Interactions of the C terminus of metabotropic glutamate receptor type 1alpha with rat brain proteins: evidence for a direct interaction with tubulin.
Metabotropic glutamate receptors (mGluRs) are coupled to G protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. As part of a strategy for defining the mechanisms for the specific targeting of mGluR1alpha, rat brain proteins which interact with the intracellular carboxy terminus of mGluR1alpha have been characterized, using affinity chromatography on a glutathione S-transferase fusion protein that contains the last 86 amino acids of mGluR1alpha. Three of the proteins specifically eluted from the affinity column yielded protein sequences, two of which were identified as glyceraldehyde-3-phosphate dehydrogenase and beta-tubulin; the other was an unknown protein. The identity of tubulin was confirmed by western immunoblotting. Using a solid-phase binding assay, the mGluR1alpha-tubulin interaction was shown to be direct, specific, and saturable with a KD of 2.3+/-0.4 microM. In addition, mGluR1alpha, but not mGluR2/3 or mGluR4, could be coimmunoprecipitated from solubilized brain extracts with tubulin using anti-beta-tubulin antibodies. However, mGluR1alpha could not be coimmunoprecipitated with the tubulin binding protein gephyrin, nor could it be coimmunoprecipitated with PSD95. Collectively these data demonstrate that the last 86 amino acids of the carboxyl-terminal tail of mGluR1alpha are sufficient to determine its interaction with tubulin and that there is an association of this receptor with tubulin in rat brain.
Ciruela
F
F
Medical Research Council Anatomical Neuropharmacology, Oxford, England, UK.
Robbins
M J
MJ
Willis
A C
AC
McIlhinney
R A
RA
D000818:Animals; D001923:Brain Chemistry / Q000502:physiology*; D015151:Immunoblotting; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism; D014404:Tubulin / Q000032:analysis / Q000378:metabolism*
D018094:Receptors, Metabotropic Glutamate; D014404:Tubulin; C104077:metabotropic glutamate receptor type 1