Plasticity of Agonist Binding Sites in Hetero-Oligomers of the Unitary Glutamate Receptor Subunit XenU1 |
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Authors: | †Mikhail M Soloviev ‡Ketevan Abutidze §Ian Mellor ¶Peter Streit †Eugene V Grishin §Peter N Usherwood Eric A Barnard |
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Institution: | Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London, and; Department of Life Science, University of Nottingham, Nottingham, England;; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia;; Institute of Physiology, Georgian Academy of Sciences, Tbilisi, Republic of Georgia;and; Brain Research Institute, University of Zurich, Zurich, Switzerland |
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Abstract: | Abstract: Two subunits from Xenopus , XenNR1G and the "short" subunit XenU1, have previously been coexpressed to form a unitary (NMDA/non-NMDA type) glutamate receptor. We now show that an antibody to XenNR1G or an antibody to XenU1 precipitates the binding sites of both XenNR1G and XenU1, with the recombinant subunits or with solubilised Xenopus brain membranes, i.e., the combination occurs in vivo. The expressed XenU1 subunits are in the cell membrane and oriented correctly. XenU1 binds not only kainate with high affinity ( K D 1.2 n M at 25°C), but also the glycine site antagonist 5,7-dichlorokynurenic acid (DCKA). DCKA, GTP, or GTPγS displaces competitively all of the bound 3H]kainate, but glycine has no effect. The results suggest that a common binding site for kainate, DCKA, and GTP can exist on XenU1. In the XenNR1G/XenU1 complex, the kainate affinity is lowered eightfold, whereas the DCKA affinity is considerably increased ( K D 147 n M ). Only 18% of the binding to the complex has the properties of the NMDA receptor glycine site, the rest being due to switching of the high-affinity kainate site of XenU1 (low-affinity DCKA) to a high-affinity DCKA (low-affinity kainate) conformation. Surprisingly, a mammalian NR2 subunit can also combine with XenU1, and this introduces similar reciprocal changes in the binding of kainate and DCKA. The combined evidence suggests a common basic mode of agonist site formation in different subunit types of the ionotropic glutamate receptors. |
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Keywords: | Unitary glutamate receptor Xenopus XenU1 subunit XenNR1G subunit Mouse NR2A subunit |
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