Immunoprecipitation, immunoblotting, and immunocytochemistry studies suggest that glutamate receptor δ subunits form novel postsynaptic receptor complexes

Abstract

An antibody was made to a C-terminus peptide of the glutamate receptor δ2 subunit and used to study the distribution, biochemical properties, and developmental expression of the δ receptor in rat brain. The antibody recognizes both δ1 and δ2 but not AMPA, kainate, NMDA, and mGluR1α glutamate receptor subunits based on Western blot analysis of transfected HEK-293 cells. Western blot analysis of brain showed a single immunoreactive band, migrating at M1 = 114,000. Immunoprecipitation of detergent- solubilized cerebellar membranes was done to determine if δ is associated with other glutamate receptor subunits and if it binds any of the common excitatory amino acid ligands. Based on results of these studies, AMPA, kainate, NMDA, and mGluR1α subunits do not coassemble with δ subunits, and 3H-glutamate, 3H-AMPA and 3H-kainate do not bind to the δ receptor complex, Western blot and immunocytochemical analyses showed marked expression of δ in the cerebellum while lower levels were detected in other regions of the brain. A dramatic increase of δ1/2 immunoreactivity was observed in the cerebellum between the ages of 10 and 15 d postnatal. Light and electron microscopy, respectively, demonstrated dense immunostaining in Purkinje cells and in postsynaptic densities of the adult parallel fiber- Purkinje spine synapse. The prominent δ 1/2 immunoreactivity found in the parallel fiber-Purkinje spine synapse, and the temporal correlation of the development of this synapse with the major increase in δ 1/2 immunoreactivity, suggest a major functional role for the δ subunits in cerebellar circuitry.