[3H]-L-2-amino-4-phosphonobutyrate labels a metabotropic glutamate receptor, mGluR4a.

1. The ligand binding site of subtype mGluR4a of the metabotropic glutamate receptor family was characterized by using [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding. 2. Specific [3H]-L-AP4 binding to membranes prepared from baby hamster kidney (BHK) cells transfected with a vector encoding mGluR4a accounted for 60-70% of the total binding whereas no specific binding of [3H]-L-AP4 was observed to membranes prepared from BHK cells expressing the vector only. 3. Specific binding of [3H]-L-AP4 to mGluR4a was detectable at 0 degree C, was saturated with 10 min and enhanced by Cl(-)-ions but not by divalent cations (Mg2+, Ca2+, Mn2+). 4. [3H]-L-AP4 binding showed a maximal binding density (Bmax) of 3.0 +/- 0.5 pmol mg-1 protein and an affinity (KD) of 441 nM. A modest decrease in affinity was observed in the presence of 0.1 mM guanosine-5'-O-(3-thio)trisphosphate-gamma-S, the KD being 761 nM and the Bmax 3.4 +/- 0.6 pmol mg-1 protein. 5. The following rank order of affinity for mGluR4a was observed: L-AP4 = L-serine-O-phosphate > glutamate = (2S,1S,2S)-2-(carboxycyclopropyl)-glycine > 1-amino-3-(phosphonomethylene)cyclobitanecar-boxylate > > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate = quisqualate > ibotenate. 6. A highly significant correlation was observed between the potencies of the compounds to inhibit forskolin-stimulated cyclic AMP-formation in BHK cells expressing mGluR4a and the affinity for displacement of [3H]-L-AP4 binding from mGluR4a suggesting that this binding site is functionally relevant. 7. In conclusion, [3H]-L-AP4 is a suitable radioligand for characterizing mGluR4a when expressed in BHK cells. Interestingly, a significant correlation was found between the ability of various compounds to displace [3H]-L-AP4 binding from mGluR4a and the previously observed potencies for inhibition of synaptic transmission via L-AP4 sensitive glutamatergic pathways. These data support the hypothesis that the L-AP4 receptor is contained within the mGluR family.