Distribution of metabotropic glutamate receptor DmGlu-A in Drosophila melanogaster central nervous system.

L-glutamate is the excitatory neurotransmitter at neuromuscular junctions in insects. It may also be involved in neurotransmission within the central nervous system (CNS), but its function therein remains elusive. The roles of glutamatergic synapses in the Drosophila melanogaster CNS were investigated, with focus on the study of DmGluRA, a G-protein-coupled glutamate receptor. In a first attempt to determine the function of this receptor, we describe its distribution in the larval and adult Drosophila CNS, using a polyclonal antibody raised against the C-terminal sequence of the protein. DmGluRA is expressed in a reproducible pattern both in the larva and in the adult. In particular, DmGluRA can be found in the antennal lobes, the optic lobes, the central complex, and the median bundle in the adult CNS. However, DmGluRA-containing neurons represented only a small fraction of all CNS neurons. DmGluRA immunoreactivity was not detected at the larval neuromuscular junction nor in the body wall muscles. The correlations between DmGluRA distribution and previously described glutamate-like immunoreactivity patterns, as well as the implications of these observations concerning the possible functions of DmGluRA in the Drosophila CNS, are discussed.

The Partner of Inscuteable/Discs-large complex is required to establish planar polarity during asymmetric cell division in Drosophila.

Frizzled (Fz) signaling regulates cell polarity in both vertebrates and invertebrates. In Drosophila, Fz orients the asymmetric division of the sensory organ precursor cell (pI) along the antero-posterior axis of the notum. Planar polarization involves a remodeling of the apical-basal polarity of the pI cell. The Discs-large (Dlg) and Partner of Inscuteable (Pins) proteins accumulate at the anterior cortex, while Bazooka (Baz) relocalizes to the posterior cortex. Dlg interacts directly with Pins and regulates the localization of Pins and Baz. Pins acts with Fz to localize Baz posteriorly, but Baz is not required to localize Pins anteriorly. Finally, Baz and the Dlg/Pins complex are required for the asymmetric localization of Numb. Thus, the Dlg/Pins complex responds to Fz signaling to establish planar asymmetry in the pI cell.

Constitutively active mutants of 5-HT4 receptors are they in unique active states?

Somatic mutations leading to constitutively active G-protein coupled receptors (GPCRs) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5-HT4 receptors (mutated or truncated in the C-terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55 degrees C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C-terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.

Rapsynoid/partner of inscuteable controls asymmetric division of larval neuroblasts in Drosophila.

Asymmetric cell division generates daughter cells with different developmental fates. In Drosophila neuroblasts, asymmetric divisions are characterized by (1) a difference in size between the two daughter cells and (2) an asymmetric distribution of cell fate determinants, including Prospero and Numb, between the two daughter cells. In embryonic neuroblasts, the asymmetric localization of cell fate determinants is under the control of the protein Inscuteable (Insc), which is itself localized asymmetrically as an apical crescent. Here, we describe a new Drosophila protein, Rapsynoid (Raps), which interacts in a two-hybrid assay with the signal transduction protein Galpha(i). We show that Raps is localized asymmetrically in dividing larval neuroblasts and colocalizes with Insc. Moreover, in raps mutants, the asymmetric divisions of neuroblasts are altered: (1) Insc is no longer asymmetrically localized in the dividing neuroblast; and (2) the neuroblast division produces two daughter cells of similar sizes. However, the morphologically symmetrical divisions of raps neuroblasts still lead to daughter cells with different fates, as shown by differences in gene expression. Our data show that Raps is a novel protein involved in the control of asymmetric divisions of neuroblasts.

Conservation of the ligand recognition site of metabotropic glutamate receptors during evolution.

Mammalian metabotropic glutamate receptors (mGluRs) are classified into 3 groups based on their sequence similarity and ligand recognition selectivity. Recently, we identified a Drosophila mGluR (DmGlu(A)R) which is about equidistant, phylogenetically, from the 3 mGluR groups. However, both the G-protein coupling selectivity and the pharmacological profile of DmGlu(A)R, as analysed with mutated G-proteins and a few compounds, look similar to those of mammalian group-II mGluRs. In the present study we carefully examined the pharmacological profile of DmGlu(A)R, and compared it to those of the rat mGlu(1a), mGlu(2) and mGlu(4a) receptors, representative of group-I, II and III respectively. The pharmacological profile of DmGlu(A)R was found to be similar to that of mGlu(2)R, and only very small differences could be identified at the level of their pharmacophore models. These data strongly suggest that the binding sites of these two receptors are similar. To further document this idea, a 3D model of the mGlu(2) binding domain was constructed based on the low sequence similarity with periplasmic amino acid binding proteins, and was used to identify the residues that possibly constitute the ligand recognition pocket. Interestingly, this putative binding pocket was found to be very well conserved between DmGlu(A)R and the mammalian group-II receptors. These data indicate that there has been a strong selective pressure during evolution to maintain the ligand recognition selectivity of mGluRs.

Mutagenesis and modeling of the GABAB receptor extracellular domain support a venus flytrap mechanism for ligand binding.

The gamma-aminobutyric acid type B (GABAB) receptor is distantly related to the metabotropic glutamate receptor-like family of G-protein-coupled receptors (family 3). Sequence comparison revealed that, like metabotropic glutamate receptors, the extracellular domain of the two GABAB receptor splice variants possesses an identical region homologous to the bacterial periplasmic leucine-binding protein (LBP), but lacks the cysteine-rich region common to all other family 3 receptors. A three-dimensional model of the LBP-like domain of the GABAB receptor was constructed based on the known structure of LBP. This model predicts that four of the five cysteine residues found in this GABAB receptor domain are important for its correct folding. This conclusion is supported by analysis of mutations of these Cys residues and a decrease in the thermostability of the binding site after dithiothreitol treatment. Additionally, Ser-246 was found to be critical for CGP64213 binding. Interestingly, this residue aligns with Ser-79 of LBP, which forms a hydrogen bond with the ligand. The mutation of Ser-269 was found to differently affect the affinity of various ligands, indicating that this residue is involved in the selectivity of recognition of GABAB receptor ligands. Finally, the mutation of two residues, Ser-247 and Gln-312, was found to increase the affinity for agonists and to decrease the affinity for antagonists. Such an effect of point mutations can be explained by the Venus flytrap model for receptor activation. This model proposes that the initial step in the activation of the receptor by agonist results from the closure of the two lobes of the binding domain.

Comparative effect of L-CCG-I, DCG-IV and gamma-carboxy-L-glutamate on all cloned metabotropic glutamate receptor subtypes.

In a previous study we reported that the addition of a carboxylic group to the mGlu receptor agonist aminocyclopentane-1,3-dicarboxylate (ACPD) changes its properties from agonist to antagonist at both mGlu1 and mGlu2 receptors, and resulted in an increase in affinity at mGlu4 receptors, with isomers being either agonists or antagonists. In the present study, the effect of gamma-carboxy-L-glutamic acid (Gla) and (2S,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), two carboxylic derivatives of non-selective agonists, were examined on all cloned mGlu receptors. We found that this additional carboxylic group on glutamate prevents its interaction with group-I mGlu receptors and generates a potent group-II antagonist (K(B) = 55 microM on mGlu2). At group-III mGlu receptors, Gla was found to be either an antagonist (mGlu7 and mGlu8 receptors) or a partial agonist (mGlu4 and mGlu6 receptors). We show here that L-CCG-I is a general mGlu receptor agonist activating all cloned receptors. We also confirm that DCG-IV, which corresponds to L-CCG-I with an additional carboxylic group, is a selective group-II agonist. However, this additional COOH group changes the properties of L-CCG-I from an agonist to an antagonist at all group-III receptors, making this compound one of the most potent group-III mGlu receptor antagonist known so far. These observations will be useful for the development of more potent and selective mGlu receptor agonists and antagonists.

The G protein-coupling profile of metabotropic glutamate receptors, as determined with exogenous G proteins, is independent of their ligand recognition domain.

Metabotropic glutamate (mGlu), Ca2+-sensing, gamma-aminobutyric acidB, and a large number of pheromone receptors constitute a peculiar family of G protein-coupled receptors. They possess a large extracellular domain that has been proposed to constitute their ligand binding domain. The aim of the current study was to examine whether this large ligand binding domain had any influence on the G protein-coupling selectivity of the receptor, and vice versa. We chose mGlu receptors, which are classified into three groups according to their sequence homology and pharmacology, as representatives of this receptor family. To define a G protein-coupling profile for these receptors, we used a set of exogenous phospholipase C-activating G proteins in the same way that synthetic ligands are used to define agonist and antagonist pharmacological profiles. This set includes Galpha15, Galpha16, Galphaq, and chimeric Galphaq proteins with the last few amino acids of either Galphai2 (Galphaqi), Galphao (Galphaqo), or Galphaz (Galphaqz). Cotransfection of mGlu receptors with these G proteins and examination of their coupling to phospholipase C revealed that group I, II, and III receptors have distinct G protein-coupling profiles. By swapping the extracellular domains of the most distantly related mGlu receptors (the rat group I mGlu1a and the Drosophila melanogaster group II DmGluA receptors), we show that the extracellular domain determines the agonist pharmacological profile and that this domain does not modify the G protein-coupling profile determined by the seven-transmembrane-domain region of mGlu receptors.

Cloning and functional expression of a Drosophila metabotropic glutamate receptor expressed in the embryonic CNS.

The excitatory neurotransmitter glutamate plays important roles in the mammalian brain, ranging from synaptic plasticity to memory. To mediate these functions, glutamate activates two types of receptors: ligand-gated channels and metabotropic receptors coupled to G-proteins. Both families of glutamate receptors share no sequence homology and possess original structural features compared with other ligand-gated channels and G-protein-coupled receptors, respectively. Glutamate-gated receptor-channel subunits have already been characterized in invertebrates. Here we report the cloning and functional characterization of an invertebrate metabotropic glutamate receptor (DmGluRA) isolated from Drosophila melanogaster. This receptor displays 45 and 43% amino acid sequence identity with its mammalian homologs mGluR3 and mGluR2, respectively. Moreover, its pharmacology and transduction mechanisms are surprisingly similar to those of mGluR2 and mGluR3. DmGluRA is expressed in the CNS of the late embryo. These results indicate that the original structural features of both glutamate receptor types are conserved from insects to mammals and suggest that the functions of these receptors have been highly conserved during evolution.

Coupling of metabotropic glutamate receptors 2 and 4 to G alpha 15, G alpha 16, and chimeric G alpha q/i proteins: characterization of new antagonists.

Together with the calcium-sensing receptor, the metabotropic glutamate receptors (mGluRs) share no sequence homology with the other G protein-coupled receptors (GPCRs) and therefore constitute a new family of receptors. Recently, it was reported that G alpha 15 and G alpha 16 subunits allow many GPCRs to activate phospholipase C (PLC). Furthermore, the exchange of a few carboxyl-terminal residues of G alpha q by those of G alpha 12 or G alpha o allows the resulting chimeric G alpha subunits (G alpha ql and G alpha qol respectively) to couple Gi-coupled receptors to PLC. We report that mGluR2 and mGluR4, two receptors negatively coupled to adenylyl cyclase, activate PLC when coexpressed with G alpha 15, G alpha ql or G alpha qo. This indicates that the carboxyl-terminal end of the G alpha subunit also plays an important role in the specific interaction between mGluRs and the G proteins. In addition, the measurement of PLC activation by Gi-coupled mGluRs coexpressed with these G alpha subunits constitutes an easy functional assay for the pharmacological characterization of these receptors. The rank order of potency of antagonists was found to be (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine approximately (R,S)- alpha-methyl-4-phosphonophenylglycine > (R,S)-alpha-methyl-4-sulfonophenylglycine > (R,S)-alpha-methyl-4-tetrazolylphenylglycine = (S)-2-amino-2-methyl-4-phosphonobutyrate for mGluR2 and to be (R,S)-alpha-methyl-4-phosphonophenylglycine > or = (S)-2-amino-2-methyl-4-phosphonobutyrate > > (R,S)-alpha-methyl-4-sulfonophenylglycine [(R,S)-alpha-methyl-4-tetrazolylphenylglycine and (2S,3S,4S)-2-methyl-2-(carboxycyclopropyl)glycine being inactive at 1 mM] for mGluR4. Using this functional assay, (R,S)-alpha-methyl-4-phosphonophenylglycine was found to have a similar KB value for mGluR2 and mGluR4.

Transected axons of adult hypothalamo-neurohypophysial neurons regenerate along tanycytic processes.

In the intact hypothalamo-neurohypophysial system, oxytocinergic or vasopressinergic neurons project their axons throughout the internal layer of the median eminence towards the blood vessels of the hypophysial neural lobe. When transected at the level of the median eminence, these axons undergo massive sprouting towards the external layer of the organ and the underlying perivascular region containing hypophysial portal vessels. The present study was designed to explore the possible roles of median eminence glial cells in such a reorganization of transected neurohypophysial axons. The relationships between regenerating axons and glial cells were studied by laser scanning confocal microscopy and electron microscopy on vibratome sections immunostained with specific antibodies against neurohypophysial peptides and/or against glial markers. All along the intact median eminence, two main types of glial cells were identified: (1) tanycytes immunoreactive to vimentin and slightly immunoreactive to glial fibrillary acidic protein, and (2) classical astrocytes immunoreactive to glial fibrillary acidic protein but vimentin-negative. In the intact median eminence, neurohypophysial axons were associated with astrocytic processes located in the internal layer. After a lesion of the hypophysial stalk, peptidergic regenerating axonal sprouts were found to project massively towards the external layer and to penetrate the underlying perivascular region in close association with tanycytic-like processes immunoreactive to both vimentin and to glial fibrillary acidic protein. In contrast, regenerating sprouts were absent from those regions of the lesioned median eminence containing astrocytic processes immunoreactive to glial fibrillary acidic protein but vimentin-negative. When fixed lesioned median eminences were treated by placing crystals of the lipophilic dye DiI on their ventricular surface, regenerating axons were found to be closely associated with DiI-labelled tanycytic-like end feet terminating in the external layer, and with connected thin processes projecting through the external vascular region. These data indicate that in the median eminence of the adult rat, lesioned neurohypophysial axons regenerate in close association with tanycytic processes.