Biochemical and assembly properties of GluR6 and KA2, two members of the kainate receptor family, determined with subunit-specific antibodies.

To examine subunit assembly and biochemical properties of two members of the kainate family of glutamate receptors (GluR), antibodies were made to synthetic peptides corresponding to the carboxyl termini of GluR6 and KA2. Immunoblot analysis of membranes from human embryonic kidney cells transfected with glutamate receptor cDNAs showed that these antibodies are selective for their respective receptor subunit except that the antibody to GluR6 also recognizes GluR7, which is expected due to the sequence homology between the two subunits at the carboxyl terminus. In transfected cell membranes, immunoblot analysis with the antibody to GluR6 showed a major immunoreactive band at 118 kDa and minor bands at 103 and 28 kDa. The 103-kDa band appears to be a deglycosylated form of GluR6 since deglycosylation eliminates staining at 118 kDa and increases staining of the 103-kDa band. Immunoblot analysis of KA2 transfected cell membranes shows a major band at 123 kDa and minor bands at 109 and 37 kDa. Deglycosylation converts the 123-kDa band into a 109-kDa band. Analysis of brain tissues shows that both antibodies label single major bands which migrate at the same molecular masses as those from transfected cell membranes, 118 and 123 kDa for GluR6 and KA2, respectively. Immunoprecipitation studies showed that antibodies to GluR6 and KA2 selectively immunoprecipitated [3H]kainate binding activity, but not 3H-labeled alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) binding activity, from Triton X-100-solubilized rat brain membranes. Furthermore, each antibody coimmunoprecipitated GluR6 and KA2 from cells co-transfected with GluR6 and KA2 cDNAs and from detergent-solubilized rat brain membranes, indicating that these two subunits can coassemble into a molecular complex. Interestingly, GluR1 and GluR2, subunits of the AMPA receptor, also co-immunoprecipitated with GluR6 in cells co-transfected with GluR6 and GluR1 or GluR2 cD-NAs. Such complexes appear to be present to a limited extent in the brain.