The Putative Glutamate Receptors from Plants Are Related to Two Superfamilies of Animal Neurotransmitter Receptors via Distinct Evolutionary Mechanisms

Frank J. Turano, Ganesh R. Panta, Marc W. Allard and Peter van Berkum

Department of Biological Sciences, George Washington University
Soybean and Alfalfa Research Laboratory, United States Department of Agriculture, Agricultural Research Service, Beltsville, Maryland

Animal ionotropic glutamate receptors (iGLRs) and members of subfamily C (Kolakowski 1994Citation ) or IV (Parmentier et al. 1998Citation ) of the G-protein-coupled receptors (subC-GPCRs), which contains the metabotropic glutamate receptors (mGLRs) and {gamma}-aminobutyric acidB receptors (GABA-BRs), have distinct pharmacological and structural characteristics (Sutcliffe, Wo, and Oswald 1996Citation ; Walker, Brooks, and Holden-Dye 1996Citation ; Sutcliffe et al. 1998Citation ). Likeness among members of the two superfamilies has been noted in the literature. The iGLRs share sequence similarity with regions of the mGLRs (Walker, Brooks, and Holden-Dye 1996Citation ), and mGLRs share sequence similarity with GABA-BRs (Kaupmann et al. 1997Citation ). In addition, all of the receptors, iGLRs (Wo and Oswald 1995Citation ), mGLRs (O'Hara et al. 1993Citation ), and GABA-BRs (Galvez et al. 1999Citation ), have sequence similarities to portions of the bacterial periplasmic binding proteins. An evolutionary link between the iGLRs and the periplasmic binding proteins has been demonstrated (Chiu et al. 1999Citation ) via a newly identified family of genes encoding for putative GLRs in plants (Lam et al. 1998Citation ). Despite the noted similarities among the iGLRs and members of subC-GPCRs, namely, the mGLRS or GABA-BRs, there are no data to support an evolutionary link between any member of the two superfamilies, including one between members of the mGLRs and iGLRs (Walker, Brooks, and Holden-Dye 1996Citation ). We provide evidence for a link between the iGLRs and members of subC-GPCRs by incongruence length difference, parsimony, and bootstrap analyses. We demonstrate that the N-terminal regions of the putative GLRs from plants are related to members of subC-GPCRs and that the C-terminal regions of the putative GLRs from plants are related to iGLRs. From this discordant relationship, we conclude that the ancestors of the plant GLRs may be the evolutionary progenitors of members of both superfamilies of highly specialized animal receptors via distinct evolutionary mechanisms. A model outlining the evolution of the receptors is presented.

Parsimony and bootstrap analyses have been used to provide evidence that plant GLRs and iGLRs shared a common ancestor prior to the divergence of iGLRs, including N-methyl-d-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate (AMPA/KA) subtypes (Chiu et al. 1999Citation ). We used a similar approach to examine relationships between the putative GLRs from plants and members of two superfamilies of animal neurotransmitter receptors, the iGLRs and members of subC-GPCRs, specifically the mGLRs and GABA-BRs.

Based on our preliminary results from BLAST (Altschul et al. 1997Citation ) and PHI-BLAST (Zhang et al. 1998Citation ) searches (data not shown), we considered the possibility of a recombination event during the evolutionary history of the loci. To test this hypothesis, we conducted an incongruence length difference analysis (Farris et al. 1994, 1995Citation ) in which we compared the relatedness of the approximate first third (N-terminal regions), and the last two thirds (C-terminal regions) of the peptides separately. In an analysis that contained members of the iGLRs, mGLRs, GABA-BRs, and plant GLRs, there was a significant difference (P = 0.001) in the relatedness of the two portions of the peptide, whereas there was not a significant difference (P > 0.1) when the plant GLRs were removed. Furthermore, these results are supported by results from parsimony analysis in which the N- and C-termini of the putative plant receptors were separated (fig. 1 ). The C-terminal regions of the plant GLRs are related to members of the iGLRs. It can be concluded that the C-terminal regions of the GLRs and iGLRs arose from an ancestral locus that predated the divergence of animal AMPA/KA and NMDA receptors. These results are in agreement with those from a recently published analysis (Chiu et al. 1999Citation ). However, our analyses of the N-terminal regions support a different scenario. The N-terminal regions of the Arabidopsis GLRs are related to GABA-BRs, which are members of the subC-GPCRs, and not to members of the iGLRs. The results of both the incongruence length difference and the parsimony analyses support the occurrence of a recombination event at the GLR locus. We conclude that the extant iGLRs and members of subC-GPCRs evolved from ancestral plant GLRs via distinct mechanisms. The iGLRs arose via a series of point mutations and selection, whereas the members of subC-GPCRs arose from a gene conversion or recombination event.



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Fig. 1.—Relationships between the N- and C-terminal regions of the plant glutamate receptors (GLRs) and other eukaryotic receptors based on parsimony analysis. The Escherichia coli leucine-binding protein (EcoLEUBP, accession number J05516) was used as an outgroup. Sequences included in the phylogenetic reconstruction were a bacterial periplasmic binding protein (BPBP), members of the ionotropic glutamate receptors (iGLRs) (including alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate and N-methyl-d-aspartate [NMDA] subtypes, members of subfamily C of the G-protein-coupled receptors (subC-GPCRs) (including metabotropic glutamate [mGLR], {gamma}-aminobutyric acidB [GABA-BR], calcium-sensing [CaSenR] and vomeronasal organ type 2 [VNtype2] receptors, and putative GLRs from plants). Similar results were obtained with nearest-neighbor analyses (not shown). The abbreviations for the iGLR, subC-GPCR, and GLR sequences are as follows: Homo sapiens GABA-BR subunit 1a (HsaGABAB1a, accession number AJ012185); H. sapiens GABA-BR subunit 1b (HsaGABABR1b, accession number AF056085); H. sapiens GABA-BR subunit 2 (HsaGABABR2, accession number AF056085); H. sapiens CaSenR (HsaCaSenR, accession number U20759); Bos taurus extracellular CaSenR (BtaExtCaSenR, accession number S67307); Mus musculus VNtype2, receptor, 4 (MmuVNtype2, accession number AF011414); H. sapiens mGLR 1 alpha (HsamGLR1a, accession number U31215); H. sapiens mGLR 2 (HsamGLR2, accession number L35318); H. sapiens mGLR 4 (HsamGLR4, accession number X80818); H. sapiens mGLR 5a (HsamGLR5, accession number D28538); H. sapiens GLR1 (HsaGLR1, accession number M64752); Oryzias latipes GLR (OlaGLR, accession number X60086); Drosophila melanogaster GLR1 (DmeGLR1, accession number M97192); D. melanogaster NMDAR1 (DmeNMDAR1, accession number X71790); H. sapiens excitatory amino acid receptor 2 (HsaEAAR2, accession number S40369); Rattus norvegicus GLR6 (RnoGluR6, accession number Z11548); R. norvegicus NMDA-1a (RnoNMDAR1, accession number X63255); M. musculus NMDA (MmuNMDAR, accession number D10028); Caenorhabditis elegans iGLR (CelGLR1, accession number U34661); C. elegans putative GLR (CelGLR, accession number Z75545); Arabidopsis putative GLR1 (AthGLR1N or AthGLR1C, accession number AF079998); Arabidopsis putative GLR2a (AthGLR2aN or AthGLR2aC, accession number AF079999); Arabidopsis putative GLR2b (AthGLR2bN or AthGLR2bC, accession number AF038557); Arabidopsis putative GLR3 (AthGLR3N or AthGLR3C, accession number AF007271); Arabidopsis putative GLR4 (AthGLR4N or AthGLR4C, accession number AC000098); Arabidopsis putative GLR5 (AthGLR5N or AthGLR5C, accession number AF210701); and Arabidopsis putative GLR6 (AthGLR6N or AthGLR6C, accession number AF170494). For the Arabidopsis sequences, the uppercase N designates the N-terminal regions, from approximately amino acid residues 1–320, whereas the uppercase C designates the C-terminal regions from approximately amino acid residue 321 to the last residue in each deduced amino acid sequence. Sequences were aligned using ClustalX, version 1.8, with default parameters except for the following: gap separation distance = 10, protein weight matrix = Blosum 30, and delay divergent sequence = 25%. Only one parsimonious tree was obtained by heuristic search methods using PAUP*, version 4.0B4a, with parameters set for maximum trees automatically increased by 100 to insure full examination, sequences were added randomly for 1,000 replicates with two trees held during stepwise addition. The tree length was 11,404 steps, consistency index = 0.756, retention index = 0.696, and rescaled consistency index = 0.526. Support of the clades (>50%) is indicated by bootstrap values using the parameters as in parsimony analysis and 1,000 replicates. Bootstrap values <50% are not shown in the figure

 
An important question arises from these observations: at what time in evolutionary history did the gene conversion or recombination event occur between the N-terminal region of plant GLRs and a gene encoding for a peptide with seven-transmembrane domains (i.e., a gene encoding for GPCR-like protein)? A gene encoding for a GPCR-like peptide has been identified in Arabidopsis (Josefsson and Rask 1997Citation ; Plakidou-Dymock, Dymock, and Hooley 1998Citation ). The deduced amino acid sequence of the GPCR-like sequence does not have similarity to the iGLRs or GLRs (data not shown), but it does contain a seven-transmembrane domain region and is related to members of the GPCR superfamily (Josefsson 1999Citation ). On the basis of finding, we propose that the gene conversion or recombination event that gave rise to members of subC-GPCRs occurred after the divergence of the plant and animal kingdoms between ancestors of the putative GLR and GPCR-like loci. This hypothesis is supported by the fact that extensive searches through nucleic acid and protein databases failed to identify plant GABA-BRs or mGLRs, i.e., peptides that contained both a putative GABA or glutamate-binding domain and a region of seven transmembrane domains. However, similar searches identified GABA-BR and mGLRs in both invertebrates and vertebrates.

A proposed phylogenetic reconstruction (fig. 2 ) demonstrates the relationships among members of the superfamilies and the proposed recombination event. We proposed that an ancestral GLR was the precursor to the iGLRs and members of subC-GPCRs. The iGLRs arose via a series of point mutations and selection, whereas members of subC-GPCRs arose from a gene conversion or recombination event between an ancestral GLR locus and a gene encoding for a protein that contains seven-transmembrane domains, a GPCR-like locus. In summary, the ancestors of the extant GLRs are the evolutionary progenitors of both the iGLRs and members of subC-GPCRs via distinct mechanisms, and thus this finding represents the previously unidentified evolutionary link between the two superfamilies of animal neurotransmitter receptors.



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Fig. 2.—Proposed evolutionary history of the bacterial periplasmic binding proteins (BPBP) and the plant glutamate receptor (GLR), ionotropic glutamate receptor (iGLR), metabotropic glutamate receptor (mGLR), and {gamma}-aminobutyric acidB receptor (GABA-BR) genes. An ancestral plant GLR evolves from a BPBP. An ancestral GLR evolves into extant plant GLR, iGLR, and members of subfamily C of the G-protein-coupled receptors (subC-GPCR) via distinct routes. The GLR and iGLR superfamilies evolve by a series of point mutations and selection. The iGLR superfamily diverges into distinct subtypes, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate (AMPA/KA) and N-methyl-d-aspartate (NMDA). An ancestral subC-GPCR arose from a gene conversion or recombination event between the 5' end of an ancestral GLR and a gene encoding for a peptide with seven-transmembrane domains, perhaps a gene encoding for a G-protein-coupled receptor-like (GPCR-like) protein (16–18). The ancestral subC-GPCR evolves into mGLR and GABA-BR by a series of point mutations and selection. Solid black or dark-gray lines represent iGLR, plant GLR, subC-GPCR, GABA-BR, or mGLR peptides. Small solid boxes represent transmembrane domains, half-shaded boxes represent putative amino-acid-binding domains, and large open rectangles represent membranes. The putative GLRs from plants and the iGLRs, both subtype NMDA and subtype AMPA/KA, contain three transmembrane domains plus the pore, or "P," domain, which does not traverse the membrane. The GPCR-like peptide and the subC-GPCRs, GABA-BRs, and mGLRs contain seven transmembrane domains.

 

Acknowledgements

We thank Drs. Robert Donaldson, Benjamin Matthews, and Alan Kinnersley for their critical review of this manuscript. F.J.T. gratefully acknowledges financial support from the Auxein Corporation, Lansing, Michigan.

Footnotes

Jeffrey C. Long, Reviewing Editor

1 Present address: Department of Pharmacology, University of Tennessee, Memphis, Tennessee. Back

1 Abbreviations: GABA-BRs, {gamma}-aminobutyric acidB receptors; iGLRs, ionotropic glutamate receptors; mGLRs, metabotropic glutamate receptors; subC-GPCRs, subfamily C of the G-protein-coupled receptors. Back

2 Keywords: Arabidopsis G-protein-coupled receptor GABA receptor gene conversion glutamate receptor incongruence length difference recombination event Back

3 Address for correspondence and reprints: Frank J. Turano, Department of Biological Sciences, George Washington University, 2030 G Street, NW, Lisner Hall, Room 340, Washington, D.C. 20052. E-mail: fturano{at}gwu.edu Back

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Accepted for publication March 15, 2001.