Origin of GPCRs Figure 6. Schematic presentation of the origin, evolution and lineage-specific losses of the five main GRAFS families, cAMP receptor family and the Fungi kingdom specific GPCRs. The eukaryotic evolutionary tree is constructed with references from the tree of life Web project (http://tolweb.org/tree/phylogeny.html). Each branch shows the presence (colored circles) and the loss (colored cross symbol) of the five main GRAFS families and the cAMP receptor family. The presence and absence of the N-terminal domains of the Glutamate and the Adhesion family is indicated with a tick mark and a crossed circle, respectively. The presence of Fungi kingdom specific GPCRs were represented by colored star symbols, and their absence with a line segment in black against the respective colored star symbol. The putative connection and origin of the Glutamate and cAMP receptor family is indicated with dotted lines at the bottom. The horizontal gene transfer of the Adhesion receptor family from Fungi to Alveolata is indicated with dotted lines in red. Branch lengths are not drawn to represent actual evolutionary distances. doi:10.1371/journal.pone.0029817.g006 Rhodopsin and cAMP receptor family We provide convincing evidence that the split of the large Rhodopsin family from the cAMP receptor family can be placed at the emergence of the common ancestor of Opisthokonts (Fungi and Metazoa) and not in Placozoa (basal lineage of Metazoa) as earlier assumed [8]. Interestingly, we found that the newly identified sequences of the Rhodopsin and the cAMP receptor family in the basal eukaryotic genomes share conserved motifs between them. This includes the important DRY and NPXXY motifs of the Rhodopsin family that are crucial to the functional integrity of these receptors [25,26]. These motifs are present as ERY and NPXXX in the 12 novel Rhodopsin family sequences in Fungi and N(S)XY and NS(A)XXY in the cAMP receptors from Fungi and Alveolata, respectively (Figure 1). This conservation within the 7TM region strongly links these families. In addition, the phylogenetic tree clearly clusters (PP.90%, 466/500) all the novel sequences from the Rhodopsin family and cAMP receptor family into two separate groups and suggest a common origin between these two families (Figure 2). Also, the scatter plot (Figure 3) clearly demonstrates the similarity between the Pfam HMM models of the Rhodopsin and the cAMP receptor family. This is in good agreement with our earlier report that the Rhodopsin family and the cAMP receptor family share an HHsearch (HMM- HMM search) homology probability of 99.4% [8]. Also, we noticed insensitivity of the Pfam HMM models to distinguish between the two families prevails right at the point where the split of the Rhodopsin family from cAMP receptor family have occurred. Further, the cAMP receptor family is present in plants, Alveolata, Amoebozoa and Fungi but subsequently become redundant in the Metazoan lineage as it is found in 3 invertebrate species and lost in the vertebrates (Figure 6). Taken all these findings together, it is evident that the cAMP receptor family gave rise to the Rhodopsin family in an event close to the common ancestor of Opisthokonts and then persisted and evolved as a large Rhodopsin family in the Metazoan lineage. Adhesion receptor family We have traced the origin of the Adhesion family to the ancestor of Unikonts (Figure 6). Here we provide the first evidence for the presence of Adhesion receptors in Filasterea (C. owczarzaki), Choanoflagellata (S. rosetta) and in two of the four major phyla of the Fungi kingdom. Also, our search emphasize that Adhesions are not found in eukaryotic lineages before the split of D. discoideum. The sole representative of Adhesions from Alveolata is a possible horizontal gene transfer from Fungi, since it cluster with the Adhesion receptors found in Ascomycota of Fungi (PP 99%) (Figure S3). This is also supported by a pair wise identity of about 50% in the 7TM region (Figure S8), which is relatively high for lineages that are as distant as Fungi and Alveolata. Considering these findings together with our earlier report showing an HHsearch homology probability of 99.8% with the cAMP receptor family [8], we validate that the Adhesions evolved from the ancient cAMP receptor family in an event close to the split of Unikonts from the common ancestor of eukaryotes. Intriguingly, the complexity of the Adhesion receptors with long N-termini containing multiple functional domains observed in the Metazoan lineage is absent in receptors from Fungi, Amoebozoa and Alveolata, whereas the 7TM region is conserved in species ranging from unicellular Alveolates to human (Figure 4). These results may suggest that ligand binding and signaling mechanisms of the Adhesion receptors in Fungi and other basal eukaryotes could be confined within the 7TM region. However, the conservation of these motifs in the Metazoan lineage suggests that they are important for the signaling mechanisms along with the long N- termini with multiple functional domains. The complex multi- domain N-termini that emerged close to the radiation and diverged within the Metazoan lineage might reflect the need for a broader signal transduction system in the intricate cell to cell communication that is characteristic for Metazoan multicellular- ity. Prime examples are the emergence of the characteristic Adhesion family domains, GPS and Calx-beta domain in Filasterea and EGF-CA domain in Choanoflagellata, which prevailed in the multicellular Metazoan species (Figure 6). In addition, we extended the knowledge of the N-terminal region of the Adhesions by adding at least three novel domains TIG, TLD and RLD to the known 31 unique domains [14]. Glutamate receptor family Our results suggest that the origin of the ancient Glutamate receptor lies in the common ancestor of eukaryotes, since we found them in Chromalveolata, Unikonts and Opisthokonts. For the first time, we have found 120 novel Glutamate receptors in the basal Opisthokont lineages which include Fungi and the close relatives of animals- Choanoflagellata, Filasterea and Porifera. We have also shown that they are also present in D. discoideum and T. pseudonana, which diverged before the Opisthokont lineage [8]. We found that the ancient Glutamate family is present in almost all eukaryotic lineages except in plants and in the Fungi subkingdom Dikarya, which include Ascomycota and Basidiomycota (see Figure 6). Interestingly, the conserved length and the nature of the functional domains within the N-termini across all eukaryotic lineages in which the Glutamate receptors were found suggest that the endogenous ligand binding or so called Venus flytrap mechanism (VFTM) and the subsequent signal transduction mechanism emerged early in the eukaryotic life (Figure 5). This is in contrast with the Adhesion receptor family where the N-termini complexity is absent in the basal eukaryotes. Further, we found that .90% of the functional domains in the N-termini of the Glutamate receptors in diverse eukaryotic lineages, belong to the periplasmic binding protein superfamily. This includes the domain ANF Receptor and the four novel domains, BMP, LysR_Sub- strate, SBP_bac_1 and OpuAC that have not previously been identified in the Glutamate receptors. Taken together, it suggests a conserved fundamental role and remarkable adaptability of these periplasmic binding protein superfamily domains in regulating the signaling mechanisms of the Glutamate receptors from unicellular organisms to human. PLoS ONE | www.plosone.org 12 January 2012 | Volume 7 | Issue 1 | e29817