Pharmacological characterization of metabotropic glutamate receptor-mediated high-affinity GTPase activity in rat cerebral cortical membranes

1 Activation of heterotrimeric guanine nucleotide-binding regulatory proteins (G-proteins) functionally coupled to metabotropic glutamate receptors (mGluRs) was assessed by agonist-induced high-affinity GTPase (EC3.6.1.-) activity in rat cerebral cortical membranes. 2 L-Glutamate (1 mM) stimulated high-affinity GTPase activity to the same extent throughout the incubation period up to 20 min, in a Mg2+-dependent manner. The addition of 1 mM L-glutamate augmented V-max of the enzyme activity (1670 to 3850 pmol mg(-1) protein 15 min(-1)) with slight increase in K-M value (0.26 to 0.63 mu M). 3 The high-affinity GTPase activity was stimulated by the following compounds with a rank order of potency of (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) > (2S,1'S,2'S)-2-(carboxycyclopyropyl)glycine (L-CCG-I) > L-glutamate greater than or equal to 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate [(2R, 4R)-APDC] > 1S,3R-1-aminocyclopentane-1,3-dicarboxylate [(1S,3R)-ACPD] > (S)-4-carboxy-3-hydroxyphenylglycine [(S)-4C3HPG] > (S)-3-carboxy-4-hydroxyphenylglycine [(S)-3C4HPG] > ibotenate, but not by L-(+)-2-amino-4-phosphonobutyrate (L-AP4), (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG], quisqualate, or L-serine-O-phosphate (L-SOP), indicative of involvement of group II mGluRs, in particular mGluR2. 4 (2S)-alpha-Ethylglutamate (EGLU), a presumably selective antagonist against group II mGluRs, inhibited DCG-IV-stimulated high-affinity GTPase activity in a competitive manner with an apparent K-B of 220 mu M. 5 L-Glutamate-stimulated activity was eliminated by pretreatment of the membranes with sulfhydryl alkylating agent N-ethylmaleimide (NEM) at 30-50 mu M, indicating that G-proteins of the G(i) family are involved. 6 These results indicate that mGluR agonist-induced high-affinity GTPase activity in rat cerebral cortical membranes may be used to detect the functional interaction between group II mGluRs, in particular mGluR2, and NEM-sensitive G(i) proteins.