Pharmacological characterization of type 1alpha metabotropic glutamate receptor-stimulated [35S]-GTPgammaS binding

Br J Pharmacol. 1997 Jul;121(6):1203-9. doi: 10.1038/sj.bjp.0701238.


1. The activation of G proteins by type 1alpha metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1alpha receptor has been studied by use of a [35S]-guanosine 5'-[gamma-thio]triphosphate ([35S]-GTPgammaS) binding assay. 2. L-Glutamate increased the rate of [35S]-GTPgammaS binding in a concentration-dependent manner (-logEC50 (M) 5.25 +/- 0.07), with an optimal (62.4 +/- 1.6%) increase over basal binding being observed following 60 min incubation at 30 degrees C with 70 pM [35S]-GTPgammaS, 1 microM GDP, 10 mM MgCl2, 100 mM NaCl and 100 microg membrane protein ml(-1). The L-glutamate (100 microM)-stimulated increase in [35S]-GTPgammaS binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 microM). 3. Quantitative analysis of the affinity and number of G proteins activated by a maximally effective concentration of L-glutamate revealed an equilibrium dissociation constant (K(D)) for [35S]-GTPgammaS binding of 0.76 +/- 0.20 nM and a maximal number of GTPgammaS-liganded G proteins (Bmax) of 361 +/- 30 fmol mg(-1) protein. 4. Metabotropic glutamate receptor agonists, quisqualate (-logEC50 (M) 6.74 +/- 0.06), 1S,3R-ACPD (4.64 +/- 0.08) and (S)-3,5-dihydroxyphenylglycine (5.16 +/- 0.23) also increased [35S]-GTPgammaS binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists. 5. (+)-alpha-Methylcarboxyphenylglycine (300 microM) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [35S]-GTPgammaS binding, allowing an antagonist equilibrium dissociation constant (K(D)) of 34.0 +/- 7.8 microM to be calculated for this mGluR antagonist. 6. Pretreatment of BHK-mGluR1alpha cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100 ng ml(-1), 24 h) before membrane preparation resulted in a marked decrease in agonist-stimulated [35S]-GTPgammaS binding (by 66.0 +/- 0.9%), and an altered concentration-effect relationship for agonist-stimulated [35S]-GTPgammaS binding by the residual PTX-insensitive G-protein population. 7. The modulation of [35S]-GTPgammaS binding by agonists and antagonists in membranes from recombinant cells provides an excellent system in which to study mGluR interactions with PTX-sensitive and -insensitive G proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cricetinae
  • Excitatory Amino Acid Agonists / pharmacology
  • GTP-Binding Proteins / metabolism
  • Guanosine 5'-O-(3-Thiotriphosphate) / metabolism*
  • Kinetics
  • Pertussis Toxin
  • Protein Binding
  • Radioligand Assay
  • Receptors, Metabotropic Glutamate / drug effects
  • Receptors, Metabotropic Glutamate / metabolism*
  • Recombinant Proteins / metabolism
  • Sulfur Radioisotopes
  • Virulence Factors, Bordetella / pharmacology


  • Excitatory Amino Acid Agonists
  • Receptors, Metabotropic Glutamate
  • Recombinant Proteins
  • Sulfur Radioisotopes
  • Virulence Factors, Bordetella
  • Guanosine 5'-O-(3-Thiotriphosphate)
  • Pertussis Toxin
  • GTP-Binding Proteins