Interactions of the C terminus of metabotropic glutamate receptor type 1 alpha with rat brain proteins: Evidence for a direct interaction with tubulin

Metabotropic glutamate receptors (mGluRs) are coupled to G protein second m essenger pathways and modulate glutamate neurotransmission in the brain, wh ere they are targeted to specific synaptic locations. As part of a strategy for defining the mechanisms for the specific targeting of mGluR1 alpha, ra t brain proteins which interact with the intracellular carboxy terminus of mGluR1 alpha have been characterized, using affinity chromatography on a gl utathione S-transferase fusion protein that contains the last 86 amino acid s of mGluR1 alpha. Three of the proteins specifically eluted from the affin ity column yielded protein sequences, two of which were identified as glyce raldehyde-3-phosphate dehydrogenase and beta-tubulin; the other was an unkn own protein. The identity of tubulin was confirmed by western immunoblottin g. Using a solid-phase binding assay, the mGluR1 alpha-tubulin interaction was shown to be direct, specific, and saturable with a K-D of 2.3 +/- 0.4 m u M. In addition, mGluR1 alpha, but not mGluR2/3 or mGluR4, could be coimmu noprecipitated from solubilized brain extracts with tubulin using anti-beta -tubulin antibodies. However, mGluR1 alpha could not be coimmunoprecipitate d with the tubulin binding protein gephyrin, nor could it be coimmunoprecip itated with PSD95. Collectively these data demonstrate that the last 86 ami no acids of the carboxyl-terminal tail of mGluR1 alpha are sufficient to de termine its interaction with tubulin and that there is an association of th is receptor with tubulin in rat brain.