Characterization of [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxy-cyclopropyl)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, [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes. 2. [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax 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 [3H]-DCG IV binding. 3. Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nNM. 4. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPgamma35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 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).
Cartmell
J
J
Pharma Division Preclinical CNS Research, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Adam
G
G
Chaboz
S
S
Henningsen
R
R
Kemp
J A
JA
Klingelschmidt
A
A
Metzler
V
V
Monsma
F
F
Schaffhauser
H
H
Wichmann
J
J
Mutel
V
V
D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D003521:Cyclopropanes / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014162:Transfection; D014316:Tritium
D003521:Cyclopropanes; D018094:Receptors, Metabotropic Glutamate; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D014316:Tritium; C082313:2-(2,3-dicarboxycyclopropyl)glycine; D005576:Colforsin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000242:Cyclic AMP; D005998:Glycine