Molecular cloning and characterisation of a novel membrane receptor gene from the lobster Jasus edwardsii
Department of Zoology, University of Canterbury, Christchurch 1, Private Bag 4800, New Zealand
* Present address: Biotechnology Center, University of Connecticut, Storrs, Connecticut, USA
Author for correspondence at present address: Department of Zoology, University of Canterbury, Private Bag 4800, Christchurch 1, New Zealand (e-mail: f.sin{at}zool.canterbury.ac.nz)
Accepted June 13, 2001
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Summary |
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Key words: signal transducer, G protein-coupled receptor, membrane protein, lobster, Jasus edwardsii, eyestalk
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Introduction |
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In the crustacean, the eyestalk X-organ sinus gland complex is an important site for the production of several endocrine factors, which are implicated in almost every aspect of crustacean physiology. These include the pigment concentration and dispersion hormones, and members of the CHH/MIH/VIH peptide family, which consists of the crustacean hyperglycemic hormone (CHH), a putative moult-inhibiting hormone (MIH), and the vitellogenesis-inhibiting hormone (VIH). These are responsible for the regulation of glucose levels in the haemolymph, and inhibition of moulting and gonad development, respectively (Quackenbush, 1986; Keller, 1992). Precisely how these hormones transmit their messages to the cell is not known.
In a recent study, in order to isolate factors that are involved in the regulation of growth in the lobster, Jasus edwardsii, we constructed cDNA libraries using mRNAs from the eyestalk and a genomic library using DNA from gill tissues. Using a novel peptide sequence (NPS) (Khoo and Sin, 1999) isolated from the eyestalk as a probe in library screenings we isolated a number of cDNA clones. In the present study, we describe the characterisation of two novel protein genes from the lobster. We show that these genes encode proteins that are associated with membranes and share characteristics with the GPCRs of other organisms.
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Materials and methods |
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Extraction of poly(A)+ RNA
Total RNA was extracted from 66 eyestalks (45 µg per eyestalk), and also from the epithelial tissue, gill, heart, hepatopancreas, and abdominal flexor muscle tissue of a single individual, by the acid guanidinium thiocyanatephenolchloroform extraction procedure (Chomczynski and Sacchi, 1987). Poly(A)+ RNA was extracted from total RNA using the polyATtract® mRNA Isolation System (Promega, Madison, WI, USA).
Cell-free translation
The integrity of the mRNA isolated from the eyestalks was tested by cell-free translation using the wheat germ translation kit according to the manufacturers instructions (Boehringer, Mannheim). The proteins were analysed by SDS-polyacrylamide gel electrophoresis on 12 % gels, as described previously (Laemmli, 1970). After electrophoresis, the gels were treated in Amplify (Amersham Pharmacia Biotech, Little Chalfont, UK), dried under vacuum, and exposed to X-ray film for 3 days.
cDNA library construction
Using oligo(dT)1218 primer, cDNA was synthesized from 5 µg of eyestalk poly(A)+ RNA using the TimeSaver cDNA synthesis kit (Amersham Pharmacia Biotech, Little Chalfont, UK). The cDNA, which appeared as a smear between 0.46 and 2.3 kilobases (kb) in an agarose gel, was cloned into the EcoRI site of gt 10 vector, and packaged in vitro (Rosenberg, 1987) to give several libraries with a combined size of 5.6x106 plaque-forming units (p.f.u.) µg1 cDNA.
cDNA library screening
The cDNA library was screened initially by in situ plaque hybridisation (Sambrook et al., 1989), using a genomic DNA clone (Khoo, 1996) containing the NPS sequence (Khoo and Sin, 1999). Phage DNA was prepared from plate lysate stocks of phage samples showing positive hybridisation to the genomic clone (Santos, 1991; Sambrook et al., 1989), and analysed by Southern hybridisation to NPS. For library screening, Hybond-N nylon filters were washed to a final stringency of 1xSSC (0.15 mol l1 NaCl, 0.015 mol l1 sodium citrate)/0.1 % SDS at 65°C, and exposed for autoradiography for 15 days. The DNA probes (10100 ng) were labelled with [-32P]dCTP (1.8 MBq, 3000 Ci mmol) to 108109 c.p.m. µg1 DNA using the random priming kit (NEBlot kit, New England Biolabs, MA, USA).
For sequencing, DNA from the NPS-positive cDNA clones was extracted using the WIZARD prep kit (Promega, Madison, WI, USA) according to the manufacturers instructions. The purified DNA was digested with NotI restriction endonuclease (New England Biolabs, MA, USA). The digested DNA fragments were gel-purified and cloned into the NotI restriction site of a cDNA cloning plasmid vector, pSPORT 1 (Gibco BRL, New York).
Sequence analysis of cDNA clones
Double-stranded plasmid DNA templates were isolated by the boiling method (Sambrook et al., 1989) and were sequenced manually from both ends by the dideoxy sequencing method (Sanger et al., 1977) using the T7Sequencing kit (Amersham Pharmacia Biotech, Little Chalfont, UK). Nucleotide sequences were analysed using DNASIS, and the NCBI BLAST tool (Altschul et al., 1997).
Northern blot analysis of mRNAs
13 µg of poly(A)+ RNA isolated from epithelial, eyestalk, gill, heart, hepatopancreas and abdominal muscle tissue were fractionated on a 1.2 % agarose gel as described previously (Khoo and Sin, 1999), and transferred by capillary action in 10x SSC onto Hybond-N (Amersham Pharmacia Biotech, Little Chalfont, UK). Heat-denatured HindIII digested DNA was included as a molecular size marker. Following transfer, the membrane was dried and the RNA was crosslinked to the membrane by UV transillumination.
Northern hybridisation was carried out as described by Lee et al. (Lee et al., 1992), but with a final post-hybridisation wash in 0.1x SSC/0.1 % SDS at 65°C for 15 min. The membrane was exposed to X-ray film at 80°C with intensifying screens for 2 h, 4 h and 14 h. The membrane was probed with the two cDNA sequences isolated. Prior to hybridisation with the second probe, the membrane was stripped by washing twice in 0.1x SSC/0.5 % SDS at 95°C for 20 min, then once in 0.1 % SDS at 100°C for 20 min. After stripping, the membrane was then exposed to an X-ray film to determine whether the stripping of the radioactive materials was efficient. When no radioactivity was detected on the X-ray film after exposure for 16 h, the membrane was used for hybridisation with the second probe.
Tissue expression of cDNA sequences using in situ hybridisation
In situ hybridisation was carried out on epithelial tissue dorsal to the heart, eyestalk, gill, heart and abdominal flexor muscle in the cephalothorax region as described previously (Bloch et al., 1986; Khoo and Sin, 1999).
cDNA inserts were excised from the pSPORT plasmid with NotI restriction endonuclease, and gel purified by electroelution. 25 ng of cDNA insert were labelled with [-32P]dCTP (1.5 MBq, 3000 Ci mmol), to 108 c.p.m. µg1 using the random prime labelling kit (Boehringer, Mannheim). The radiolabelled probe was purified by column chromatography prior to use as probes in northern blot analysis and in situ hybridisation.
As controls, RNase-digested tissue sections were included in each set of experiments. Control tissue sections were incubated in 2x SSC containing 100 µg ml1 RNase A at 37°C for 60 min prior to in situ hybridization. Both control and experimental slides were developed after a 3-day exposure period, which was the time required to produce an autoradiographic image according to Bloch et al. (Bloch et al., 1985).
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Results |
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Sequence analysis of cDNA clones
The nucleotide and deduced amino acid (aa) sequences of the 568 bp cDNAs of clones peJK2 and peJK3 are shown in Fig. 2. peJK2 and peJK3 shared 96.6 % sequence identity over a stretch of 558 bp. peJK2 contained an open reading frame encoding a 110-aa polypeptide with a deduced molecular mass of 11,177 Da, and a translation stop codon at nucleotide (nt) 401 followed by an untranslated region, which ended with a poly(A)+ tail 146 nt downstream. The polyadenylation signal AATAAA (Birnstiel et al., 1985) was located between nucleotides 523 and 530, 26 nucleotides upstream from the poly(A)+ tail, which was 19 residues long.
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Analysis of the deduced peJK proteins
The crustacean eyestalk neuropeptides CHH, VIH and MIH are considered to be related (Keller, 1992; de Kleijn and Van Herp, 1995). Multiple sequence alignment of the putative peptides of peJK2 and peJK3 with these sequences, retrieved from the BLASTP searches, and published crustacean eyestalk neuropeptide sequences (Lacombe et al., 1999) was performed using CLUSTALW (Thompson et al., 1994) on the European Bioinformatics Institute (ebi) server. Alignment of the sequences showed that the motifs present in the CHH/VIH family (Lacombe et al., 1999) were absent from the deduced protein encoded by peJK2 and peJK3. The invariant six cysteine residues, which form the three disulfide bridges, and the conserved aa residues located in the vicinity of the six cysteine residues (de Kleijn and Van Herp, 1995) were absent from the deduced proteins.
The hydrophobicity plots of peJK2 and peJK3 show that the N-terminal and central domain of these putative peptides were hydrophobic, with a high proportion of consecutive valine and alanine residues (Fig. 3). The hydrophobic domain suggests that this domain was buried in the membrane (von Heijne, 1987; Lehninger et al., 1993). The theoretical pI values of peJK2 and peJK3 were calculated to be 10.0 and 10.79 (Bjellqvist et al., 1993).
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The deduced proteins were further characterised using TopPred 2 (von Heijne, 1992), which predicted two transmembrane alpha helices present in the deduced proteins encoded by peJK 2 and peJK3 (Fig. 4). In peJK2 protein, helix 1 is between aa 17 and 37, and helix 2 between aa 43 to 63. Similar helices were found in peJK3, between aa 17 and 37, and aa 46 and 66. Motifs were detected using PROSITE (Hofman et al., 1999). peJK2 had one GPCR motif II, VAVVVAMAATVARGN, located in the deduced aa sequence in the second transmembrane helix, but a similar sequence (VAIVVAMAATMARAN) was identified in peJK3 in a similar region of the deduced peptide. A protein kinase C phosphorylation site (TNK) (Kishimoto et al., 1985) was also detected in position 76 in peJK2 and position 79 in peJK3.
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Discussion |
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Sequence analyses suggest that the deduced proteins encoded by these two cDNA clones have two transmembrane alpha helices. Analyses of the aa sequences using four different programmes, TopPred 2 (von Heijne, 1992), SOSUI (Hirokawa et al., 1998), DAS (Cserzo et al., 1997) and ALOM (Klein et al., 1985; Nakai and Kanehisa, 1992), all indicate the presence of two transmembrane helices (Fig. 4). Analysis of the aa sequence of the deduced proteins further suggests the presence of a signal peptide in these peptides. The signal peptide itself is a transient N-terminal signal sequence found in most secretory proteins, and serves to initiate export across the endoplasmic reticulum (von Heijne, 1986; Lehninger et al., 1993). Thus, it is highly likely that these peptides are synthesised and transported to the membrane where the signal peptide is cleaved and the remaining peptide forms part of the membrane structure.
Further, peJK2 has a GPCR motif in the second transmembrane helix, while peJK3 had a sequence in the same region showing high homology to the GPCR motif. The presence of a GPCR motif strongly indicates that these peptides may function as signal transducers. This, however, is in contrast to the rhodopsin-like GPCRs, a widespread protein family that includes hormones, neurotransmitter and light receptors, which have seven GPCR motifs (Lameh et al., 1990; Attwood and Findlay, 1993, Bockaert and Pin, 1999). In order for the peptides encoded by peJK2 and peJK3 to function as a signal transducer, they would have to form a complex structure consisting of multiple units of the peptides. The variation of the GPCR motif-like sequence in peJK3 may suggest that this is an error resulting from sequencing or cloning, or it might truly represent an allelic form of the GPCR motif in the lobster.
The presence of a kinase C phosphorylation site in these deduced proteins strongly suggests that they are phosphoproteins and their function may be mediated by protein kinase C (Pitcher et al., 1998). Phosphorylation of the receptor molecules after interaction with ligands is a known mechanism in GPCR regulation (Daaka et al., 1997; Carman et al., 1998).
At present, there is no explanation for the presence of a nuclear localisation signal in the carboxyl terminal of both deduced proteins. Detailed functional analyses are required to determine the functional significance and the mechanism of function of these proteins.
From the evolutionary perspective, the identified features of these deduced peptides suggest that the proteins encoded by the peJK genes may be related to, or derived from, the same ancestral gene(s) for the GPCR family of proteins. Their evolutionary relationship to other GPCR proteins will await the elucidation of the function and mechanism of action of these proteins.
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Acknowledgments |
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