Plasticity of agonist binding sites in hetero-oligomers of the unitary glutamate receptor subunit XenU1.
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 nM at 25 degrees C), but also the glycine site antagonist 5,7-dichlorokynurenic acid (DCKA). DCKA, GTP, or GTPgammaS 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 nM). 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.
Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London, England, UK.
M M
MM
Soloviev
K
K
Abutidze
I
I
Mellor
P
P
Streit
E V
EV
Grishin
P N
PN
Usherwood
E A
EA
Barnard
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007608:Kainic Acid / Q000378:metabolism; D007736:Kynurenic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D011233:Precipitin Tests; D017470:Receptors, Glutamate / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins
D017470:Receptors, Glutamate; C101562:XenU1 protein, Xenopus; D029867:Xenopus Proteins; D006160:Guanosine Triphosphate; D007736:Kynurenic Acid; D007608:Kainic Acid; C066192:5,7-dichlorokynurenic acid