Origin of GPCRs Figure 5. Schematic presentations of N-terminal domains of the Glutamate receptor family across the eukaryotic tree. The figure illustrates N-terminal domain architecture of the Glutamate receptors across different kingdoms of eukaryotes. The domains were identified with Pfam search and verified with RPS-Blast with a cutoff e-value of 0.1. For comparison, we represent known domain architecture of 22 Glutamate receptors from Homo sapiens for Metazoan lineage and 17 from D. discoideum for Amoebozoa and 1 from T. pseudonana for Alveolata. The illustration displays newly identified sequences from other eukaryotic lineages like Porifera (G. cydonium) a sister group to Metazoans, Choanoflagellata (M. brevicollis and S. rosetta), Filasterea (C. owczarzaki) and 4 genomes of Fungi; 1 for Blastocladiomycota (A. macrogynus) 2 representing for Chytridiomycota (B. dendrobatidis, S. punctatus) and 1 for Zygomycota (R. oryzae). The sequences are grouped respective to the major lineages across the eukaryotic tree. The colored boxes indicate the sequences to which phylum they belong to. The loss of Glutamate receptors in Dikarya which descended from a common ancestor of Fungi is indicated in red. The numbers at the top of each connective thread along domain symbols indicates the length of the N-termini. Each domain is marked with a symbol and abbreviated in the lower right corner. The domains are NCD3G (Nine Cysteines Domain of family 3 GPCR), ANF receptor (Receptor family ligand binding region), BMP (Basic membrane protein), LysR_Substrate (LysR substrate binding domain), SBP_bac_1 (Bacterial extracellular solute-binding protein), OpuAC (Substrate binding domain of ABC-type glycine betaine transport system), Pentaxin domain, 5_nucleotid_C domain, TIG (this family consists of a domain that has an immunoglobulin like fold), Cache_1 (Cache domain) and GCC2_GCC3. doi:10.1371/journal.pone.0029817.g005 The Frizzled receptor family The Frizzled family of GPCRs consists of 10 receptors (FZD1– 10) in human [10]. They are mostly known as receptors for Wnt proteins and play key role in tissue polarity and cell signaling [1]. The family members are characterized by the CRD_FZ domain or FZ domain which has 10 conserved cysteine residues. The ancient member or origin of this family can be traced back to D. discoideum [8]. Novel homologues. Our analysis identified 6 new Frizzled receptor sequences, distributed as 2 in Fungi (S. punctatus) and 4 in sponges (3 sequences in species Amphimedon queenslandica, 1 in Suberites domuncula), which belong to phylum Porifera. However, we did not find any Frizzled receptors in Choanoflagellata, Filasterea, Fungi phyla Ascomycota and Basidiomycota and in Alveolata. Conserved features. Multiple sequence alignment of the known Frizzled receptors in human and 6 novel sequences in Fungi and Porifera revealed that they are conserved with an identity of about 40%. Multiple cysteine residues along the sequence of the frizzled receptors were found to be completely conserved in most, and partially in a few cases (Figure S6). Phylogenetic analysis. Phylogenetic analysis of the Frizzled family was performed using the sequences from human, N. vectensis, T. adhaerens and D. discoideum and compared to the novel homologues from Fungi and Porifera (Figure S7). The tree is rooted with the node clustering the ancient members of the family in D. discoideum. The analysis revealed that the two sequences SPPG_01720 and SPPG_06172 in S. punctatus of Fungi cluster together (PP 100%) and placed basal to the node which cluster (PP 100%, 492/500) the Metazoan homologues of the Frizzled family. Furthermore, the tree demonstrates that the two sequences ADO16580.1 and ADO16569.1 from phylum Porifera (sponges), the closest relatives or sister group to the Metazoans do not show orthologous relationship with Metazoan receptors. In contrast, the other two sequences ADO16570.1 and CAD97575.1 identified in Porifera, cluster (PP 98%) together along with the sequences FZD4, FZD9 and FZD10 from humans, TA_31674 from T. adhaerens, NV_168924 and NV_139208 from N. vectensis. Fungi specific GPCRs In this study, we also searched for the previously known Fungi kingdom specific GPCRs that involve in pheromone sensing (Ste2, Ste3), sensors for glucose (Git3), and nitrogen sensors (stm1) among others. We identified 619 fungal specific GPCRs across the 79 species analyzed in this study (Table S3). Surprisingly, we could not find reliable homologues for Ste2, Ste3 and Git3 in Phylum Chytridiomycota that instead has homologues for mammalian like GRAFS families. Similarly, Zygomycetes R. oryzae has no homologues except for the nutrient sensing receptors. This observation is in line with the previous report on R. oryzae [18]. It is noteworthy that a widespread representation of these unique Fungi specific GPCRs was found only in Ascomycota and Basidiomycota that thoroughly lack the mammalian like GRAFS families. Discussion Here we provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi. The evidence is convincing for all the families and we were able to create alignments of the human and fungal members for each of the families. The results are further supported by a number of motifs that seem remarkably conserved and these are likely to be crucial for the function of the family specific properties of these receptors. Also, we identified GPCRs in T. pseudonana and P. tetraurelia, which belong to the eukaryotic kingdom known as Chromalveolata. Based on this comprehensive study covering 83 species, we establish that the origin of the Rhodopsin family could be traced back to the ancestor of Opisthokonts (,1100 MYA), the Adhesion and the Frizzled family to Unikonts (,1275 MYA) and the Glutamate and the cAMP receptor family to the common ancestor of Alveolates and Unikonts, early in eukaryotic evolution (.1400 MYA) (Figure 6). We provide higher resolution and deduced precise timeframe of the origin and diversification of the GRAFS families than previous reports [8]. We establish that the Frizzled and the Adhesion family evolved from the cAMP receptor family before the split of Unikonts from the common ancestor of eukaryotes. The Rhodopsin family evolved from the cAMP receptor family in the common ancestor of Opisthokonts, after the divergence of D. discoideum. It is interesting to note the similarities between the cAMP and Rhodopsin families, suggesting that the Rhodopsin family is an expansion of the more ancient cAMP branch perhaps taking over its functions in more complex organisms while cAMP receptors then become redundant. Later, the Secretin family evolved from the Adhesion family in an event that happened between the split of Cnidaria (N. vectensis) and the split of Nematoda (C. elegans) in the Metazoan lineage, as we previously reported [14]. Also, we have traced the lineage and species specific losses of these families across the eukaryotic tree (Figure 6). This study also highlights the importance of an ancient role of the N- terminal functional domains of the Adhesion and the Glutamate families across the diverse eukaryotic lineages. Intriguingly, the widespread distribution of these classical membrane receptors across the eukaryotic domain demonstrates that evolutionary divergent eukaryotes like the unicellular Alveolates and the complex multicellular organisms of Metazoan lineage share a basal signal transduction system that was present already in early eukaryotic evolution. PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e29817