Genome wide survey of G protein-coupled receptors in Tetraodon nigroviridis
© Metpally and Sowdhamini; licensee BioMed Central Ltd. 2005
Received: 25 February 2005
Accepted: 15 July 2005
Published: 15 July 2005
The G-protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane proteins. They play a central role in physiological processes affecting almost all aspects of the life cycle of an organism. Availability of the complete sets of putative members of a family from diverse species provides the basis for cross genome comparative studies.
We have defined the repertoire of GPCR superfamily of Tetraodon complement with the availability of complete sequence of the freshwater puffer fish Tetraodon nigroviridis. Almost all 466 Tetraodon GPCRs (Tnig-GPCRs) identified had a clear human homologue. 189 putative human and Tetraodon GPCR orthologous pairs could be identified. Tetraodon GPCRs are classified into five GRAFS families, by phylogenetic analysis, concurrent with human GPCR classification.
Direct comparison of GPCRs in Tetraodon and human genomes displays a high level of orthology and supports large-scale gene duplications in Tetraodon. Examples of lineage specific gene expansions were also observed in opsin and odorant receptors. The human and Tetraodon GPCR sequences are analogous in terms of GPCR subfamilies but display disproportionate numbers of receptors at the subfamily level. The teleost genome with its expanded set of GPCRs provides additional and interesting comparators to study both evolution and function of these receptors.
The G-protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane proteins, accounting for 1–2% of the vertebrate genome. GPCRs are characterized by the presence of highly conserved molecular architecture encoding seven transmembrane (TM) hydrophobic regions linked by three extracellular loops that alternate with three intracellular loops . The extracellular N-terminus is usually glycosylated and the cytoplasmic C-terminus is generally phosphorylated. The extracellular side of these receptors contains residues that are specifically recognized by ligands and is therefore involved in ligand-specific binding. The endogenous ligands for GPCRs have exceptionally high chemical diversity. They include biogenic amines, glycoproteins, ions, lipids, nucleotides, peptides and proteases. Moreover, the sensation of exogenous stimuli such as light, odor and taste is also mediated via this superfamily of receptors. Ligand-induced activation of all GPCRs leads to a conformational change of the receptor and triggers a family of heterotrimeric GTP binding proteins (G proteins) and modulates several cellular signaling pathways.
GPCRs have been aggressively pursued as drug targets due to their central role in physiological processes affecting almost all aspects of the life cycle of an organism . Almost half of the GPCRs are likely to encode sensory receptors and the rest of receptors could be considered as potential drug targets . It is estimated that about 50% of all current drug targets are GPCRs and are the most successful of any target class in terms of therapeutic benefit [4, 5]. A major goal of GPCR research is to expand the knowledge of GPCR structure/function in order to validate additional GPCR family members as tractable drug targets. Much effort, therefore, has been made to identify novel GPCRs and their ligands with potential therapeutic value [6–8].
The completion of several other vertebrate and invertebrate genome sequencing projects paves the way for "functional genomics". The quest for assigning function to putative gene products exploits the sequence and structural similarities to known genes and further could be elucidated using molecular biology techniques [9, 10]. Such studies have important implications in biology and in understanding the evolution of distinct organisms. Sequencing of the model organisms can be an important source of information on the function of human target class members. For example, evolutionary comparison of GPCR sequences between species can help to identify conserved motifs and may recognize key functional residues [11–13]. The majority of GPCR functional data have been derived from studies in genetic models such as mice, rat, worm and Drosophila; additional species provide new comparators for GPCR studies. Teleost fish, Tetraodon nigroviridis is one of the smallest known vertebrate genomes. It has all the specialized functions of higher vertebrates and can be a good vertebrate model system to study [14, 15]. The first available nearly complete sequence of T. nigroviridis genome now allows for the identification and analysis of its full set of GPCRs. Here, we describe the genome wide survey of Tnig-GPCR repertoire and a detailed analysis of opsin, fish-odorant receptors (FOR) and taste receptors (T1R).
Results and discussion
G protein-coupled receptors of Tetraodon nigroviridis (Tn). The numbers predicted in each family and sub-family are shown in comparison to humans (Hs)
1+3 = 4
2+2 = 4
1+2 = 3
1+2+1 = 4
1+4+1+4 = 10
1+2+1+1 = 5
Rhodopsin family in Tetraodon has up to one and half times the number of receptors compared with human (excluding olfactory receptors), whereas about two fold as many GPCR sequences as in fugu and about three fourth of the zebrafish GPCRs . Tetraodon also has similar numbers of frizzled receptors as expected in mammals and fish genomes. Some of the gene families in Tetraodon like opsins and fish odorant receptors have shown species-specific expansions similar to trace amine receptors in zebrafish . However, taste receptors type 2 (TAS2) and mas related (MRG) receptors seem to be absent in Tetraodon like other known fish genomes .
23 candidate odorant receptors (OR) were identified in fish odorant receptor (FOR) subfamily of rhodopsins in Tetraodon. These OR genes are found in clusters of 3–4 members in the Tetraodon genome, located on different chromosomes. They display higher sequence identity within a cluster suggesting tandem duplication events might be responsible for OR gene family expansion in Tetraodon as observed in the genomes of every vertebrate organism investigated earlier, including zebrafish, mice and humans . Phylogenetic analysis of Tetraodon ORs with fish odorant receptor subfamily members (mainly zebrafish, channel catfish, Japanese pufferfish, medaka fish, goldfish etc) grouped them into six clusters of orthologues with very high boot strap support (Figure 5). In teleost lineage, different members of FOR subfamily have shown species specific gene expansion. For example, there is a large group of FORs with 18 zebrafish members, 6 catfish members, 4 medaka fish and one each of Tetraodon and channel catfish. Another group consists of 12 Tetraodon members, 2 medaka fish members and one each of goldfish and Japanese pufferfish (Figure 5). High differences in numbers of OR genes in specific fish reflect creature-specific lifestyle and these receptors are responsible for binding ligands important to a particular species [18–20, 25].
We have identified and analyzed repertoire of Tetraodon GPCRs and found high level of orthology with human counterparts. The human and Tetraodon GPCR sequences are analogous in terms of GPCR subfamilies, but display disproportionate number of receptors at the subfamily level. The teleost genome, with its expanded set of GPCRs, provides an additional and interesting model to study both evolution and function of these receptors. The availability of repertoire of Tetraodon GPCRs will facilitate further studies through "functional genomics" and "reverse pharmacological" strategies to match their cognate ligands and to elucidate biological functions. Systematic mutation of Tetraodon GPCRs will help to determine their neural, developmental and behavioral roles. They might also yield novel insights into the physiological functions and mutational pathologies of their human homologues in particular and other vertebrate homologues in general.
Identification of Tnig-GPCRs
Sequences of the Tetraodon nigroviridis are obtained from NCBI and Genoscope Tetraodon Genome Browser . HumanGPCR sequences were identified using GPCRDB  (Release 8.1) and based on earlier studies [7, 19, 23, 31]. GPCRs were identified using comprehensive approach (Figure 1) that includes RPS-BLAST  (using CDD v2.01 : SMART , Pfam  and COG Databases; E-value cut-off 10-5), Hmmpfam of HMMER 2.3.2  (using Pfam15; E-value cut-off 0.01) and BLASTP  homology comparisons against GPCRDB. Putative GPCR sequences were manually checked for GPCR specific patterns and presence of 7TM domain (at least 70% or more of Pfam 7TM should be aligned with each of the sequence). This is followed by secondary structure (transmembrane helix(TMH)) predictions using one or more methods like HMMTOP , SOSUI , MEMSTAT  and TMHMM2 . A range of 6–8 predicted TM helices acquired maximum coverage (96 percent; please see Additional data file 4 for details) when tested on a dataset of 327 annotated human GPCRs. A similar range was set to recognize acceptable tetraodon protein sequences containing transmembrane domain. Other examples, that either have under predicted or over predicted number of TM helices are earmarked separately ('#' symbol) in the current analysis. Splice variants, polymorphism and duplicates were eliminated by applying 90% sequence identity cut-off using CD-hit  and also checked manually. The corresponding genomic DNA sequences were also searched against the EST database at NCBI using BLASTN with a cutoff E-value of 1e12 . We could not obtain any Tetraodon nigroviridis EST hits, as there were few or no Tetraodon nigroviridis EST sequences available in the database.
Two genes, A from genome GA and B from GB, were considered orthologs if B is the best match of gene A in GB and A is the best match of B in GA using BLASTP .
Preliminary phylogenetic analysis  was performed using neighbor joining method with fewer number of bootstrap replicas and no randomization of sequence order. This was sufficient to separate GPCR sequences into rhodopsin like receptors and non rhodopsin like receptors. Rhodopsin like receptor and non-rhodopsin like receptor sequence datasets (separately full length and 7TM domain only), along with respective human GPCRs, were separately randomized twenty times with regard to sequence input order using a script called RandSeq (available upon request). These twenty datasets of different sequence order were aligned using clustalX 1.83  using multiple sequence alignment parameters with protein weight matrix BLOSUM series, gap opening penalty 10.0 and gap extension penalty 0.05 and delay divergence of 35 percent. To obtain unrooted trees, each alignment was bootstrapped 50 times and neighbor joining method (NEIGHBOR; Phylip package ) was employed to obtain tree topology using distance matrices obtained from alignments by PRODIST . Consensus tree was obtained from 1000 neighbor trees using CONSENSE . Only 500 boot strap replicas were used for rhodopsin like receptors due to limitations in the CONSENSE program and the trees were generated using Treeview . Maximum-likelihood tree of non-rhodopsin like receptors were also inferred from the alignment using TREE-PUZZLE . 10,000 quartet-puzzling steps were performed to obtain support values (reliability) for each internal branch.
R.S. is a Senior Research Fellow of the Wellcome Trust, UK. M.R.P.R. is a Senior Research Fellow of the Council of Scientific & Industrial Research (CSIR), India. We thank Ms. G. Mahima (BITs, Pilani) for GPCR pattern work and Mr. Nitin Gupta (UCSD) for coding a Java script to generate figure 2. We thank Tetraodon Sequencing Project for public availability of sequencing data. We also thank NCBS-TIFR for infrastructural support.
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