The Origin of GPCRs: Identification of Mammalian like Rhodopsin , Adhesion , Glutamate and Frizzled GPCRs in Fungi . . Arunkumar Krishnan , Markus Sa¨ llman Alme´ n , Robert Fredriksson, Helgi B. Schio¨ th* Department of Neuroscience, Functional Pharmacology, Uppsala University, Uppsala, Sweden Abstract G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily. Citation: Krishnan A, Alme´n MS, Fredriksson R, Schio¨ th HB (2012) The Origin of GPCRs: Identification of Mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in Fungi. PLoS ONE 7(1): e29817. doi:10.1371/journal.pone.0029817 Editor: Chaoyang Xue, University of Medicine & Dentistry of New Jersey - New Jersey Medical School, United States of America Received September 16, 2011; Accepted December 6, 2011; Published January 4, 2012 Copyright: 2012 Krishnan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The study was supported by the Swedish Research Council, The Novo Nordisk Foundation, Swedish Royal Academy of Sciences, and Magnus Bergvall Foundation. RF was supported by the Swedish Research Council. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: helgi.schioth@neuro.uu.se . These authors contributed equally to this work. Introduction G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors with about 800 members in the human genome serving as targets for many drugs [1,2]. Over the years, the repertoire of GPCRs has been analyzed in several species which are from the phyla Chordata, Echinodermata, Arthropoda, Nematoda, Cnidaria, Placozoa and Amoebozoa [3–8]. At first, in 1994, Attwood and Findlay categorized the superfamily into six classes (A–F) based on sequence homology and functional similarity [9]. Later, our comprehensive phylogenetic analysis of the human repertoire provided the GRAFS classification. This system grouped the mammalian GPCR repertoire into five main families; Glutamate (Class C), Rhodopsin (Class A), Adhesion (Class B), Frizzled (Class F), and Secretin (Class B) [10]. The Rhodopsin family is the largest with 683 members in humans [8], characterized by short N-termini and interactions with a broad variety of ligands. The Glutamate family is distinguished with long N-termini which act as the endogenous ligand binding region. The Adhesion receptors have long N-termini which contain a plethora of multiple domains while the Frizzled receptors have long cysteine- rich N-termini [1]. Mining of GPCRs has revealed that each of the five main mammalian families have long evolutionary histories. Recently, our group has shown the connections between the GPCR families and provided strong evidence that the Rhodopsin, Adhesion, Frizzled and Secretin share a common origin with the cAMP receptors (Class E) and dismissed relationship with families like the insect odorant receptors, insect gustatory receptors, trehalose receptors and DUF267, which in many publications were considered to be GPCRs [8]. Our previous study [8] also showed an early presence of Rhodopsin (7tm_1) family members in the phylum Placozoa (Trichoplax adhaerens), an invertebrate which forms the basal group of Metazoa [11], while Adhesion (7tm_2) and Frizzled family members were found in the phylum Amoebozoa (Dictyostelium discoideum), which is a basal group to both Fungi and Metazoa [12]. The Glutamate (7tm_3) family members were found in the phylum Heterokontophyta (Thalassiosira pseudonana), a diatom classified under the Chromalveolata kingdom [13] while the earliest occurrence of Secretin family members, which evolved from Adhesion PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29817