Neurotransmitter 2014; 1: e388. doi: /nt.388; 2014 by Takashi Hayashi

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1 BRIEF REPORT Evolutionarily conserved palmitoylation-dependent regulation of ionotropic glutamate receptors in vertebrates Takashi Hayashi 1, 2 1 Section of Cellular Biochemistry, Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP) Ogawa-Higashi, Kodaira, Tokyo , Japan 2 Department of Molecular Neurobiology and Pharmacology, Graduate School of Medicine, The University of Tokyo Hongo, Bunkyo-ku, Tokyo , Japan Correspondence: T. H. thayashi@ncnp.go.jp Received: October 26, 2014 Published online: December 01, 2014 In the vertebrate central nervous system, glutamate is the major excitatory neurotransmitter. Ionotoropic glutamate receptors (iglurs) are responsible for the glutamate-mediated postsynaptic excitation of neurons. Regulation of glutamatergic synapses in vertebrate central nervous system is critical for higher brain functions such as neural communication, memory formation, learning, emotion and behavior. Previous studies showed that post-translational protein palmitoylation, the only reversible covalent attachment of lipid to protein, regulates synaptic expression, intracellular localization and membrane trafficking of iglurs in mammalian neurons. Here, I further focus on conservation of palmitoylation sites found in iglur vertebrate orthologs. Analysis of databases shows that every palmitoylation sites of iglurs have been completely conserved during evolution in the vertebrate lineage, in spite of the divergence of iglurs amino acid sequences. Namely, palmitoylated cysteine residues of AMPA receptors, NMDA receptors and KA receptors, are evolutionarily conserved against mutation pressure throughout vertebrate species without exception. This finding suggests that the dynamic regulation of glutamatergic synapses made possible by this reversible palmitoylation of iglurs is critical for the vertebrate-specific refined functions of complex nervous systems. Keywords: palmitoylation; AMPA receptor; NMDA receptor; KA receptor; excitatory synapses; vertebrate To cite this article: Takashi Hayashi. Evolutionarily conserved palmitoylation-dependent regulation of ionotropic glutamate receptors in vertebrates. Neurotransmitter 2014; 1: e388. doi: /nt.388. Introduction Glutamate is the major excitatory neurotransmitter in the vertebrate central nervous system (CNS). The ionotropic glutamate receptors (iglurs) are classified into four groups, namely, -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type, kainate (KA)-type, (delta)-type and N-methyl-D-aspartate (NMDA)-type receptors. Of these iglurs, AMPA receptors mediate the majority of fast excitatory synaptic transmission in the mammalian brain [1, 2]. The expression level of post-synaptic AMPA receptors is closely linked to excitatory synaptic strength. Quantitative control of synaptic AMPA receptor amount is thus not only critical for basal synaptic transmission, but also for mammalian synaptic plasticity and higher brain function [3-5]. AMPA receptors trafficking to and from synapses is dynamically regulated by post-translational protein modification, both of receptors themselves, and of scaffold proteins, which bind receptors and direct them to specific subcellular locations [6-9]. NMDA receptors are the main triggers for the induction of synaptic plasticity, synaptogenesis and excitotoxicity [5, 10-12]. KA receptors play roles distinct from other iglur families by changing neural Page 1 of 10

2 Figure 1. Vertebrate iglurs palmitoylation sites are evolutionarily conserved. Schematic of iglurs, showing location of palmitoylation sites (left). Summary of evolutionary subclasses and the presence or absence of iglur palmitoylation sites (right). +: Cys for palmitoylation site; : substituted residues at corresponding sites;?: residues not determined. Species numbers in each subclass are shown. (A) AMPA receptor subunits GluA1-4, (B) NMDA receptor subunits GluN2A and GluN2B, (C) KA receptor subunit GluK2. circuit activity through modulation of excitatory and inhibitory transmission and neuronal excitability [13]. One key modification of mammalian iglurs is the reversible addition of the lipid palmitate to intracellular cysteine residues. This process, post-translational protein Page 2 of 10

3 Table 1. The BLAST alignments of vertebrate iglurs. (A) GluA1 (AMPA receptor subunit), (B) GluN2A (NMDA receptor subunit) and (C) GluK2 (KA receptor subunit). Percent identity among orthologs across any two species were obtained by performing BLAST search (with BLOSUM62) with full length amino acid sequences of vertebrate iglur orthologs. Homo sapiens (human), Mus musculus (mouse), Gallus gallus (chicken), Chrysemys picta bellii (painted turtle), Xenopus laevis (African clawed frog), Danio rerio (zebrafish) are compared as representative of each vertebrate class. palmitoylation, acts as a sticky tag that can direct receptors to specific regions of the plasma membrane, or to specific intracellular membranes or vesicles [14-16]. Genetic evidence strongly links impaired palmitoylation to abnormal mammalian brain development and/or function, including human neuropsychiatric disorders [17-22]. We have previously shown that palmitoylation regulate the synaptic expression and localization of AMPA receptors and NMDA receptors [23-26]. All four subunits of the mammalian AMPA receptor, GluA1-4 (also known as GluR1-4, GluRA-D or GluR 1-4) and the regulatory subunits of the mammalian NMDA receptor, GluN2A and GluN2B (also known as NR2A, NR2B or GluR 1, GluR 2) are palmitoylated at their two distinct sites. Palmitoylation of one site causes AMPA receptors and NMDA receptors to accumulate in the Golgi apparatus. Palmitoylation thus appears to act as a quality control mechanism to ensure correct receptor maturation. In contrast, second palmitoylation sites in AMPA receptors and NMDA receptors are implicated in quantitative regulation of synaptic receptor numbers, which relate to complex neuronal events such as synaptic plasticity and higher brain function. In addition, surface insertion and stabilization of KA receptor subunit GluK2 (also known as GluR6 or GluR 2) also occurs in its palmitoylation-dependent manner [27, 28]. Summarizing the above, post-translational protein palmitoylation appears uniquely suited to create dynamic controls of the synaptic expression and intracellular trafficking of mammalian iglurs in the CNS. Many neurotransmitter receptors, including iglurs, Page 3 of 10

4 appear to be evolutionarily conserved; orthologs with identical domains and transmembrane topology are found in organisms from worms to man [29]. Here, I focused on the palmitoylation sites of iglurs in the vertebrate lineage and sequence analysis of databases provides evidence for the complete conservation of palmitoylation mechanism in iglur regulations during vertebrate evolution. Materials and methods For analysis of the iglur orthologs, available cdna sequences, protein sequences, expressed sequence tags (ESTs) and genomic sequences are obtained by searching the NCBI databases, Genbank, EST banks, elephant shark genome project ( Joint Genome Institute ( and the Ensembl database ( by sequence homologies. Results Completely conserved palmitoylation sites in iglurs Recent progress in genome analyses revealed that many animal species possess orthologs of mammalian iglurs [29-32]. Our previous examination of the evolutionary conservation of the AMPA receptor palmitoylated cysteines revealed that the first palmitoylation site, which commonly lies in GluA1-4 transmembrane domain (TMD) 2, is present in every animal species examined except for nematodes [23, 33]. In contrast, the second palmitoylation site, located in the GluA1-4 C-terminal region, present only in all vertebrate AMPA receptor orthologs, whereas this site is absent in the majority of invertebrate AMPA receptor homologs; the only exception being a subset of insect species [23, 33]. The emergence and conservation of the second palmitoylation site in vertebrates suggests that AMPA receptor C-terminal palmitoylation may have evolved to allow additional regulation of synaptic AMPA receptor trafficking that may be required for higher brain function (Fig. 1A). Furthermore, palmitoylation sites in both Cys cluster I and Cys cluster II of NMDA receptor subunits GluN2A and GluN2B are completely conserved in all reported vertebrate orthologs (Fig. 1B). Only vertebrates GluN2A and GluN2B have the long intracellular C-terminal domain, carrying both palmitoylation sites, and they are lost in invertebrates [34]. While palmitoylation at Cys cluster I regulates synaptic expression of NMDA receptors, palmitoylation at Cys cluster II accumulates the receptors in the Golgi apparatus, like AMPA receptors [25]. Additional analysis of sequence databases shows that palmitoylation sites in GluK2 (Cys 858, 871), which affects surface localization of KA receptors in neurons, are also completely conserved in every vertebrate species (Fig. 1C). See supplementary information for full details of data sources. Divergence of amino acid sequence in iglurs full length The homology comparison of full length amino acid sequences of AMPA receptor orthologs show ~98% identity among mammalian species, ~93% identity between mammals and birds, ~93% identity between mammals and reptiles, ~88% identity between mammals and amphibians, ~74% identity between mammals and fishes (Table 1A). NMDA receptor orthologs show ~95% identity among mammalian species, ~82% identity between mammals and birds, ~80% identity between mammals and reptiles, ~74% identity between mammals and amphibians, ~67% identity between mammals and fishes (Table 1B). Higher homology scores are obtained among KA receptor orthologs, ~99 identity among mammalian species, ~98% identity between mammals and birds, ~98% identity between mammals and reptiles, ~94% identity between mammals and amphibians, ~91% identity between mammals and fishes (Table 1C). In conclusion, random mutations are observed all over vertebrate iglur sequences during vertebrate evolution. Discussion Generally, structurally or functionally important amino acid residues are conserved during molecular evolution against mutation pressure. Despite divergences in amino acid sequences among iglurs, this study reveal that palmitoylation sites in AMPA receptors, NMDA receptors and KA receptors are completely conserved throughout vertebrate species without exception, which strongly suggest critical roles of the multi-layer regulation of vertebrate glutamatergic excitatory synapses by palmitoylation of iglurs. In other words, vertebrates commonly use palmitoylation in excitatory synapses to control their CNS. Especially, synaptic palmitoylation site in the AMPA receptor C-termini are completely conserved in the vertebrate lineage from the Agnatha superclass (jawless fishes, such as hagfish and lamprey) to the Gnathostomata (jawed vertebrate) superclass. This modification site is readily acquired in the vertebrate common ancestor by simple gain-of-function mutations. In contrast, invertebrate species have non-cysteine residues at the corresponding sites of AMPA receptor homologs presumably because of random mutations in molecular evolution [33]. These facts suggest that the palmitoylation-dependent regulation of the AMPA receptor was established only in the common vertebrate ancestor around 500 million years ago in the late Cambrian to the early Ordovician periods. The vertebrate specific palmitoylation mechanism has been completely maintained up to the present time. On the other hand, the origin of Page 4 of 10

5 evolutionary acquirements of vertebrate NMDA receptors and KA receptors palmitoylation still remains unclear, because sequence information of primitive receptor orthologs of jawless fishes and species in the Urochordata subphylum (ascidians or sea squirts) or in the Cephalochordata subphylum (amphioxus and lancelet) is currently unavailable. Future genome analyses will answer detailed processes of acquisition of the post-translational protein palmitoylation in vertebrate glutamatergic excitatory synapses. Conflict of Interest The author declares that he has no conflicting interests. Acknowledgements This work was supported in part by the Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), the Takeda Science Foundation, the Mitsubishi Foundation and the Brain Science Foundation. References 1. Hollmann M, Heinemann S. Cloned glutamate receptors. Annu Rev Neurosci 1994; 17: Seeburg PH. The TINS/TiPS Lecture. The molecular biology of mammalian glutamate receptor channels. Trends Neurosci 1993; 16: Kessels HW, Malinow R. Synaptic AMPA receptor plasticity and behavior. Neuron 2009; 61: Shepherd JD, Huganir RL. The cell biology of synaptic plasticity: AMPA receptor trafficking. Annu Rev Cell Dev Biol 2007; 23: Collingridge GL, Isaac JT, Wang YT. Receptor trafficking and synaptic plasticity. Nat Rev Neurosci 2004; 5: Anggono V, Huganir RL. Regulation of AMPA receptor trafficking and synaptic plasticity. Curr Opin Neurobiol 2012; 22: Lu W, Roche KW. Posttranslational regulation of AMPA receptor trafficking and function. Curr Opin Neurobiol 2012; 22: Thomas GM, Hayashi T, Chiu SL, Chen CM, Huganir RL. Palmitoylation by DHHC5/8 targets GRIP1 to dendritic endosomes to regulate AMPA-R trafficking. Neuron 2012; 73: Thomas GM, Hayashi T, Huganir RL, Linden DJ. DHHC8-dependent PICK1 palmitoylation is required for induction of cerebellar long-term synaptic depression. J Neurosci 2013; 33: Mori H, Mishina M. Structure and function of the NMDA receptor channel. Neuropharmacology 1995; 34: Lau CG, Zukin RS. NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders. Nat Rev Neurosci 2007; 8: Mori H, Mishina M. Roles of diverse glutamate receptors in brain functions elucidated by subunit-specific and region-specific gene targeting. Life Sci 2003; 74: Contractor A, Mulle C, Swanson GT. Kainate receptors coming of age: milestones of two decades of research. Trends Neurosci 2011; 34: Fukata Y, Fukata M. Protein palmitoylation in neuronal development and synaptic plasticity. Nat Rev Neurosci 2010; 11: Linder ME, Deschenes RJ. Palmitoylation: policing protein stability and traffic. Nat Rev Mol Cell Biol 2007; 8: Nadolski MJ, Linder ME. Protein lipidation. FEBS J 2007; 274: Fallin MD, Lasseter VK, Wolyniec PS, McGrath JA, Nestadt G, Valle D, et al. Genomewide linkage scan for bipolar-disorder susceptibility loci among Ashkenazi Jewish families. Am J Hum Genet 2004; 75: Mansouri MR, Marklund L, Gustavsson P, Davey E, Carlsson B, Larsson C, et al. Loss of ZDHHC15 expression in a woman with a balanced translocation t(x;15)(q13.3;cen) and severe mental retardation. Eur J Hum Genet 2005; 13: Mukai J, Dhilla A, Drew LJ, Stark KL, Cao L, MacDermott AB, et al. Palmitoylation-dependent neurodevelopmental deficits in a mouse model of 22q11 microdeletion. Nat Neurosci 2008; 11: Otani K, Ujike H, Tanaka Y, Morita Y, Kishimoto M, Morio A, et al. The ZDHHC8 gene did not associate with bipolar disorder or schizophrenia. Neurosci Lett 2005; 390: Raymond FL, Tarpey PS, Edkins S, Tofts C, O'Meara S, Teague J, et al. Mutations in ZDHHC9, which encodes a palmitoyltransferase of NRAS and HRAS, cause X-linked mental retardation associated with a Marfanoid habitus. Am J Hum Genet 2007; 80: Young FB, Butland SL, Sanders SS, Sutton LM, Hayden MR. Putting proteins in their place: palmitoylation in Huntington disease and other neuropsychiatric diseases. Prog Neurobiol 2012; 97: Hayashi T, Rumbaugh G, Huganir RL. Differential regulation of AMPA receptor subunit trafficking by palmitoylation of two distinct sites. Neuron 2005; 47: Lin DT, Makino Y, Sharma K, Hayashi T, Neve R, Takamiya K, et al. Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and palmitoylation. Nat Neurosci 2009; 12: Hayashi T, Thomas GM, Huganir RL. Dual palmitoylation of NR2 subunits regulates NMDA receptor trafficking. Neuron 2009; 64: Mattison HA, Hayashi T, Barria A. Palmitoylation at two cysteine clusters on the C-terminus of GluN2A and GluN2B differentially control synaptic targeting of NMDA receptors. PLoS One 2012; 7:e Pickering DS, Taverna FA, Salter MW, Hampson DR. Palmitoylation of the GluR6 kainate receptor. Proc Natl Acad Sci U S A 1995; 92: Page 5 of 10

6 28. Copits BA, Swanson GT. Kainate receptor post-translational modifications differentially regulate association with 4.1N to control activity-dependent receptor endocytosis. J Biol Chem 2013; 288: Okamura Y, Nishino A, Murata Y, Nakajo K, Iwasaki H, Ohtsuka Y, et al. Comprehensive analysis of the ascidian genome reveals novel insights into the molecular evolution of ion channel genes. Physiol Genomics 2005; 22: Chiu J, DeSalle R, Lam HM, Meisel L, Coruzzi G. Molecular evolution of glutamate receptors: a primitive signaling mechanism that existed before plants and animals diverged. Mol Biol Evol 1999; 16: Sprengel R, Aronoff R, Volkner M, Schmitt B, Mosbach R, Kuner T. Glutamate receptor channel signatures. Trends Pharmacol Sci 2001; 22: Chen YC, Kung SS, Chen BY, Hung CC, Chen CC, Wang TY, et al. Identifications, classification, and evolution of the vertebrate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunit genes. J Mol Evol 2001; 53: Thomas GM, Hayashi T. Smarter neuronal signaling complexes from existing components: how regulatory modifications were acquired during animal evolution: evolution of palmitoylation-dependent regulation of AMPA-type ionotropic glutamate receptors. Bioessays 2013; 35: Ryan TJ, Emes RD, Grant SG, Komiyama NH. Evolution of NMDA receptor cytoplasmic interaction domains: implications for organisation of synaptic signalling complexes. BMC Neurosci 2008; 9:6. Page 6 of 10

7 Supplementary information Figure 1. Vertebrate iglurs palmitoylation sites are evolutionarily conserved. The Vertebrata subphylum consists of two superclasses, Gnathostomata (jawed vertebrate) and Agnatha (jawless fish). This figure represents sequence data from the following vertebrate species. (A) All cloned and predicted AMPA receptor othologs in each class are shown below. GluA1-4 subunits have two distinct palmitoylation sites in their C-termini and TMD2 region: GluA1 Cys811, Cys585; GluA2 Cys836, Cys610; GluA3 Cys841, Cys615; GluA4 Cys817, Cys611 (amino acid residues in the mouse GluA sequence as representative). Gnathostome AMPA receptor subunits In the Mammalia class: Homo sapiens (human), Pan paniscus (bonobo), Pan troglodytes (chimpanzee), Gorilla gorilla (western lowland gorilla), Pongo abelii (Sumatran orangutan), Nomascus leucogenys (northern white-cheeked gibbon), Macaca fascicularis (crab-eating macaque), Macaca mulatta (rhesus monkey), Papio anubis (olive baboon), Callithrix jacchus (white-tufted-ear marmoset), Saimiri boliviensis (black-capped squirrel monkey), Tarsius syrichta (tarsier), Microcebus murinus (mouse lemur), Otolemur garnettii (northern greater galago), Tupaia belangeri (northern treeshrew), Spermophilus tridecemlineatus / Ictidomys tridecemlineatus (thirteen-lined ground squirrel), Mesocricetus auratus (golden hamster), Rattus norvegicus (Norway rat), Mus musculus (house mouse), Dipodomys ordii (kangaroo rat), Cavia porcellus (guinea pig), Octodon degus (degu), Heterocephalus glaber (naked mole rat), Oryctolagus cuniculus (rabbit), Ochotona princeps (American pika), Erinaceus europaeus (European hedgehog), Sorex araneus (European shrew), Pteropus vampyrus (megabat), Pteropus alecto (black flying fox), Myotis lucifugus (little brown bat), Myotis brandtii (Brandt's bat), Myotis davidii (mouse-eared bat), Felis catus (cat), Canis lupus familiaris (dog), Mustela putorius furo (ferret), Ailuropoda melanoleuca (giant panda), Leptonychotes weddellii (Weddell seal), Equus caballus (horse), Ceratotherium simum (white rhinoceros), Bos taurus (cattle), Ovis aries (sheep), Capra hircus (goat), Pantholops hodgsonii (chiru/tibetan antelope), Tursiops truncates (dolphin), Orcinus orca (killer whale), Sus scrofa (wild boar), Camelus ferus (wild Bactrian camel), Vicugna pacos (alpaca), Choloepus hoffmanni (two-toed sloth), Dasypus novemcinctus (armadillo), Loxodonta africana (African elephant), Trichechus manatus latirostris (Florida manatee), Procavia capensis (rock hyrax), Echinops telfairi (lesser hedgehog tenrec), Chrysochloris asiatica (Cape golden mole), Elephantulus edwardii (Cape elephant shrew), Macropus eugenii (wallaby), Sarcophilus harrisii (Tasmanian devil), Monodelphis domestica (gray short-tailed opossum), Ornithorhynchus anatinus (platypus). In the Aves class (birds): Gallus gallus (chicken), Meleagris gallopavo (turkey), Anas platyrhynchos (mallard/duck), Columba livia (domestic pigeon), Falco peregrinus (peregrine falcon), Falco cherrug (saker falcon), Pseudopodoces humilis (ground tit), Ficedula albicollis (collared flycatcher), Taeniopygia guttata (zebra finch), Geospiza fortis (medium ground-finch), Zonotrichia albicollis (white-throated sparrow), Melopsittacus undulatus (budgerigar). In the Reptilia class: Trachemys scripta (red-eared slider turtle), Chrysemys picta bellii (painted turtle), Pelodiscus sinensis (Chinese softshell turtle), Chelonia mydas (green sea turtle), Anolis carolinensis (green anole), Alligator mississippiensis (American alligator), Alligator sinensis (Chinese alligator). In the Amphibia class: Rana catesbeiana (bullfrog), Xenopus tropicalis (western clawed frog), Xenopus laevis (African clawed frog). In the Actinopterygii class (ray-finned fishes); Fugu rubripes (Japanese pufferfish), Tetraodon fluviatilis (green spotted pufferfish), Tetraodon nigroviridis (a similar brackish species of green spotted pufferfish), Gadus morhua (Atlantic cod), Morone chrysops x Morone saxatilis (white bass x striped sea-bass), Gasterosteus aculeatus (stickleback), Oreochromis mossambicus (Mozambique tilapia), Oreochromis niloticus (Nile tilapia), Danio rerio (zebrafish), Carassius auratus (goldfish), Carassius carassius (crucian carp), Oryzias latipes (Japanese medaka), Astyanax mexicanus (Mexican tetra/blind cave fish), Poecilia formosa (Amazon molly), Haplochromis burtoni (Burton's mouthbrooder), Pundamilia nyererei (cichlid), Xiphophorus maculatusi (platyfish), Lepisosteus oculatusi (spotted gar). In the Sarcopterygii class (lobe-finned fishes): Latimeria chalumnae (coelacanth). In the Chondrichthyes class (cartilaginous fishes): Squalus acanthias (spiny dogfish shark), Callorhinchus milii (elephant shark). Agnatha AMPA receptor subunits In the Myxini class: Paramyxine yangi (brown hagfish). In the Petromyzontida class: Petromyzon marinus (sea Page 7 of 10

8 lamprey). (B) All cloned and predicted NMDA receptor regulatory subunits GluN2 othologs in each class are shown below. GluN2A and GluN2B subunits have two distinct palmitoylation sites in their C-termini: GluN2A Cys cluster I (Cys 848, 853, 870), GluN2A Cys cluster II (Cys1214, 1217, 1236, 1239) and GluN2B Cys cluster I (Cys 849, 854, 871), GluN2B Cys cluster II (Cys1215, 1218, 1239, 1242, 1245) (amino acid residues in the mouse GluN2A and GluN2B sequence as representative). Gnathostome NMDA receptor subunits In the Mammalia class: Homo sapiens (human), Pan paniscus (bonobo), Pan troglodytes (chimpanzee), Gorilla gorilla (western lowland gorilla), Pongo pygmaeus (Borneo orangutan), Pongo abelii (Sumatran orangutan), Hylobates muelleri (Müller's Bornean gibbon), Nomascus leucogenys (northern white-cheeked gibbon), Cercopithecus ascanius (red-tailed monkey), Cercopithecus diana (Diana monkey), Cercopithecus wolfi (Wolf's mona monkey), Allenopithecus nigroviridis (Allen's swamp monkey), Miopithecus talapoin (Angolan talapoin), Macaca fascicularis (crab-eating macaque), Macaca mulatta (rhesus monkey), Macaca fuscata (Japanese macaque), Chlorocebus sabaeus (green monkey), Papio hamadryas (hamadryas baboon), Papio anubis (olive baboon), Colobus angolensis (Angola colobus), Trachypithecus cristatus (silvery lutung), Trachypithecus vetulus (purple-faced langur), Pygathrix nemaeus (douc langur), Nasalis larvatus (proboscis monkey), Callithrix jacchus (white-tufted-ear marmoset), Callithrix pygmaea (pygmy marmoset), Saimiri boliviensis (black-capped squirrel monkey), Alouatta caraya (black howler monkey), Ateles fusciceps (black-headed spider monkey), Lagothrix lagotricha (brown woolly monkey), Callicebus moloch (dusky titi), Tarsius syrichta (tarsier), Microcebus murinus (mouse lemur), Lemur catta (ring-tailed lemur), Varecia rubra (red ruffed lemur), Otolemur garnettii (northern greater galago), Galeopterus variegatus (Sunda flying lemur), Tupaia belangeri (northern treeshrew), Tupaia chinensis (Chinese treeshrew), Spermophilus tridecemlineatus / Ictidomys tridecemlineatus (thirteen-lined ground squirrel), Cricetulus griseus (Chinese hamster), Mesocricetus auratus (golden hamster), Microtus ochrogaster (prairie vole), Rattus norvegicus (Norway rat), Mus musculus (house mouse), Jaculus jaculus (lesser Egyptian jerboa), Peromyscus maniculatus bairdii (deer mouse), Chinchilla lanigera (chinchilla), Dipodomys ordii (kangaroo rat), Cavia porcellus (guinea pig), Octodon degus (degu), Heterocephalus glaber (naked mole rat), Oryctolagus cuniculus (rabbit), Ochotona princeps (American pika), Erinaceus europaeus (European hedgehog), Sorex araneus (European shrew), Condylura cristata (star-nosed mole), Eptesicus fuscus (big brown bat), Pteropus vampyrus (megabat), Pteropus alecto (black flying fox), Myotis lucifugus (little brown bat), Myotis brandtii (Brandt's bat), Myotis davidii (mouse-eared bat), Felis catus (cat), Panthera tigris altaica (Amur tiger), Canis lupus familiaris (dog), Mustela putorius furo (ferret), Ursus maritimus (polar bear), Ailuropoda melanoleuca (giant panda), Leptonychotes weddellii (Weddell seal), Odobenus rosmarus divergens (walrus), Equus caballus (horse), Equus przewalskii (Przewalski's horse), Ceratotherium simum (white rhinoceros), Bos taurus (cattle), Bos mutus (yak), Bubalus bubalis (water buffalo), Ovis aries (sheep), Capra hircus (goat), Moschus moschiferus (Siberian musk deer), Elaphurus davidianus (Père David's deer), Pantholops hodgsonii (chiru/tibetan antelope), Hippopotamus amphibius (hippopotamus), Tursiops truncates (dolphin), Orcinus orca (killer whale), Kogia sima (dwarf sperm whale), Physeter catodon (sperm whale), Mesoplodon densirostris (Blainville's beaked whale), Platanista gangetica (river dolphin), Lipotes vexillifer (baiji), Delphinus capensis (common dolphin), Delphinapterus leucas (beluga whale), Stenella attenuata (pantropical spotted dolphin), Sousa chinensis (Chinese white dolphin), Grampus griseus (Risso's dolphin), Lipotes vexillifer (Yangtze River dolphin), Balaenoptera acutorostrata (minke whale), Balaenoptera omurai (Omura's whale), Neophocaena phocaenoides (finless porpoise), Sus scrofa (wild boar), Sus scrofa domesticus (pig), Camelus bactrianus (Bactrian camel), Camelus ferus (wild Bactrian camel), Vicugna pacos (alpaca), Choloepus hoffmanni (two-toed sloth), Dasypus novemcinctus (armadillo), Loxodonta africana (African elephant), Trichechus manatus latirostris (Florida manatee), Procavia capensis (rock hyrax), Echinops telfairi (lesser hedgehog tenrec), Chrysochloris asiatica (Cape golden mole), Elephantulus edwardii (Cape elephant shrew), Orycteropus afer (aardvark), Macropus eugenii (wallaby), Sarcophilus harrisii (Tasmanian devil), Monodelphis domestica (gray short-tailed opossum), Ornithorhynchus anatinus (platypus). In the Aves class (birds): Gallus gallus (chicken), Meleagris gallopavo (turkey), Anas platyrhynchos (mallard/duck), Columba livia (domestic pigeon), Falco peregrinus (peregrine falcon), Falco cherrug (saker falcon), Corvus brachyrhynchos (American crow), Pseudopodoces humilis (ground tit), Ficedula albicollis (collared flycatcher), Taeniopygia guttata (zebra finch), Geospiza fortis (medium ground-finch), Zonotrichia albicollis (white-throated sparrow), Melopsittacus undulatus (budgerigar), Calypte anna (Anna's hummingbird). Page 8 of 10

9 In the Reptilia class: Chrysemys picta bellii (painted turtle), Pelodiscus sinensis (Chinese softshell turtle), Chelonia mydas (green sea turtle), Anolis carolinensis (green anole), Alligator mississippiensis (American alligator), Alligator sinensis (Chinese alligator), Python bivittatus (Burmese python), Ophiophagus hannah (king cobra). In the Amphibia class: Xenopus tropicalis (western clawed frog), Xenopus laevis (African clawed frog). In the Actinopterygii class (ray-finned fishes); Fugu rubripes (Japanese pufferfish), Tetraodon nigroviridis (a similar brackish species of green spotted pufferfish), Gadus morhua (Atlantic cod), Gasterosteus aculeatus (stickleback), Oreochromis niloticus (Nile tilapia), Danio rerio (zebrafish), Carassius auratus (goldfish), Oryzias latipes (Japanese medaka), Poecilia reticulata (guppy), Astyanax mexicanus (Mexican tetra/blind cave fish), Maylandia zebra (zebra mbuna), Poecilia formosa (Amazon molly), Haplochromis burtoni (Burton's mouthbrooder), Pundamilia nyererei (cichlid), Xiphophorus maculatusi (platyfish), Apteronotus leptorhynchus (brown ghost knifefish), Lepisosteus oculatusi (spotted gar), Cynoglossus semilaevis (tongue sole). In the Sarcopterygii class (lobe-finned fishes): Latimeria chalumnae (coelacanth). In the Chondrichthyes class (cartilaginous fishes): Callorhinchus milii (elephant shark). Agnatha NMDA receptor subunits In the Petromyzontida class: Petromyzon marinus (sea lamprey), the corresponding residues of palimitoyation sites at Cys cluster II have not been determined. (C) All cloned and predicted KA receptor subunit GluK2 orthologs in each class are shown below. GluK2 subunit has two distinct palmitoylation sites in their C-termini: GluK2 Cys858, Cys671 (amino acid residues in the mouse GluK2 sequence as representative). Gnathostome KA receptor subunit GluK2 In the Mammalia class: Homo sapiens (human), Pan paniscus (bonobo), Pan troglodytes (chimpanzee), Gorilla gorilla (western lowland gorilla), Pongo abelii (Sumatran orangutan), Nomascus leucogenys (northern white-cheeked gibbon), Macaca fascicularis (crab-eating macaque), Macaca mulatta (rhesus monkey), Chlorocebus sabaeus (green monkey), Papio anubis (olive baboon), Callithrix jacchus (white-tufted-ear marmoset), Saimiri boliviensis (black-capped squirrel monkey), Tarsius syrichta (tarsier), Otolemur garnettii (northern greater galago), Galeopterus variegatus (Sunda flying lemur), Tupaia belangeri (northern treeshrew), Tupaia chinensis (Chinese treeshrew), Spermophilus tridecemlineatus / Ictidomys tridecemlineatus (thirteen-lined ground squirrel), Cricetulus griseus (Chinese hamster), Mesocricetus auratus (golden hamster), Microtus ochrogaster (prairie vole), Rattus norvegicus (Norway rat), Mus musculus (house mouse), Jaculus jaculus (lesser Egyptian jerboa), Peromyscus maniculatus bairdii (deer mouse), Chinchilla lanigera (chinchilla), Dipodomys ordii (kangaroo rat), Cavia porcellus (guinea pig), Octodon degus (degu), Heterocephalus glaber (naked mole rat), Oryctolagus cuniculus (rabbit), Ochotona princeps (American pika), Erinaceus europaeus (European hedgehog), Sorex araneus (European shrew), Condylura cristata (star-nosed mole), Eptesicus fuscus (big brown bat), Pteropus vampyrus (megabat), Pteropus alecto (black flying fox), Myotis lucifugus (little brown bat), Myotis brandtii (Brandt's bat), Myotis davidii (mouse-eared bat), Felis catus (cat), Panthera tigris altaica (Amur tiger), Canis lupus familiaris (dog), Mustela putorius furo (ferret), Ursus maritimus (polar bear), Ailuropoda melanoleuca (giant panda), Leptonychotes weddellii (Weddell seal), Odobenus rosmarus divergens (walrus), Equus caballus (horse), Equus przewalskii (Przewalski's horse), Ceratotherium simum (white rhinoceros), Bos taurus (cattle), Bos mutus (yak), Bubalus bubalis (water buffalo), Ovis aries (sheep), Capra hircus (goat), Pantholops hodgsonii (chiru/tibetan antelope), Tursiops truncates (dolphin), Orcinus orca (killer whale), Physeter catodon (sperm whale), Lipotes vexillifer (baiji), Lipotes vexillifer (Yangtze River dolphin), Balaenoptera acutorostrata (minke whale), Sus scrofa (wild boar), Camelus ferus (wild Bactrian camel), Vicugna pacos (alpaca), Choloepus hoffmanni (two-toed sloth), Dasypus novemcinctus (armadillo), Loxodonta africana (African elephant), Trichechus manatus latirostris (Florida manatee), Procavia capensis (rock hyrax), Echinops telfairi (lesser hedgehog tenrec), Chrysochloris asiatica (Cape golden mole), Elephantulus edwardii (Cape elephant shrew), Orycteropus afer (aardvark), Macropus eugenii (wallaby), Sarcophilus harrisii (Tasmanian devil), Monodelphis domestica (gray short-tailed opossum), Ornithorhynchus anatinus (platypus). In the Aves class (birds): Gallus gallus (chicken), Meleagris gallopavo (turkey), Anas platyrhynchos (mallard/duck), Columba livia (domestic pigeon), Falco peregrinus (peregrine falcon), Falco cherrug (saker falcon), Corvus brachyrhynchos (American crow), Pseudopodoces humilis (ground tit), Ficedula albicollis (collared flycatcher), Taeniopygia guttata (zebra finch), Geospiza fortis (medium ground-finch), Zonotrichia albicollis (white-throated sparrow), Melopsittacus undulatus (budgerigar), Calypte anna (Anna's hummingbird). Page 9 of 10

10 In the Reptilia class: Chrysemys picta bellii (painted turtle), Pelodiscus sinensis (Chinese softshell turtle), Chelonia mydas (green sea turtle), Anolis carolinensis (green anole), Alligator mississippiensis (American alligator), Alligator sinensis (Chinese alligator), Python bivittatus (Burmese python), Ophiophagus hannah (king cobra). In the Amphibia class: Xenopus tropicalis (western clawed frog), Xenopus laevis (African clawed frog). In the Actinopterygii class (ray-finned fishes); Fugu rubripes (Japanese pufferfish), Tetraodon nigroviridis (a similar brackish species of green spotted pufferfish), Gadus morhua (Atlantic cod), Gasterosteus aculeatus (stickleback), Oreochromis niloticus (Nile tilapia), Danio rerio (zebrafish), Oryzias latipes (Japanese medaka), Poecilia reticulata (guppy), Astyanax mexicanus (Mexican tetra/blind cave fish), Maylandia zebra (zebra mbuna), Poecilia formosa (Amazon molly), Haplochromis burtoni (Burton's mouthbrooder), Pundamilia nyererei (cichlid), Xiphophorus maculatusi (platyfish), Lepisosteus oculatusi (spotted gar), Cynoglossus semilaevis (tongue sole). In the Sarcopterygii class (lobe-finned fishes): Latimeria chalumnae (coelacanth). In the Chondrichthyes class (cartilaginous fishes): Callorhinchus milii (elephant shark). Agnatha KA receptor subunit GluK2 In the Myxini class: Paramyxine yangi (brown hagfish), the corresponding residues of palimitoyation sites have not been determined. In the Petromyzontida class: Petromyzon marinus (sea lamprey), the corresponding residues of palimitoyation sites have not been determined. Page 10 of 10