Identification of group ii metabotropic glutamate receptor-interacting proteins: their role in receptor function in neurons

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Project objective

The principal objective of the proposal is to identify metabotropic glutamate 2 and 3 (mGlu 2/3) receptor interacting proteins and to specify their roles in receptor neurotic targeting, membrane trafficking and signaling in central mammalian neurons. The so-called proteomic method will be used to identify mGlu2/3 receptor interacting proteins. The C-terminus of the mGlu 2/3 receptor contains a specific amino acid sequence, which has the recognition motif specific to PDZ domain proteins. Therefore, the C-terminus sequence of the mGlu 2/3 receptor will be used to fish interacting proteins from brain extract. These proteins will then be isolated, separated by two-dimensional electrophoresis, analyzed by mass spectrometry and identified from protein data-base. Cerebellar granule cells will be transfected with the cDNA of the identified proteins either alone or co-transfected with mGlu 2/3 receptors.Immunocytochemical studies will be used to elucidate the localization in membranes versus cytosol of the receptor-protein complex and their distribution in sub cellular (axonal, somato-dendritic, synaptic) compartments. Electrophysiological and calcium fluorescent measurements will be assessed to examine the effect of these interacting proteins in receptor signaling. Furthermore, dialysis of mimetic peptides into neurons and the expression of oligonucleotide antisense of the protein will serve to confirm the role of the endogenenous proteins in receptor targeting and signaling.

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Participants

• CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE · PARISCoordinatorFrance

Links

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