Pergamon Neuroscience Vol. 73, No. 4, pp. 903 906, 1996 Copyright© 1996IBRO. Publishedby Elsevier ScienceLtd Printed in Great Britain 0306-4522/96 $15.00+ 0.00 PII: S0306-4522(96)00157-1 Letter to Neuroscience THE C-TERMINAL DOMAIN OF GLUTAMATE RECEPTOR SUBUNIT 1 IS A TARGET FOR CALPAIN-MEDIATED PROTEOLYSIS X. BI,*t V. CHANG,* E. MOLNAR,:~ R. A. J. MclLHINNEY~: and M. BAUDRY* *Neuroscience Program, USC, Los Angeles, CA 90089-2520, U.S.A. :~Medical Research Council, Anatomical Neuropharmacology Unit, Mansfield Road, Oxford, OX1 3TH, U.K. Key words: glutamate receptors, calpain, plasticity, neurodegeneration. The AMPA receptors are glutamate-gated ion chan- nels mediating synaptic transmission at the majority of excitatory synapses in the mammalian CNS. They are composed of four subunits (GluR1-4) which exist in two alternatively spliced variants (flip and flop) and are generally considered to form pentameric receptors.7,15 The transmembrane structure of the receptors remains a matter of controversy as some data suggest a transmembrane topology consisting of five,t5 four, 7 or three 3's membrane spanning regions. Some receptor properties have been shown to be regulated by phos- phorylation processes as well as by the phospholipid environment. ~'t°'t3 More recently, we have shown that calcium treatment of thin (10 pm) frozen-thawed brain sections resulted in profound modifications of the immunochemical properties of the AMPA receptors.5 More specifically, immunolabelling of the AMPA re- ceptors with antibodies directed against the C-terminal domain of GluR1 and GluR2/3 was markedly decreased in dendritic felds following such treatment at 35°C. This effect was temperature-dependent and com- pletely blocked by inhibitors of the calcium-dependent proteases calpains, and we suggested that calpains are involved in the regulation of AMPA receptor proper- ties. The results of the present study demonstrate that calpain activation produces a partial proteolysis in the C-terminal domain of the receptors and generates a new receptor species with an apparent molecular weight of 103,000 tool. wt. Sequence analysis of the GIuR1 tTo whom correspondence should be addressed. Abbreviations: AMPA, ~t-amino-3-hydroxy-4-methyl-5- isoxazole propionic acid; EDTA, ethylenediaminetetra- acetic acid; EGTA, ethyleneglycol bis (fl-amino- ethylether)N,N,N',N'-tetraacetic acid; GIuR, glutamate receptor subunit; PBS, phosphate-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TBS, Tris-buffered saline; TPCK, N-tosyl-l-phenylalanine-choromethyl ketone. C-terminal domain suggests a couple of cleavage sites for calpains. These results are of particular interest considering the body of evidence implicating calpains and changes in excitatory amino acid receptors in mechanisms of synaptic plasticity as well as in neuro- degenerative processes. 1'2'4't2 Copyright © 1996 IBRO. Published by Elsevier Science Ltd. Two different site directed antipeptide antibodies against GluR1 subunits were used in our studies. The first one recognizes an epitope in the C-terminal domain, residues 877-88918 of the subunit (C-anti- body), while the second one recognizes an epitope near the N-terminal domain, residues 253-267 H (N-antibody). Following calcium treatment of thin frozen-thawed brain sections, the tissues were col- lected and processed for western blots with both antibodies (Fig. 1). As previously reported, 5 calcium treatment resulted in a large decrease (about 80%) in the amount of the 110,000 mol. wt protein labelled with the C-antibody without the comcomitant appearance of any lower molecular weight species. This effect was completely blocked by calpain inhibi- tor I. The effect of calcium was markedly different when GluR1 subunits were labelled with the N- antibody. The amount of the 110,000 mol. wt protein was decreased and a new species with an estimated molecular weight of about 103,000 appeared. Quanti- tative analysis indicated that the amount of this new species was about one third the amount of the original l l0,000mol, wt species in the control conditions whereas the l l0,000mol, wt species was decreased by about 30%. This effect was also completely blocked by calpain inhibitor I. We evaluated the effect of calcium treatment on the immunoreactivity of GluR1 subunits with the N- and C-antibodies (Fig. 2). As previously observed, 5 calcium treatment resulted in a marked decrease in GIuRI immunoreactivity with the C-antibody. With 903