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Originally published In Press as doi:10.1074/jbc.M512723200 on March 9, 2006

J. Biol. Chem., Vol. 281, Issue 18, 12336-12343, May 5, 2006
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A Metabotropic Glutamate Receptor Family Gene in Dictyostelium discoideum*

Hideo Taniura1, Noriko Sanada2, Nobuyuki Kuramoto3, and Yukio Yoneda

From the Laboratory of Molecular Pharmacology, Graduate School of Natural Science and Technology, Kanazawa University, Kakumamachi, Kanazawa, Ishikawa 920-1192, Japan

Received for publication, November 29, 2005 , and in revised form, March 3, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {alpha}-carboxylic and {alpha}-amino groups of glutamate were well conserved in DdmGluPR, but the residues interacting with the {gamma}-carboxylic group of glutamate were not. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABAB 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, DdmGluPR-null 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, have been reported in Drosophila (7, 8). DmGluRA shares a very similar pharmacological profile with its mammalian orthologues, mGluR2 and mGluR3, although DmXR is insensitive to glutamate or any other standard amino acids. A sequence analysis of the LBD of Drosophila DmXR revels that the residues contacting the amino acid moiety of glutamate are conserved, whereas the residues interacting with the {gamma}-carboxylic group are not.

The social ameba Dictyostelium discoideum is a unicellular eukaryote and an ideal model organism to investigate developmental regulation. A proteome-based phylogeny shows that Dictyostelium diverged from the animal-fungal lineage after the plant-animal split but before the divergence of the fungi (9). D. discoideum has an ability to alternate between unicellular and multicellular forms. Dictyostelium cells grow vegetatively as individual amebae and gather with neighboring cells to form aggregates upon starvation. After aggregation, cells differentiate to specific cell types and finally culminate into a fruiting body consisting of spores on top of a supporting stalk. During this process, extracellular cAMP signaling and cAMP-specific GPCRs are involved in its multicellular development. One of four highly homologous cAMP receptors, cAR1, is essential for aggregation and subsequent development. cAR1 mediates cAMP relay response by activating an adenylyl cyclase (10, 11). Other signaling factors, prestarvation factor and conditioned medium factor (CMF), also help to initiate the chemoattractant signaling relay system through G-protein-dependent and -independent pathways (12–14).

Here, we screened the Dictyostelium genome using the BLAST program with the rat mGluR1 LBD as bait, and identified a new receptor, DdmGluPR (Dictyostelium mGlu precursor receptor), belonging to the mGluR family. The predicted protein contains a domain with homologous sequence to the LBD of mGluRs and a seven-transmembrane region. A sequence comparison between DdmGluPR and mGluRs revealed that only part of the residues involved in the glutamate binding was conserved like Drosophila DmXR. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABABRs split but before mGluR family divergence. We propose that DdmGluPR was the evolutionary precursor to mGluRs. We also demonstrated the important roles of the DdmGluPR gene in aggregates formation during Dictyostelium development.


Figure 1
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  		FIGURE 1.
Deduced amino acid sequence of DdmGluPR. A, the DdmGluPR gene encodes an 816-amino-acid protein. A putative signal sequence (amino acid residues 1–27) is shown with underline. Predicted seven transmembrane segments (TM1, TM2, TM3, TM4, TM5, TM6, and TM7) are shown with bold and underline. B, hydropathy plot of the deduced amino acid sequence of the transmembrane region (amino acid residues 438–680). Predicted seven transmembrane segments are shown.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Growth and Development—Vegetative cells of D. discoideum Ax-2 were grown axenically in PS-medium (1% peptone, 0.7% yeast extract, 1.5% D-glucose, 0.11% KH2PO4, 0.05% Na2HPO4, 40 ng/ml vitamin B12, 80 ng/ml folic acid) at 22 °C. DdmGluPR-null cells were grown in PS-medium supplemented with 10 µg/ml blasticidin S (Invitrogen). To allow cells to differentiate, cells were washed in 10 mM Na-potassium phosphate buffer (10 mM Na-potassium phosphate, 0.2 mM MgSO4, and 1 mM CaCl2, pH 6.1), plated on non-nutrient agar (1.5% agar in 10 mM Na-K phosphate buffer) at a density of 107 cells/ml, and incubated at 22 °C. Conditioned medium was prepared by starving wild-type or DdmGluPR-null cells at 5 x 106 cells/ml in 10 mM Na-potassium phosphate buffer in shaking culture for 20 h. To examine the effect of conditioned medium on cell aggregation, cells were starved in submerged culture at densities 8, 4, 2, 1, and 0.5 x 105 cells/ml in 10 mM Na-potassium phosphate buffer.

Sequence Analysis—BLAST searches were performed using the data base for Dictyostelium and for other organisms (http://ncbi.nlm.nih.gov/). The signal sequence and transmembrane regions of DdmXR were predicted by using PSORTII and SOSUI. The sequence alignment between DdmGluPR, the Drosophila DmXR, and the mouse mGluR3 was produced using ClustalW. The phylogenetic tree was constructed using class III GPCR sequences from various species retrieved from the data base. Sequences were aligned using ClustalW. The resulting multi-alignment was then used for the construction of an evolutionary tree by the Neighbor Joining method using MEGA2 program.

Northern Blot Analysis—cDNAs for DdmGluPR, cAR1, aca, psA spiA, discoidin, and Ig7 were obtained by RT-PCR, cloned into pT7 Blue (Novagen) or pBluescript SK (Stratagene) and confirmed by sequencing. The ecmB gene was kindly provided from Dr. R. Escalante at Instituto de Investigaciones Biomedicas, Spain. All of the cDNAs were transcriptionally labeled by T3 or T7 RNA polymerases to make the antisense digoxigenin-labeled riboprobes. The extracted total RNA (5 µg) were resolved on 1% formaldehyde-agarose gels and transferred onto positively charged nylon transfer membranes (Hybond N+, Amersham Biosciences). Hybridizations were carried out using digoxigenin-labeled riboprobe.

Expression of GFP-DdmGluPR—The expression vector pDXA-GFP2 was kindly provided by Dr. Thomas T. Egelhoff at Case Western Reserve School of Medicine (15). GFP was fused at the N terminus of DdmGluPR. The construct was introduced into Dictyostelium by electroporation, and stable transformants were selected on G418.

DdmGluPR Gene Inactivation—The DdmGluPR-null strains were generated by gene targeting. cDNA encompassing the entire coding region (~2.5 kbp) with restriction sites EcoRI and XbaI at 5' and 3', respectively, was obtained by RT-PCR and cloned into pBluescript SK. The targeting construct contains the digested fragment with EcoRI and HindIII (~1.7 kbp), and the Bsr cassette was inserted at the KpnI site. The Bsr cassette was kindly provided by Drs. Y. Tanaka and T. Morio at University of Tsukuba, Japan. The targeting construct was digested with EcoRI and HindIII and introduced into wild-type Ax-2 cells. Transformants were selected for blasticidin resistance. Clone C2 was identified as the DdmGluPR-null strain and used for the experiments.


Figure 2
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  		FIGURE 2.
Multiple alignment of DdmGluPR, mouse mGluR3, and Drosophila DmXR. A, amino acid sequences of DdmGluPR, mouse mGluR3, and Drosophila DmXR were aligned using ClustalW. Identical residues are shaded black and similar residues are shaded gray. The residues involved in glutamate binding conserved in mGluRs compared with the residues at equivalent positions in DdmGluPR and DrosophilaDmXRareindicatedbyanarrowhead.The domain presumed for the binding of the {alpha}-carboxylic and {alpha}-amino groups of glutamate in mGluRs is grouped by a square. B, comparison of the conserved residue in mouse mGluR3 and the residues at equivalent positions in DdmGluPR for the glutamate binding. Numbers show the position in mouse mGluR3 conserved residues for the glutamate binding.

 
Assay for Chemotaxis toward cAMP—Prior to assay, Dictyostelium cells were starved in 10 mM Na-K buffer, pH 6.1, at 22 °C for 4 h. Small wells in the center of 1.5% non-nutrient agar plates were filled with cAMP solution (5 µl of 10 µM solution), and the starved cells were spotted on the agar plates 5 mm from the edge of the well at 107 cells/ml. The plates were further incubated for 5 h at 22°C, and the resultant chemotaxis toward the wells was observed under a phase contrast microscope. To examine the effects of most of natural amino acids including Glu, Arg, Lys, Asp, His, Gly, Gln, Pro, Ser, Ala, Asn, Thr, Leu, Met, Val, and GABA on starvation-induced chemotactic response toward cAMP, all of the reagents were dissolved in water at 100 mM (pH 6.1). The cells were starved, and the chemotactic response to cAMP was observed in the presence of each reagent at 1 mM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Analysis of Dictyostelium DdmGluPR Homologous to mGluRs—We screened the Dictyostelium genome data base using the BLAST program with the rat mGluR1 LBD (amino acid residue 33–522) as a bait and identified a new receptor homologous to mGluRs, called DdmGluPR (Dictyostelium mGlu precursor receptor), in D. discoideum (DDB0231976 GenBankTM accession number DQ447637 [GenBank] ). The DdmGluPR gene encodes an 816-amino-acid protein as shown in Fig. 1A. The N terminus of the predicted amino acid sequence of DdmGluPR contained a potential signal sequence, and seven possible transmembrane regions were predicted from hydrophobicity algorithms for a plasma membrane protein (Fig. 1B). These structural features suggest that DdmGluPR is a class of GPCR. The mGluRs share sequence similarity with the GABAB receptors (GABABRs), the calcium-sensing receptor, and pheromone receptors and constitute the class III GPCR family. The extracellular region of DdmGluPR showed the closest sequence similarity to mouse mGluR3 and Drosophila DmXR among the class III GPCR family. The extracellular region of mGluRs contains the LBD and the cysteine-rich region. The cysteine-rich region is conserved among all mGluR subtypes and the calcium-sensing receptor but not in the GABABRs. Because DdmGluPR has no cysteine-rich regions, an alignment of the derived amino acid sequences from the extracellular region of DdmGluPR and the LBDs of mouse mGluR3 and Drosophila DmXR is provided in Fig. 2A. The analysis of crystal structures of the LBD of mGluR1 identified several key residues involved in the glutamate binding (2). Ser-165 and Thr-188 and Asp-208, Tyr-236, and Asp-318 in mGluR1 are involved in the binding of the {alpha}-carboxylic and {alpha}-amino groups of glutamate, respectively. In addition, Arg-78 and Lys-409 in mGluR1 are involved in the binding of the {gamma}-carboxylic group of glutamate. Although the residues of DdmGluPR involved in the binding of the {alpha}-carboxylic and {alpha}-amino groups of glutamate were well conserved, the residues interacting with the {gamma}-carboxylic group of glutamate were not (Fig. 2). As shown in an alignment, DdmGluPR has extensive homology at the regions presumed for the binding of the {alpha}-carboxylic and {alpha}-amino groups of glutamate (indicated as a box in Fig. 2A) with mouse mGluR3 and Drosophila DmXR (27.5 and 26.9% identical and 49.1 and 52.1% similarity with mouse mGluR3 and Drosophila DmXR, respectively). Although, the transmembrane region of DdmGluPR showed closest sequence similarity to rat or human GABABR2 among the class III GPCR family (30 and 18% identical, and 60 and 46.5% similarity with human GABABR2 and mouse mGluR3, respectively) (data not shown). The residues of Drosophila DmXR for interacting with the {gamma}-carboxylic group of glutamate are also missing, and glutamate cannot activate Drosophila DmXR (8), suggesting that DdmGluPR is not a glutamate receptor.


Figure 3
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  		FIGURE 3.
Phylogenetic analysis among DdmGluPR, Drosophila DmXR, and class III GPCRs. The sequences of the indicated class III GPCRs (accession numbers indicated) from human, rat, mouse, bovine, chick, C. elegans, goldfish, salmon, fugu, sea urchin, Drosophila, and DdmGluPR were aligned, and an evolutionary tree was constructed by the Neighbor Joining method. Bootstrap values for branch are indicated. DdmGluPR and Drosophila DmXR are shown with bold.

 
To further analyze the DdmGluPR sequence, a multiple alignment of DdmGluPR and many other members of the class III GPCRs from various species was generated and used to construct a phylogenetic tree (Fig. 3). The phylogenetic tree revealed three main subclasses corresponding to GABABRs, calcium-sensing and pheromone receptors, and mGluRs. DdmGluPR diverged after the mGluR family-GABABRs split but before mGluR family divergence. Although, Drosophila DmXR diverged after mGluRs calcium-sensing and pheromone receptors split. The same phylogenetic relations were obtained with or without excluding positions with gaps. Because DdmGluPR has a hybrid structure with an extracellular region similar to mGluRs and a transmembrane region similar to GABABRs, we propose that DdmGluPR was the evolutionary precursor to mGluRs.

Expression and Localization of DdmGluPR—We isolated DdmGluPR cDNA encompassing the entire coding region (~2.5 kbp) by RT-PCR. Northern blot was carried out to determine the temporal regulation of DdmGluPR expression during development (Fig. 4A). DdmGluPR mRNA was expressed in vegetative cells and throughout development. The level of expression was relatively high until 4 h after starvation and gradually decreased thereafter, suggesting that it functions during the differentiation phase. To examine the localization of DdmGluPR, we expressed the DdmGluPR protein carrying a GFP tag at the N terminus. As shown in Fig. 4B, GFP-DdmGluPR was localized to the plasma membrane of axenically grown Ax-2 cells.

Inactivation of the DdmGluPR Gene—To determine the functional importance of DdmGluPR, the DdmGluPR gene was inactivated by homologous recombination. The targeting construct containing a part of the coding region with an insertion of a blasticidin cassette (Fig. 5A) was introduced into the Dictyostelium wild-type strain Ax-2. Individual clones were selected, and positive clones were identified by the presence of an ~3-kb PCR product that is ~1.5 kb in wild-type cells (Fig. 5B, left panel). The disruption of DdmGluPR was further confirmed by the absence of a PCR product from an oligo(dT)-primed RT-PCR (Fig. 5B, right panel).


Figure 4
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  		FIGURE 4.
Northern blot analysis of DdmGluPR mRNA expression during development. A, total RNA (5 µg) was prepared from either growing cells or from cells developed on non-nutrient agar plates for 0, 4, 8, 16, 20, and 24 h. The blot was hybridized with the probes shown. cAR1; a cAMP receptor, aca; an adenylyl cyclase, spiA; the spore-specific marker, ecmB; the prestalk/stalk-specific marker, psA; the prespore-specific marker. Mitochondrial rRNA Ig7 was used as a loading control. B, localization of GFP-DdmGluPR in axenically grown Ax-2 cells. Expression of GFP-DdmGluPR was visualized under fluorescence microscope (right panel, GFP-DdmGluPR). The left panel shows the phase image of the same field of cells (Phase). Scale bar:50 µm.

 
Cell Growth and Prestarvation Response in DdmGluPR-null Mutant—Because DdmGluPR was expressed in vegetative phase cells, we examined the cell growth of the DdmGluPR-null mutant. DdmGluPR-null cells grew faster at high cell density compared with wild-type Ax-2 cells and reached higher densities than Ax-2 cells (Fig. 6A). The absence of DdmGluPR appeared to enhance vegetative growth. A part of the differentiation program has been shown to begin before starvation (prestarvation response); premature expression of early developmental genes such as discoidin and cAR1 is induced as the density of growing cells increased (12). However, no significant differences in the expressions of discoidin and cAR1 were detected between growing DdmGluPR-null cells and wild-type Ax-2 cells (Fig. 6B). Thus, the regulation of prestarvation gene expression does not require DdmGluPR.

Development of DdmGluPR-null Mutant—To investigate the defect in the developmental process of the DdmGluPR-null mutant, development on non-nutrient agar plates was monitored microscopically. As shown in Fig. 7, DdmGluPR-null cells exhibited delayed aggregates formation compared with wild-type Ax-2 cells. We observed faint aggregates in wild-type cells at 5 h after starvation, and the aggregates grew larger and increased density thereafter, whereas the aggregates in DdmGluPR-null cells were not prominent even after 7 h and showed low density at 8 h. To analyze the relation of delayed aggregates formation in DdmGluPR-null cells to early gene expressions such as cAR1 and aca, we examined the expression patterns of these genes during development in DdmGluPR-null cells and wild-type Ax-2 cells by Northern blot analysis (Fig. 8A). cAR1 and aca were rapidly induced and peaked 2–4 h upon starvation and decreased thereafter in wild-type cells, whereas the expressions of these genes were reduced, sustained, and peaked at ~8 h in DdmGluPR-null cells compared with wild-type cells. We next examined chemotaxis in response to cAMP of DdmGluPR-null cells. The movement of cells toward a source of cAMP was observed microscopically. Fig. 8B showed directional movement of DdmGluPR-null cells toward cAMP, but it was inefficient. The migration distance from the spot of DdmGluPR-null cells was 66% of that of wild-type cells. Our findings suggest that the delayed aggregate formation of DdmGluPR-null cells phenotype is caused by inefficient chemotaxis toward cAMP with aberrant expression pattern of cAR1 and aca during early development. We further tested the ability of DdmGluPR expression to rescue the phenotype of DdmGluPR-null cells. The null cells that expressed GFP-DdmGluPR showed restorations of the delayed aggregates formation and the inefficient chemotaxis toward cAMP almost similar to those of Ax-2 cells (Fig. 9, A and B).


Figure 5
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  		FIGURE 5.
Isolation of DdmGluPR-null mutant. A, schematic representation of the DdmGluPR gene disruption. cDNA encompassing the entire coding region (~2.5 kbp) with restriction sites EcoRI and XbaI at 5' and 3', respectively was obtained by RT-PCR. The targeting construct contains the digested fragment with EcoRI and HindIII (~1.7 kbp), and the Bsr cassette was inserted into at KpnI site. The arrows denote two primers (primer U and L) for PCR analysis. Primer L recognizes part of the region that is not present in the targeting construct. B, identification of DdmGluPR disruptants by PCR analysis. Genomic DNA was isolated from several clones after transformation. PCR analysis using primers U and L is predicted to yield an ~3 kbp for a DdmGluPR gene disruptant and a product ~1.5 kbp for an intact gene (left panel). To detect the DdmGluPR transcript, total RNA was prepared from the transformants and RT-PCR analysis was performed using the same primers U and L (right panel). W, wild-type Ax-2; C2, C7, C8, and C9, blasticidin-resistant clones.

 


Figure 6
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  		FIGURE 6.
Cell growth and prestarvation response in DdmGluPR-null mutant. A, growth of wild-type Ax-2 (Wild) and DdmGluPR-null (KO) cells in an axenic suspension culture. Cells were diluted to 1 x 105 cells/ml in PS-medium and rotated at 160 rpm at 22 °C. B, expression pattern of prestarvation genes, cAR1 and discoidin, in wild-type Ax-2 and DdmGluPR-null cells. Cells were separately harvested at the indicated density or stationary growth phase (S), and extracted RNA was used for Northern blot analysis using cRNA probes of each genes.

 


Figure 7
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  		FIGURE 7.
Phenotype of DdmGluPR-null mutant on starvation-induced development. Wild-type Ax-2 (A, C, E, and G, Wild) and DdmGluPR-null (B, D, F, and H, KO) cells were harvested, washed, and spotted at a density of 1 x 107 cells/ml on non-nutrient agar plates. Development at 22 °C was monitored with microscope. Phase-contrast micrographs were taken after 5 (A and B), 6 (C and D), 7 (E and F), and 8 (G and H) h of starvation. Scale bar: 250 µm.

 
Our analysis of the primary structure of DdmGluPR suggests that DdmGluPR may be able to bind to an amino acid. To test the possibility of agonist or antagonist effect of an amino acid on DdmGluPR, we examined the effects of most of natural amino acids including Glu, Arg, Lys, Asp, His, Gly, Gln, Pro, Ser, Ala, Asn, Thr, Leu, Met, Val, and GABA on the starvation-induced chemotactic response toward cAMP. As shown in Fig. 9C, we could not detect any alteration of chemotactic response toward cAMP in the presence of an amino acid as indicated or GABA at 1 mM, suggesting that a simple amino acid is not a ligand for DdmGluPR. Aggregate formation on non-nutrient agar was also not affected in the presence of these amino acids or GABA (data not shown). Aggregation of starved cells requires the presence of extracellular molecules, the CMF involved in a cell density-sensing mechanism. CMF receptors appears to mediate the G-dependent and G-independent signal transductions (13, 14). To test the sensitivity to CMF, we starved wild-type and DdmGluPR-null cells at various densities in submerged culture in the presence or absence of CMF (Table 1). Both wild-type and DdmGluPR-null cells starved in the absence of CMF were able to aggregate in 24 h at densities 4 x 105 cells/ml or above. The same cells starved for 24 h in the presence of CMF prepared from either wild-type or DdmGluPR-null cells formed aggregates at lower densities. Thus, the cell density-sensing mechanism by CMF does not require DdmGluPR.


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  		TABLE 1
Ability to aggregate at different cell densities

Wild-type (Wild) and DdmGluPR-null (KO) cells were starved at various cell densities in submerged culture in the presence or absence of CMF. The fields of cells were examined after 24 h. The presence of aggregates is denoted with a plus, whereas the absence of aggregates is represented with a minus. CMF-A and CMF-B were prepared from wild-type and DdmXR-null cells, respectively.

 


Figure 8
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  		FIGURE 8.
Gene expression of cAMP-signaling components and chemotaxis response to cAMP in DdmGluPR-null cells. A, wild-type Ax-2 (Wild) and DdmGluPR-null (KO) cells were starved on non-nutrient agar plates for the time indicated. Total RNA was isolated and analyzed by northern blotting using cRNA probes for cAR1, aca, and Ig7. B, wild-type Ax-2 and DdmGluPR-null cells were starved in 10 mM Na-K buffer, pH 6.1, at 22 °C for 4 h. Small wells in the center of 1.5% non-nutrient agar plates were filled with cAMP solution (5 µl of 10 µM solution), and the starved cells were spotted on the agar plates 5 mm from the edge of the well at 1 x 107 cells/ml. The plates were further incubated for 5 h at 22 °C, and the resultant chemotaxis toward the wells was observed under a phase contrast microscope (left panel). The position of the cells spotted and the direction of cAMP gradient are indicated at the top. Chemotaxis response to cAMP was quantified by measuring the distance of cell migration of wild-type Ax-2 and DdmXR-null cells in each spot (right panel). Each value represents the mean ± S.E. (n = 6). *, significant at <0.001 compared with wild-type cells.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Glutamate receptors are categorized into ionotropic glutamate receptors (iGluRs) and mGluRs. The iGluRs internally contain ligandgated ion channels. Chen et al. (16) have identified an iGluR, GluR0, from a prokaryote, the cyanobacterium Synechocystis. GluR0 has a hybrid structure between iGluRs and potassium channels and is thought to be a common ancestor for these molecular families. The mGluRs are structurally and evolutionally distinct from the iGluRs.


Figure 9
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  		FIGURE 9.
Restoration of DdmGluPR-null mutant phenotype by ectopic expression of GFP-DdmGluPR and the effect of most of natural amino acids and GABA on cAMP chemotaxis. A, wild-type Ax-2 (Wild), DdmGluPR-null (KO), and the null mutant expressed GFP-DdmGluPR (KO + GFP-DdmGluPR) cells were harvested, washed, and spotted at a density of 1 x 107 cells/ml on non-nutrient agar plates. Phase contrast micrographs were taken after 6 h of starvation. Scale bar: 250 µm. B, wild-type Ax-2, DdmGluPR-null, and the null mutant expressed GFP-DdmGluPR cells were starved in 10 mM Na-K buffer, pH 6.1, at 22 °C for 4 h and spotted on the agar plates at 1 x 107 cells/ml, 5 mm from the edge of the well, which was filled with cAMP solution (5 µl of 10 µM solution). Chemotaxis response to cAMP was quantified by measuring the distance of cell migration. Each value represents the mean ± S.E. (n = 3). *, significant at <0.001 compared with KO cells. C, starvation-induced chemotactic response toward cAMP on non-nutrient agar in the presence of an amino acid as indicated or GABA at 1 mM concentration. Each value represents the mean ± S.E. (n = 3).

 
Here, we identified a new receptor DdmGluPR in Dictyostelium belonging to the mGluR family. The BLAST search showed that the extracellular region of DdmGluPR was closest in sequence similarity to mouse mGluR3 and Drosophila DmXR. Mitri et al. (8) has found the receptor called mXR as belonging to the mGluR subclass in insects such as the Anopheles, Apis, and Drosophila. They could not find any orthologues of mXR in C. elegans or mammalian genomes. The residues of mGluRs involved in the binding of the {alpha}-carboxylic and {alpha}-amino groups of glutamate were well conserved in both of DdmGluPR and Drosophila DmXR but the residues interacting with the {gamma}-carboxylic group of glutamate were not. Ligands for Drosophila DmXR and DdmGluPR are not determined yet, but this suggests that the natural ligand might be an amino acid-like molecule. However, a simple amino acid is unlikely as a ligand. The GABABR and the mGluR families appeared to be derived from a common ancestral receptor. The phylogenetic analysis suggests that DdmGluPR diverged after the mGluR family-GABABRs split but before mGluR family divergence. Although, Drosophila DmXR diverged after mGluR calcium-sensing and pheromone receptors split. This is in good agreement with the sequence in which the transmembrane region of DdmGluPR is similar to GABABRs while the region of DmXR is similar to mGluRs. Thus, we propose that DdmGluPR is an ancestor leading to the mGluR family divergence, and Drosophila DmXR is a direct ancestor for mGluRs divergence. mXR-type receptors would disappear during evolution in higher organisms except insects.

We also described the functional involvement of DdmGluPR in the early development of D. discoideum. Prestarvation factors and conditioned medium factors were not the ligands for DdmGluPR, because the responses against these factors showed no difference between wild-type cells and DdmGluPR-null cells. We also noted that the DdmGluPR-null mutant was not affected in sensing folate (data not shown), although folate contains a glutamate structure. DdmGluPR mRNA was expressed in vegetative cells and throughout development, but the level of the expression was relatively high until 4 h and peaked at 2 h (data not shown) after starvation, suggesting that an unknown ligand is released at early development and affects the expressions of cAR1 and aca through DdmGluPR.

The mGluRs are known to modulate synaptic properties in vertebrates. The Drosophila genome possesses another mGluR gene, DmGluRA, an mGluR2/3 ortholog (7). DmGluRA is the only functional mGluR in Drosophila. Bogdanik et al. (17) have described that DmGluRA is expressed at the glutamatergic neuromuscular junction and the null mutants display an increase in synaptic facilitation during short stimulus trains. The mutant phenotype cannot be replicated by an acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short term modulation. To understand the functional roles of DdmGluPR and mXRs will provide information for developmental roles of mGluRs in the nervous system or other tissues.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) DQ447637 [GenBank] Back

2 Present address: Faculty of Pharmaceutical Sciences, Doshisha Women's College of Liberal Arts, Kodo, Kyotanabe 610-0395, Japan. Back

3 Present address: Dept. of Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan. Back

1 To whom correspondence should be addressed. Tel.: 81-76-234-4473; Fax: 81-76-234-4473; E-mail: hideo{at}p.kanazawa-u.ac.jp.

4 The abbreviations used are: mGluR, metabotropic glutamate receptor; GPCR, G-protein-coupled receptor; LBD, ligand binding domain; CMF, conditioned medium factor; GABA, {gamma}-aminobutyric acid; GABAR, GABA receptor; RT-PCR, reverse transcription PCR; GFP, green fluorescent protein; iGluR, ionotropic GluR. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Y. Maeda at Tohoku University for providing Ax-2 cells, Drs. Y. Tanaka and T. Morio at Tsukuba University for technical support, and Dr. T. Tomiki at VALWAY Technology Center, NEC Soft, Ltd. for helpful discussion on the phylogenetic tree.



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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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