A Metabotropic Glutamate Receptor Family Gene in Dictyostelium discoideum

Abstract

Metabotropic glutamate receptors (mGluRs) are a class of G-protein-coupled receptors that possess a seven transmembrane region involved in the modulation of excitatory synaptic transmission in the nervous system. mGluR orthologs have been identified in Drosophila, Caenorhabditis elegans, and higher organisms. Drosophila possesses two mGluR genes, DmGluRA and DmXR. We screened the Dictyostelium genome data base using the ligand binding domain of rat mGluR1 as bait, and identified a new receptor, DdmGluPR, belonging to the mGluR family. Similar to Drosophila DmXR, the residues of mGluRs involved in the binding of the ␣-carboxylic and ␣-amino groups of glutamate were well conserved in DdmGluPR, but the residues interacting with the ␥-carboxylic group of glutamate were not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABA B receptors split but before mGluR family divergence. DdmGluPR mRNA was expressed in vegetative cells and throughout starvation-induced development, but the level of the expression was relatively high until 4 h after starvation. DdmGluPR was localized to the plasma membrane of axenically grown Ax-2 cells expressed as a green fluorescent protein fusion protein. DdmGluPR-null cells grew faster at high cell density and reached higher densities than wild-type cells. DdmGluPR-null cells exhibited delayed aggregates formation upon starvation and impaired chemotaxis toward cAMP. Although expressions of cAR1 and aca, cAMP-signaling components, were rapidly induced and peaked at 2-4 h in wild-type cells, DdmGluPRnull cells displayed sustained and peaked at 8 h of the expressions of these genes. Our findings suggest the involvement of DdmGluPR in the early development of Dictyostelium discoideum. Metabotropic glutamate receptors (mGluRs)4 are a class of G-protein-coupled receptors (GPCRs) that possess a seven-transmembrane region and couple with second messenger systems including phosphoinositide hydrolysis and regulation of adenylate cyclase activation. mGluRs are key receptors in the modulation of excitatory synaptic transmission in the central nervous system. The eight subtypes of mGluRs are classified into three subgroups according to sequence similarity, agonist selectivity, and effector system differences: subgroup I (mGluR1 and -5), subgroup II (mGluR2 and -3), and subgroup III (mGluR4, -6, -7, and -8) (1). The mGluR structure is divided into three regions: the extracellular region, the seven-transmembrane spanning region, and the cytoplasmic region (2). The extracellular region is further divided into the ligand binding domain (LBD) and the cysteine-rich region. They share sequence similarity with the calcium-sensing receptor (3) and the pheromone receptor (4, 5) to form the mGluR family. The primary structure and pharmacology of mGluRs are evolutionarily well conserved in Drosophila, Caenorhabditis elegans, and higher mammals. Sequence analysis of the C. elegans genome reveals the presence of one homologue for each group (6). Two kinds of mGluRs, DmGluRA and DmXR...

Systematic Analysis of γ-Aminobutyric Acid (GABA) Metabolism and Function in the Social Amoeba Dictyostelium discoideum

“…2A), with the same results acquired when cells were developed on buffer-saturated filters (data not shown). This is consistent with a previous study which reported that the presence of 1 mM GABA or glutamate had no effect on the cAMP chemotactic response (20). We also tested the ability of cells to migrate directionally toward a wide range of GABA gradients (1 nM to 10 mM) using a micropipette assay.…”

“…Briefly, vegetative cells were washed three times with 10 ml MES buffer (20 7 vegetative cells were washed twice with cold MES buffer and suspended in 1 ml of cold MES buffer. The cells were lysed by passing through an Acrodisc 5 m pore size syringe filter (Pall).…”

“…A previous study reported a delay of at least 2 h in early development of grlE Ϫ cells, and showed that this delay could be rescued by overexpressing GrlE (20). We were skeptical that GrlE functioned redundantly at the transition from growth to development and also in early development, therefore we generated our own grlE Ϫ cells and our grlE Ϫ cells exhibited a slight delay in aggregation at 6 h after development.…”

“…In addition, either disruption of gabT or direct addition of the irreversible GABA transaminase inhibitor vigabatrin promotes SDF-2 production and induces spore maturation (16). grlE Ϫ cells also exhibit rapid growth in suspension and delay in early development (20), however, the mechanism remains unknown. Functions of several other members of the GABA B receptor-like family which are induced in sporulation have also been explored.…”

“…Strikingly, 17 genes (grlA-H, grlJ-R) encoding homologues of GABA B metabotropic receptors have been reported (17)(18)(19). Among them, only GrlE shares a well conserved N-terminal ligand binding domain (20), and this has been proven to be a bona fide GABA receptor (21).…”