Origin of GPCRs Figure 4. Conserved features and structural motifs within the Adhesion receptor family in eukaryotes. The illustration represents known Adhesion receptors from Metazoan lineage with one sequence for each group I–VIII, GPR128 as others and VLGR1. Sequence DDB0231831, only member from D. discoideum represents Amoebozoa. The illustration includes newly identified sequences from other eukaryotic lineages like Choanoflagellata (S. rosetta; 8), Filasterea (C. owczarzaki; 7), Fungi (32), and Alveolata (P. tetraurelia; 1). The sequences are grouped respective to the major lineages across the eukaryotic tree. Common motifs of the Adhesion receptors were adapted from [8]. The residues which have $90% conservation across the eukaryotic tree are marked with a star at the bottom of the alignment. Interestingly, the loss of the Adhesion receptors in Basidiomycota phylum which descended from a common ancestor of Dikarya is indicated in red. The right part of the figure displays the diversity within the N-termini of the Adhesion receptors. The domains were identified with Pfam search and also verified with RPS-Blast with a cutoff e-value of 0.1. Each domain is marked with a symbol, and the explanation is found in the legend in the lower right corner. The numbers at corner of each connective thread along the domain symbols indicates the length of the N-termini. The following domains were found: GPS (GPCR proteolytic site), DUF3497 (Domain of unknown function), HBD (hormone-binding domain), OLF (olfactomedin domain), GBL (galactose-binding lectin domain), EGF_CA (calcium-binding epidermal growth factor-like domain), Ig (immunoglobulin domain), LRR (leucine-rich repeat), CA (cadherin repeats), EGF_Lam (laminin type epidermal growth factor domain), LamG (laminin G domain), Pentaxin domain, SEA (sea urchin sperm protein domain), TSP1 (thrombospondin repeats, type 1), CUB (C1r/C1s urinary epidermal growth factor and bone morphogenetic domain), Calx-beta domain, EPTP (epitempin protein domain), TLD (this domain is predicted to be an enzyme and is often found associated with pfam0147), FN3 (Fibronectin type III domain), TIG (this family consists of a domain that has an immunoglobulin like fold), RLD (Receptor L domain), CRD_FZ (CRD_domain cysteine-rich domain, also known as Fz (frizzled) domain). doi:10.1371/journal.pone.0029817.g004 66 residues with no functional domains. But the Adhesion receptors (AMAG_09540 and AMAG_13158) identified in A. macrogynus, belonging to the basal fungal lineage Blastocladiomycota, has an N-termini length of 251 and 254, respectively. The Glutamate receptor family The Glutamate family consists of 22 members in human. Most of these receptors have long N-termini that serve as an endogenous ligand binding region. Furthermore, the N-terminal region of the majority of receptors in human is also characterized with the presence of a cysteine rich domain (has 9 conserved cysteine residues) known as CRD or NCD3G. The ancient member of this family is present in species T. pseudonana. Novel homologues. Significantly, 118 novel members of the Glutamate family were found, of which 96 receptors are found in Fungi, 1 in Geodia cydonium (Porifera), and 1 each in M. brevicollis and S. rosetta and 20 in C. owczarzaki. Surprisingly, 96 putative Glutamate receptors in Fungi are from only 4 species, with a remarkable number of 78 members in A. macrogynus from phylum Blastocladiomycota (the most found in any species including human), 14 in S. punctatus and 3 in B. dendrobatidis from phylum Chytridiomycota (which also have Rhodopsin and Adhesion receptor homologues) and 1 in Rhizopus oryzae from phylum Zygomycota. Similar to the previous reports, we did not find any Glutamate receptors in the Fungi phyla Ascomycota and Basidiomycota. Conserved features. In the multiple sequence alignments there were partially or completely conserved residues across the 7TM region between the identified novel Glutamate family members and the characterized members of the Glutamate family in human (Figure S4). It is noteworthy that the 78 putative members of the Glutamate receptor family in A. macrogynus share about 27.4% identity in the 7TM regions and full length identity of just about 14.5%. This relatively low value suggests that even if these genes are a possible outcome of duplication, or local expansion, within the species, they seem to have diverged considerably. Phylogenetic analysis. Phylogenetic analysis was performed including the novel members of the Glutamate family identified in Fungi, Choanoflagellata, Filasterea and Porifera with the known Glutamate receptors in human, D. discoideum and T. pseudonana (Figure S5). The analysis revealed that the putative Glutamate receptors identified in Fungi clustered (PP.80%) separately from the homologues of the Glutamate family in Metazoa. The sequences found in C. owczarzaki forms a distinct cluster with a posterior probability .70%. Further, the tree demonstrates that the sequence GB_CAA76688.1 in species G. cydonium from phylum Porifera cluster (PP 100%) with GABBR1 (gamma amino butyric acid B receptor) in human, which suggests a close relationship with each other. N-terminal domain architecture. The N-termini of almost all identified members of the Glutamate family in Fungi and other eukaryotic lineages, was very similar in both length and the nature of the functional domains present in the Glutamate family receptors in human (Figure 5). The length of the N-termini of the novel sequences range from 57–1667 (average of 497) residues, which was similar to humans, which range from 31–621 (average of 446) residues. The functional domains included the ANF_receptor (PF01094), which is one of the members of clan Periplasmic binding protein like (CL0144; Periplas_BP) and is part of the N- terminal region for CASR, GABBR1 &2, GRMs and TAS1Rs receptors in human. Curiously, the ANF_receptor domain was also present in most of the Glutamate family members identified in Fungi, 3 sequences in C. owczarzaki and also in sequence DD231976 identified in D. discoideum. It is noteworthy that GB_CAA76688.1 from the phylum Porifera, which clusters with GABBR1 in the phylogenetic tree, lacks the ANF_receptor domain. Further, additional domains were found that belong to the same Pfam clan CL0144; Periplas_BP. This includes BMP (PF02608) domain in 13 members of the Glutamate family identified in D. discoideum and in two sequences AMAG_08984, AMAG_12053 from A. macrogynus. In addition, we found other periplasmic binding protein domains that are (grouped in a separate Pfam clan, PBP: CL0177) not found in human Glutamate receptors, but found in putative Glutamate receptors identified in Fungi. They are LysR_substrate (PF03466) domain (in AMAG_01751, 04246, SPPG_00031), PBP_like_2 (PF12849) domain (in SPPG_06488), OpuAC (PF04069) domain (in AMAG_01750, 04252, 14324, 14326, 15836 and 17562) and SBP_bac_1 (PF01547) domain in 8 sequences in A. macrogynus. But the NCD3G (PF07562) domain found in CASR, GRMs, TAS1Rs in human is absent in all identified novel non-Metazoan members. Further, domains were found that have not been reported previously in the Glutamate family like Pentaxin (PF00354) domain in CASP_EFW47134.1, 5_nucleotid_C (PF02872) domain in CASP_EFW44378.1, TIG (PF01833) domain in CAS- P_EFW40850.1 from C. owczarzaki and Cache_1 (PF02743) domain in TP_7830 in T. pseudonana from Alveolata. It is noteworthy that the Pentaxin domain is found in the Adhesion family receptors GPR144 and GPR126 in humans and TIG domain in the Adhesion receptors from S. rosetta. PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29817