Institut de
Génomique
Fonctionnelle

I- Drosophila larval neuromuscular junction : role of different components in growth and activity of this synapse

II- Insect metabotropic glutamate receptors : structure, functions and evolution

II- Insect metabotropic glutamate receptors : structure, functions and evolution

A- Situation of the research area

Metabotropic glutamate receptors (mGluRs) belong to the class C of G protein coupled receptors. MGluRs are important targets of the neurotransmitter glutamate in the mammalian brain. At least eight neuronal mGluR receptor (mGlu1 to mGlu8) have been identified in the vertebrate brain with different physiological, pharmacological properties and distribution (for review, Pin et al., 2004). We found that the complete sequenced genome of the fruit fly Drosophila melanogaster encode only two receptors homologous to mGluRs (fig.1) : DmGluRA (Parmentier et al., 1996) and an new receptor we called DmXR (see below). We had found that, surprisingly, the evolutionary distant Drosophila mGluR (DmGluAR) shares a very similar pharmacological profile with its mammalian orthologues (mGlu2R and mGlu3R) (Parmentier et al., 1998 ; Raymond et al., 1999 ; Parmentier et al., 2000). The function of DmGluRA is described in the neuromuscular junction chapter.

B- Previous data from this team:Anopheles, Apis and Drosophila mX receptors are new homologues of mGluRs with a divergent ligand binding pocket (Mitri et al., 2004, JBC)

We have shown that the DmX receptor is activated by an amino acid different from glutamate in insects. Indeed sequence comparison associated with 3D modeling of the ligand binding pocket revealed that the residues contacting the amino acid moiety of glutamate (the aCOO- and NH3+ groups) were conserved in DmXR, whereas the residues interacting with the g-carboxylic group were not. Of interest, this receptor was activated by an amino acid-like compound found in extracts from adult Drosophila head and from other insects (Anopheles, Schistocerca). Sequence orthologue to the orphan DmXR could only be found in insect genome (Anopheles gambiae, Apis mellifera). Our results suggested that the mXRs evolved to recognize an amino acid different from glutamate in insects.

Fig. 1 : Phylogenetic tree of family III GPCRs

C- Research project (methodology, techniques, planned milestones)

1.THE RECEPTOR DmX

1-1.Ligands of the DmX receptor To deorphanize DmXR, we have extracted and purified the endogenous ligand from the adult head of Drosophila using a biochemical approach (ion exchange chromatography, HPLC, Nuclear Magnetic Resonance). In 2005, we identified the endogenous ligand of the receptor. We also discovered that a plant synthetized compound, known for its insecticide properties, activates DmXR. In order to compare the mXR ligand binding pocket to the canonical mgluRs binding pocket, we plan to identify the ligand binding pocket residues involved in the interaction with the different ligands by docking of the ligands and site selected mutagenesis.

1-2. Function of the DmX receptor. To determine the function of DmXR, we are currently using a neuropharmacogenetic approach (i.g. the examination of the effects of drugs in presence of genes variants – DmXR receptors loss of function or gain of function-). Drugs (DmXRs agonists, antagonist) are administered to adult flies in their food. These pharmacological tools have introduced behavioral alterations in dose-response studies. In an ongoing study, we are studying the effects of these drugs in DmXR mutants context. Our results leaded to :

1) the discovery of one of the DmXR function, 2) the deposit of a patent covering the use of the DmXR as target for anti-insect compound.

In order to compare the mXR ligand binding pocket to the canonical mgluRs binding pocket, we plan to identify the ligand binding pocket residues involved in the interaction with the different ligands by docking of the ligands and site selected mutagenesis. In addition to the typical use of Drosophila (i.e. screening for novel genes and their function), the fruit fly is becoming a good model when a combination of gene alteration, pharmacological and functional assays of a phenotype (e.g. behavior) is needed. Such a combined approach is particularly valuable in the studies of complex system such as the CNS. Furthermore, the identification of the function of divergent receptors may open the way to the discovery of new compounds that would help in the struggle against pathogenic insects.