Glutamate is a major excitatory neurotransmitter in mammalian brain. Glutametergic neurotransmission is mediated by a family of glutamate receptors which can be grouped in two classes, ionotropic (GluR) and metabotropic (mGluR) receptors. The ionotropic GluRs can be divided into two subclasses, N-methyl-D-Aspartate (NMDA) and non-NMDA receptors. Five different forms of NMDA receptors (NMDAR1, R2A, R2B, R2C, and R2D) have been isolated. NMDAR1 is always required for the formation of functional NMDA receptors. Non-NMDA receptors can be divided into at least two subtypes, AMPA receptors which bind to methyl-4-isoxazole proprionic acid (AMPA) and kainate binding (KA or kainic acid) receptors. Multiple subunits appear to comprise the family of non-NMDA receptors. GluR1-4 receptors (also known as GluR-A, B, C, and D) preferentially bind to AMPA. KA receptors consist of five subunits: GluR5, 6, 7, KA-1, and KA-2. Functional receptors are formed by various combinations of these subunits and multiple forms of GluR are expressed in the same populations of neurons. GluR2 migrates at a molecular weight of ~102 kDa in SDS-PAGE.
Monoclonal antibody (mAb) 6C4 recognizes rat, macaque monkey, and dog GluR2. It does not cross-react with GluRs 1, 3, 4c, 5, 6, or 7. A recombinant trpE fusion protein containing the putative N-terminal portion (amino acids 175-430) of GluR2 was used as immunogen. The antibody was originally characterized by western blot analysis and immunohistochemical analysis of paraformaldehyde-fixed cells and paraformaldehyde-fixed frozen tissue sections. Its specificity for GluR2 was determined by radioimmunoassay and by western blot analysis. In western blots obtained from cells transiently transfected from with different GluRs, the antibody recognized GluR2 but not GluRs 1, 3, 4c, 5, 6, 7. Cross-species western blot reactivity was determined by using homogenates of fresh frozen tissue samples from rat, macaque monkey and dog cerebral cortex. In western blot analysis of tissue homogenates, clone 6C4 recognizes GluR2 as an ~102 kDa protein. Other proteins noted by western blot at 66 kDa and lower molecular weights appear to be breakdown products of GluR2. The specificity of 6C4 for GluR2 in rat brain tissue sections was verified by showing that antibody reactivity was blocked by preincubation with recombinant GluR2 protein but not with recombinant GluR1 or GluR3 protein. Immunohistochemical analysis using 6C4 shows that GluR2 is widely distributed at both the cellular and synaptic levels in rat hippocampus and neocortex, please refer to Vissavajjhala, et al. for additional staining information.
