Pharmacological characterization and immunoaffinity purification of metabotropic glutamate receptor from Drosophila overexpressed in Sf9 cells

Panneels, Valérie; Eroğlu, Çağla; Cronet, Philippe; Sinning, Irmgard

doi:10.1016/s1046-5928(03)00100-1

Cited by 23 publications

(28 citation statements)

References 29 publications

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“…Because DmGluRA is the only functional metabotropic glutamate receptor encoded within the sequenced Drosophila genome (Mitri et al, 2004), it seemed probable that previous experimental limitations prevented detection of the receptor at the NMJ. Panneels et al (2003) recently described a superior new monoclonal antibody (7G11) directed against the extracellular domain of DmGluRA. We used this new probe to reevaluate DmGluRA localization at NMJs of mature third instar larvae.…”

Section: Resultsmentioning

confidence: 99%

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TheDrosophilaMetabotropic Glutamate Receptor DmGluRA Regulates Activity-Dependent Synaptic Facilitation and Fine Synaptic Morphology

Bogdanik¹,

Mohrmann²,

Ramaekers³

et al. 2004

J. Neurosci.

141

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In vertebrates, several groups of metabotropic glutamate receptors (mGluRs) are known to modulate synaptic properties. In contrast, the Drosophila genome encodes a single functional mGluR (DmGluRA), an ortholog of vertebrate group II mGluRs, greatly expediting the functional characterization of mGluR-mediated signaling in the nervous system. We show here that DmGluRA is expressed at the glutamatergic neuromuscular junction (NMJ), localized in periactive zones of presynaptic boutons but excluded from active sites. Null DmGluRA mutants are completely viable, and all of the basal NMJ synaptic transmission properties are normal. In contrast, DmGluRA mutants display approximately a threefold increase in synaptic facilitation during short stimulus trains. Prolonged stimulus trains result in very strongly increased (ϳ10-fold) augmentation, including the appearance of asynchronous, bursting excitatory currents never observed in wild type. Both defects are rescued by expression of DmGluRA only in the neurons, indicating a specific presynaptic requirement. These phenotypes are reminiscent of hyperexcitable mutants, suggesting a role of DmGluRA signaling in the regulation of presynaptic excitability properties. The mutant phenotypes could not be replicated by acute application of mGluR antagonists, suggesting that DmGluRA regulates the development of presynaptic properties rather than directly controlling short-term modulation. DmGluRA mutants also display mild defects in NMJ architecture: a decreased number of synaptic boutons accompanied by an increase in mean bouton size. These morphological changes bidirectionally correlate with DmGluRA levels in the presynaptic terminal. These data reveal the following two roles for DmGluRA in presynaptic mechanisms: (1) modulation of presynaptic excitability properties important for the control of activity-dependent neurotransmitter release and (2) modulation of synaptic architecture.

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Section: Resultsmentioning

confidence: 99%

“…Df(4)O2 removes the whole DmGluRA. The epitope recognized by 7G11 antibody is part of the extracellular domain, between the LBD and the seven transmembrane domain (7TM) (Panneels et al, 2003), and is not deleted in DmGluRA 112b allele. The sequence selected for DmGluRA-RNAi constructs is indicated at the bottom.…”

Section: Generation Of Dmglura Mutantsmentioning

confidence: 99%

TheDrosophilaMetabotropic Glutamate Receptor DmGluRA Regulates Activity-Dependent Synaptic Facilitation and Fine Synaptic Morphology

Bogdanik¹,

Mohrmann²,

Ramaekers³

et al. 2004

J. Neurosci.

141

View full text Add to dashboard Cite

show abstract

“…Homologous competitive binding experiments were performed on Sf9DGR and DrDGR membranes, as described (7,8), in the presence of varying concentrations of cold L-glutamate (10 Ϫ3 to 10 Ϫ8 M) to determine the affinity of the receptor to glutamate. Membranes (20-40 g) were incubated with 1.5 M tritiated glutamate (Amersham Pharmacia Biotech) in 20 mM Tris, (pH 7.4) buffer containing 0.2% BSA for 2 h at 25°C.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we have described the overexpression of DmGluRA in Drosophila photoreceptor cells and in Sf9 cells (7,8). We have purified and reconstituted DmGluRA, and showed that the ligand binding depends on the presence of sterols in the liposomes (8).…”

Section: Dmglura Has Different Affinities To Glutamate Depending On Thementioning

confidence: 95%

“…The first Drosophila mGluR cloned, (DmGluRA), shares the structural features characteristic for mammalian mGluRs (6). Recently, we have reported the expression of DmGluRA in insect cells by recombinant baculovirus infection (7), and in transgenic Drosophila melanogaster (8), where the receptor is directed to the photoreceptor cells in the fruit fly eye, and is colocalized with rhodopsin in specialized membrane stacks, called rhabdomeres.…”

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confidence: 99%

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