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

1 The activation of G proteins by type 1 alpha metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1 alpha receptor has been studied by use of a [S-35]-guanosine 5'-[gamma-thio]triphosphate ([S-35]-GTP gamma S) binding assay. 2 L-Glutamate increased the rate of [S-35]-GTP gamma S binding in a concentration-dependent manner (-logEC(50) (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 [S-35]-GTP gamma S, I mu M GDP, 10 mM MgCl2 100 mM NaCl and 100 mu g membrane protein ml(-1). The L-glutamate (100 mu M)-stimulated increase in [S-35]-GTP gamma S binding was totally prevented in the presence of the group I mGluR antagonist (S)-4-carboxy-3-hydroxyphenylglycine (300 mu M). 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 [S-35]-GTP gamma S binding of 0.76+/-0.20 nM and a maximal number of GTP gamma S-liganded G proteins (B-max) of 361+/-30 fmol mg(-1) protein. 4 Metabotropic glutamate receptor agonists, quisqualate (-logEC(50) (M) 6.74+/-0.06), 1S,3R-ACPD (4.64+/-0.08) and (S)-3,5-dihydroxyphenylglycine (5.16+/-0.23) also increased [S-35]-GTP gamma S binding in a concentration-dependent manner, with the latter two agents behaving as partial agonists. 5 (+)-alpha-Methylcarboxyphenylglycine (300 mu M) caused a parallel rightward shift of the L-glutamate concentration-effect curve for [S-35]-GTP gamma S binding, allowing an antagonist equilibrium dissociation constant (K-D) of 34.0+/-7.8 mu M to be calculated for this mGluR antagonist. 6 Pretreatment of BHK-mGluR1 alpha 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 [S-35]-GTP gamma S binding (by 66.0+/-0.9%), and an altered concentration-effect relationship for agonist-stimulated [S-35]-GTP gamma S binding by the residual PTX-insensitive G-protein population. 7 The modulation of [S-35]-GTP gamma S 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.