Characterization of [³H]Quisqualate Binding to Recombinant Rat Metabotropic Glutamate 1a and 5a Receptors and to Rat and Human Brain Sections

Abstract

Abstract:We have investigated the binding properties of [ 3 H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K D values of 27 Ϯ 4 and 81 Ϯ 22 nM for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S)-4-Carboxyphenylglycine, (S)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [ 3 H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM [ 3 H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 M kainate, revealing the distribution of mGlu1ϩ5 receptors. The latter were almost completely inhibited by the group I agonist (S)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively. Key Words: [ 3 H]Quisqualate binding-Metabotropic glutamate 1a and 5a receptors-Radioautography. J. Neurochem. 75, 2590 -2601 (2000).Eight G protein-coupled metabotropic glutamate (mGlu) receptors have been cloned to date (for review, see Pin et al., 1999). On the basis of their sequence similarities, signal transduction, and agonist rank order of potency, these receptors have been subdivided into three groups. Group I mGlu receptors, for which several splice variants have been identified , are activated by the weak group I-selective agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (S)-3-hydroxyphenylglycine (3HPG) and the potent nonselective agonist quisqualate (Aramori and Nakanishi, 1992). This group comprises mGlu1, which is selectively blocked noncompetitively by 7-(hydroxyimino)cyclopropa [b]chromen1a-carboxylate ethyl ester (CPCCOEt) (Litschig et al., 1999), and mGlu5, which is selectively blocked, noncompetitively, with high potency by methylphenylethynylpyridine . Although precise physiological roles cannot yet be assigned to mGlu1 and mGlu5 receptors (potent, selective agonists/antagonists with good brain penetration are not yet widely available), group I receptors have, nevertheless, been implied in psychiatric and neurological diseases (see Bordi and Ugolini, 1999).

Agonist-dependent Signaling by Group I Metabotropic Glutamate Receptors Is Regulated by Association with Lipid Domains

“…The measured affinity for quisqualate was similar for myc-mGluR1 and myc-mGluR1mut-i1/i3 (mGluR1 K D 75.9 Ϯ 31.6 nM, mGluR1mut-i1/i3 K D 78.1 Ϯ 26.6 nM, n ϭ 6; Fig. 3, A and B) although lower than previously reported (24), presumably because of the presence of the tag close to agonist binding site. To examine the impact of caveolin-1 binding on receptor association with lipid rafts, HEK293 cells expressing myc-mGluR1mut-i1/i3 were incubated with GPT/pyruvate alone or challenged with 1 mM glutamate for 5 min; DRMs were prepared from control and glutamate-treated cells, and receptor localization was examined by immunoblot.…”

Sequential Inter- and Intrasubunit Rearrangements During Activation of Dimeric Metabotropic Glutamate Receptor 1

“…Concentrationresponse curves for these FRET signals yielded EC 50 values of 1.3 mM for quisqualate and 22.6 mM for glutamate (Fig. 4C), which parallels in vitro functional assays of the mGluR1 (45).…”

“…The concentration-response curve for the agonist quisqualate gave an EC 50 (median effective concentration) value of 1.05 mM (Fig. 3D), which is compatible with mGluR1 pharmacology (44,45). The increase in FRET between fluorescent proteins placed in the intracellular loops of the subunits is compatible with the assumed activation mechanism of class C receptors that involves a rearrangement of the two subunits within the receptor dimer (46)(47)(48).…”

The Metabotropic Glutamate G-protein-coupled Receptors mGluR3 and mGluR1a Are Voltage-sensitive

“…The results of the experiments conducted with the chimeric receptors indicate that also for mGluR3 and mGluR1a, as for other GPCRs (25,40), the coupling to the G-protein is involved in determining the affinity of the receptors toward their agonist (but see other reports (41,42)). Therefore, we suggest that depolarization causes a conformational change that affects the intracellular loops that couple to the G-protein, thereby regulating the likelihood of the receptors to couple to their corresponding G-protein.…”