Characterization of [3H]-(2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine ([3H]-DCG IV) binding to metabotropic mGlu2 receptor-transfected cell membranes

1 The binding of the new selective group II metabotropic glutamate receptor radioligand, [H-3]- (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([H-3]-DCG IV), was characterized in rat mGlu(Z) receptor-transfected CHO cell membranes. 2 [H-3]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a K-d value of 160 nM and a B-max value of 10 pmol mg(-1) protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl--dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [H-3]-DCG IV binding. 3 Of the compounds observed to inhibit [H-3]-DCG TV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; K-i value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; K-i value 19 nM). AS expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) inhibited [H-3]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nM. 4 A highly significant correlation was observed between the potencies of compounds able to inhibit [H-3]-DCG IV binding and potencies obtained for agonist activity in a GTP gamma(35)S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu(2) receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-alpha-methylserine-O-phosphate (MSOP).