Antibody specific for phosphorylated AMPA-type glutamate receptors at GluR2 Ser-696.
Possible phosphorylation sites on the Purkinje cell alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunits were identified using in vitro kinase assays of 17 synthetic peptides derived from the transmembrane-3 (TM3) domain to the end of C-terminal of a rat glutamate receptor 2 (GluR2). Only two peptides containing Ser-662 and Ser-696 were found to be efficiently phosphorylated by protein kinase C (PKC). The peptide including Ser-696 was also phosphorylated by protein kinase G (PKG). Another peptide containing Thr-692 of a rat GluRA, clone almost identical to GluR1, was phosphorylated by PKC but not by PKG. Antisera recognizing phosphorylated AMPA receptor subunits at GluR2 Ser-696 or the homologous sites of GluR1/3/4 were produced, and the specificity of one of them, named 12P3, was established by enzyme-linked immunosorbent assay (ELISA), immunoblot and immunoprecipitation analyses. 12P3-immunocytochemistry on cerebellar slices demonstrated an AMPA-induced transient AMPA receptor phosphorylation, which appeared in Purkinje cell dendrites as well as somata immediately after AMPA treatment and disappeared after 20 min. This antibody may be a useful tool to study the role of AMPA receptor phosphorylation in producing synaptic plasticity.
Nakazawa
K
K
Laboratory for Synaptic Function, Institute of Physical and Chemical Research (RIKEN), Saitama, Japan.
Tadakuma
T
T
Nokihara
K
K
Ito
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000302:isolation & purification; D000918:Antibody Specificity; D002531:Cerebellum / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D008297:Male; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011689:Purkinje Cells / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D018091:Receptors, AMPA / Q000276:immunology / Q000378:metabolism*; D017470:Receptors, Glutamate / Q000276:immunology / Q000378:metabolism*; D012694:Serine / Q000276:immunology / Q000378:metabolism*
D000906:Antibodies; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; D012694:Serine; D011493:Protein Kinase C