Biochemical evidence for the existence of a pool of unassembled C2 exon-containing NR1 subunits of the mammalian forebrain NMDA receptor.
Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [3H]MK-801 radioligand binding activity. The most efficient conditions were 1% Triton X-100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X-100/1 M NaCl-solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti-NR1 911-920 antibody affinity chromatography from 1% Triton X-100/1 M NaCl solubilised extracts yielded purification of the NR1 Mr 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X-100/1 M NaCl extracts with anti-NR1 911-920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti-NR1 17-35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a co-association of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti-NR1 911-920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon-containing NR1 subunits, i.e., NR1-1a, NR1-1b, NR1-2a, and NR1-2b, selectively solubilised by 1% Triton X-100/1 M NaCl.
Chazot
P L
PL
Department of Pharmaceutical and Biological Chemistry, School of Pharmacy, London, England.
Stephenson
F A
FA
D000367:Age Factors; D017398:Alternative Splicing / Q000502:physiology; D000818:Animals; D000918:Antibody Specificity; D002531:Cerebellum / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents; D005091:Exons / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D007668:Kidney / Q000166:cytology; D051379:Mice; D008970:Molecular Weight; D011233:Precipitin Tests; D016548:Prosencephalon / Q000737:chemistry*; D016194:Receptors, N-Methyl-D-Aspartate / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology; D012995:Solubility; D014162:Transfection
D003902:Detergents; D016194:Receptors, N-Methyl-D-Aspartate