Pharmacological characterization of type 1<i>α</i> metabotropic glutamate receptor‐stimulated [³⁵S]‐GTPγS binding

Abstract

The activation of G proteins by type 1α metabotropic glutamate receptors (mGluRs) in membranes from recombinant baby hamster kidney cells expressing the cloned rat mGluR1α receptor has been studied by use of a [ 35 S]‐guanosine 5′‐[γ‐thio]triphosphate ([ 35 S]‐GTPγS) binding assay. l ‐Glutamate increased the rate of [ 35 S]‐GTPγS 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 60min incubation at 30°C with 70p m [ 35 S]‐GTPγS, 1μ m GDP, 10m m MgCl 2 , 100m m NaCl and 100μg membrane proteinml −1 . The l ‐glutamate (100μ m )‐stimulated increase in [ 35 S]‐GTPγS binding was totally prevented in the presence of the group I mGluR antagonist ( S )‐4‐carboxy‐3‐hydroxyphenylglycine (300μ m ). 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 [ 35 S]‐GTPγS binding of 0.76±0.20n m and a maximal number of GTPγS‐liganded G proteins (B max ) of 361±30fmol mg −1 protein. Metabotropic glutamate receptor agonists, quisqualate (−logEC 50 ( m ) 6.74±0.06), 1 S ,3 R ‐ACPD (4.64±0.08) and ( S )‐3,5‐dihydroxyphenylglycine (5.16±0.23) also increased [ 35 S]‐GTPγS binding in a concentration‐dependent manner, with the latter two agents behaving as partial agonists. (+)‐α‐Methylcarboxyphenylglycine (300μ m ) caused a parallel rightward shift of the l ‐glutamate concentration‐effect curve for [ 35 S]‐GTPγS binding, allowing an antagonist equilibrium dissociation constant ( K D ) of 34.0±7.8μ m to be calculated for this mGluR antagonist. Pretreatment of BHK‐mGluR1α cells with a concentration of pertussis toxin (PTX) shown to be maximally effective (100ngml −1 , 24h) before membrane preparation resulted in a marked decrease in agonist‐stimulated [ 35 S]‐GTPγS binding (by 66.0±0.9%), and an altered concentration‐effect relationship for agonist‐stimulated [ 35 S]‐GTPγS binding by the residual PTX‐insensitive G‐protein population. The modulation of [ 35 S]‐GTPγS 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.