Cloning of the long intracellular loop of the AMPA-selective glutamate receptor for phosphorylation studies

It is now widely accepted that many receptors are regulated by phosphorylation. The glutamate receptor which belongs to a family of AMPA receptors has recently been cloned and contains in the long intracellular loop several consensus phosphorylation sites for Ca(2+)-calmodulin-dependent protein kinase type II and protein kinase C. In order to study the possible function of these phosphorylation sites antibodies are specifically raised against these sites. For this purpose the DNA encoding the long intracellular loop was synthesized by PCR amplification from rat cerebral cortex and hippocampus RNA. Primer sequences were taken from the published receptor cDNA and the resulting inserts showed after sequence determination the presence of different receptor isoforms. The proteins have been expressed by the T7 RNA polymerase system. These can then be used after purification to generate antibodies against the entire protein and partial peptides both in a phosphorylated and unphosphorylated form.