in the DLGR-2 gene, is the only cause of lethality in mutant P919.

Figure 9

Schematic representation of four LGR cDNAs. The regions coding for the seven-transmembrane loops are given as black bars, those for the Leu-rich repeats as gray bars. Only complete Leu-rich repeats (see Fig. 4) are taken into account. Intron positions in the four LGR genes are indicated by arrows. (A) The cDNA coding for DGLR-2. (B) The cDNA coding for DLGR-1 (Hauser et al. 1997). (C) The cDNA coding for the rat FSH receptor (Heckert et al. 1992). (D) The cDNA coding for the LGR from the sea anemone A. elegantissima (Nothacker and Grimmelikhuijzen 1993; Vibede et al. 1998).