Characterization of metabotropic glutamate receptors coupled to a pertussis toxin sensitive G‐protein in bovine brain coated vesicles

Abstract

Glutamate metabotropic receptors (mGluRs) in bovine brain coated vesicles have been characterized by pharmacological and kinetic binding experiments. Saturation experiments revealed a single binding site with a Kd = 607.9 f 78.5 nM and a B,,,,, = 6.45 f 0.88 pmol/mg protein. The specific binding of Lj3H]glutamate to mGluRs is regulated by guanine nucleotides. Guanosine-5'-triphosphate (GTP; 100 PM) shifts the agonist competition curves to the right, increasing the XC, values. Pertussis toxin treatment produces a pharmacological binding profile for quisqualate similar to that obtained in the presence of 1OOpM GTP. These results indicate the presence of metabotropic glutamate receptors in coated vesicles and its coupling to a pertussis toxin sensitive G-protein.

Distribution and Ontogeny of 1S,3R‐1‐Aminocyclopentane‐1,3‐Dicarboxylic Acid‐Sensitive and Quisqualate‐Insensitive [3H]Glutamate Binding Sites in the Rat Brain

“…1S,3R-ACPD-sensitive [3H]glutamate binding has been reported by others using clathrin-coated bovine brain vesicles (Martin et al, 1991(Martin et al, , 1993 and in rat brain sections by using autoradiographic imaging (Cha et al, 1990;Greenamyre et al, 1990;Catania et al, 1993). Although the assay conditions were somewhat different, it is probable that [3H]glutamate binding to the QUIS-sensitive sites studied here corresponds to the AMPA-insensitive, QUIS-sensitive, [3H]glutamate binding reported by Cha et al .…”

Structural insights into the activation of metabotropic glutamate receptors

“…Our studies identified ECL2 as being necessary for relaying the agonist-induced conformational changes to the 7TM domain by providing a second, rigid attachment point between the ECD and transmembrane domains. Thus, we propose that the ECL2-CRD interaction is the structural basis for the allostery that has been observed between the ECD and 7TM domains 36,37 . While our results do not fully explain how agonist binding at the VFT leads to G protein coupling and activation, they do support a model in which both inter- and intrasubunit rearrangements are required for full activity 5 .…”

Acamprosate Inhibits the Binding and Neurotoxic Effects of Trans‐ACPD, Suggesting a Novel Site of Action at Metabotropic Glutamate Receptors

“…The radioligand binding paradigm was characterized by initially determining the equilibrium binding time for [ 3 H]glutamate and performing a saturation analysis that described a K d for [ 3 H]glutamate in this preparation that was consistent with previous studies (Baudry and Lynch, 1981;Cross et al, 1986;Oillet et al, 1995). Because we were interested in the potential interactions of acamprosate at all glutamate binding sites, we chose not to add saturating concentrations of NMDA and ␣-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid in the binding studies as done by others to isolate mGluRs (Catania et al, 1994;Martín et al, 1993;Schoepp and True, 1992;Wright et al, 1994). The displacement of [ 3 H]glutamate by 1 mM unlabeled glutamate also provided a reference for comparison across all competition studies.…”

“…The interaction of acamprosate at these sites is also differentially altered in the presence of 100 M GTP as compared with the two agonists unlabeled glutamate and trans-ACPD. GTP produces a transition of mGluRs from high-to low-affinity states (Martín et al, 1993). This transition affects the ability of micromolar concentrations of Na-acamprosate to displace [ 3 H]glutamate.…”

“…GPCRs can exist in numerous conformations that are affected by the availability of guanine nucleotides in the system. In the presence of 100 M GTP [a concentration used in similar studies in bovine brain coated vesicles (Martín et al, 1993)], the ability of unlabeled glutamate and trans-ACPD, agonists at mGluRs, to displace [ 3 H]glutamate was determined in the radioligand binding paradigm. As with both unlabeled glutamate and trans-ACPD, the presence of 100 M GTP caused an increase in the abilities of the lower concentrations of these agonists to displace [ 3 H]glutamate ( Fig.…”