Identification of amino acids in the glutamate receptor, GluR3, important for antibody-binding and receptor-specific activation.
We reported (Twyman, R. E., Gahring, L. C., Speiss, J., and Rogers, S. W. (1995) Neuron 14, 755-762) that antibodies to a subregion of the glutamate receptor (GluR) subunit GluR3 termed GluR3B (amino acids 372-395), act as highly specific GluR agonists. In this study we produced additional rabbit anti-GluR3B-specific antibodies, ranked them according to their ability to function as GluR agonists and characterized the immunoreactivity using deletion and alanine substitution mutagenesis. These anti-GluR3B antibodies bound to a subset of the residues in GluR3B (amino acids 372-386), of which glutamate 375, valine 378, proline 379, and phenylalanine (Phe) 380 were preferred. The level of GluR activation correlated with the binding of antibody to Phe-380, which suggests that immunoreactivity directed toward Phe-380 is an index for the anti-GluR agonist potential. Since the identity of this residue varies between respective GluR subunits, this suggested that this residue may be important for imparting antibody subunit specificity. To test this possibility, the alanine in GluR1 was converted to a phenylalanine, which extended the subunit specificity from GluR3 to the modified GluR1. We conclude that antibody contacts with key residues in the GluR3B region define a novel GluR subunit-specific agonist binding site and impart subunit-specific immunoreactivity.
Carlson
N G
NG
Salt Lake City Veteran's Administration Medical Center and Geriatrics Research, Education and Clinical Center, Salt Lake City, Utah 84148, USA.
Gahring
L C
LC
Twyman
R E
RE
Rogers
S W
SW
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000494:pharmacology*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D004797:Enzyme-Linked Immunosorbent Assay; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D018091:Receptors, AMPA / Q000276:immunology*; D017470:Receptors, Glutamate / Q000276:immunology / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D000906:Antibodies; D018690:Excitatory Amino Acid Agonists; D010446:Peptide Fragments; D018091:Receptors, AMPA; D017470:Receptors, Glutamate; C104723:glutamate receptor ionotropic, AMPA 3; D000409:Alanine