The repertoire of G protein-coupled receptors in the sea squirt Ciona intestinalis

Abstract

Background: G protein-coupled receptors (GPCRs) constitute a large family of integral transmembrane receptor proteins that play a central role in signal transduction in eukaryotes. The genome of the protochordate Ciona intestinalis has a compact size with an ancestral complement of many diversified gene families of vertebrates and is a good model system for studying protochordate to vertebrate diversification. An analysis of the Ciona repertoire of GPCRs from a comparative genomic perspective provides insight into the evolutionary origins of the GPCR signalling system in vertebrates.

Results: We have identified 169 gene products in the Ciona genome that code for putative GPCRs. Phylogenetic analyses reveal that Ciona GPCRs have homologous representatives from the five major GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin) families concomitant with other vertebrate GPCR repertoires. Nearly 39% of Ciona GPCRs have unambiguous orthologs of vertebrate GPCR families, as defined for the human, mouse, puffer fish and chicken genomes. The Rhodopsin family accounts for ~68% of the Ciona GPCR repertoire wherein the LGR-like subfamily exhibits a lineage specific gene expansion of a group of receptors that possess a novel domain organisation hitherto unobserved in metazoan genomes.

Conclusion: Comparison of GPCRs in Ciona to that in human reveals a high level of orthology of a protochordate repertoire with that of vertebrate GPCRs. Our studies suggest that the ascidians contain the basic ancestral complement of vertebrate GPCR genes. This is evident at the subfamily level comparisons since Ciona GPCR sequences are significantly analogous to vertebrate GPCR subfamilies even while exhibiting Ciona specific genes. Our analysis provides a framework to perform future experimental and comparative studies to understand the roles of the ancestral chordate versions of GPCRs that predated the divergence of the urochordates and the vertebrates.

Figure 1

GPCR sequence analysis strategy employed. The Ciona Proteome was searched for 6/7/8 'TM' segment spanning sequences and the hits were taken for comparison against GPCRDB and against customized HMMs and PSSMs of GPCR families/subfamilies using an array of similarity/pattern search tools like BLASTP, HMMPFAM and RPS-BLAST. Subsequently, a number of phylogenetic analyses were performed on the sequences identified as putative GPCRs.

Figure 2

Phylogenetic relationship between GPCRs in Ciona and other genomes. The figure illustrates the presence of representatives of Glutamate, Rhodopsin, Adhesion, Frizzled, and Secretin family members in Ciona. Two sequences that are homologous to cAMP and Methuselah GPCRs are also represented. The position of the Rhodopsin family was established by including 15 receptors from the Rhodopsin family. The divergent, "Other/Unclassified" GPCRs known to lack reliable homologs from other species or which are fast evolving, are excluded from the final representation. For display reasons bootstrap values are not represented in the figure. Instead, the corresponding tree file in standard Newick format is provided [Additional data file 5]. For Figure 2, and Figures 3, 5 and 6, the multiple sequence alignment was built taking into account terminally truncated TM spanning regions, while the consensus phylogenetic tree was calculated using NJ method on 1000 replicas of the dataset. Ciona GPCR taxons are represented in numerals as per numbering in Additional data file 2. Abbreviations for known GPCRs are as described in [5, 6, 8, 9] and based on Swiss-prot IDs. Ciona GPCRs that deviate from the predicted 7TM structure are marked using a '#' symbol.

Figure 3

Phylogenetic relationship between non-(LDLRR-GPCR/LGR) Rhodopsin receptors in Ciona and other genomes. All members of the Ciona Rhodopsin subfamilies except for the LDRR-GPCRs/LGRs were included with closely related sequences from other genomes to construct the phylogenetic tree. To ascertain the phylogenetic position of Ciona LGR-like/LDLRR-GPCR cluster, 10 of those sequence members were added for tree reconstruction and later the branches removed from the final representation to be replaced by an arrow. The divergent "Other/Unclassified Rhodopsins" known to lack reliable homologs from other species, or which are fast evolving were excluded from the final representation. For display reasons boot strap values have not been shown. Instead, the corresponding tree file in standard Newick format is attached [Additional data file 6]. Ciona GPCRs that deviate from the predicted 7TM structure are marked using a '#' symbol.

Figure 4

Phylogenetic relationship of LDLRR-GPCR/LGR-like members of Ciona with those of Snail and Human LGRs and their domain organization. A) Maximum likelihood tree of the TM regions of the 14 Ciona LDLRR-GPCRs, the Ciona INSL3/relaxin receptor-like and the Snail and Human LGRs. The tree is rooted using a human LHCGR and a Ciona orphan LGR (ci0100151424) that are distantly related to these sequences. Support values are indicated in percentages. Taxons represented in numerals refer to Ciona GPCRs as per information in Additional data file 2. Ciona GPCRs that deviate from the predicted 7TM structure are marked using a '#' symbol. B) Schematic diagram representing the modular domain organization corresponding to the clusters identified in the phylogenetic tree.

Figure 5

Phylogenetic relationship within the Ciona GPCR set of Adhesion receptors. A phylogeny of Ciona Adhesion GPCRs shows the presence of five different paralogous clusters (group I- group V). Tree branches that support the five groups (> 75% bootstrap support) are coloured and their bootstrap values are represented. Branches that suggest gene duplication events (>98% bootstrap support) are reported with the bootstrap values in bold italics. Ciona Adhesion GPCR homologs of Human members are depicted with a '*' symbol and the GPCRs that deviate from the predicted 7TM structure are marked using a '#' symbol.