Cloning and characterization of a metabotropic glutamate receptor, mGluR4b

An alternative spliced variant of metabotropic glutamate receptor subtype mGluR4a, termed mGluR4b was isolated from a rat cDNA library. Subtype mGluR4b was identical to the previously described mGluR4a, except for the last 64 amino acids in the C-terminal region in which were replaced by 135 new amino acids in mGluR4b. Recombinant baculoviruses coding for mGluR4a and mGluR4b were expressed in Spodoptera frugiperda, Sf-9, insect cells and characterized pharmacologically by measuring [3H]-L-2-amino-4-phosphonobutyrate ([3H]-L-AP4) binding and second messenger formation. [3H]-L-AP4 binding to membranes prepared from Sf-9 cells expressing mGluR4a and mGluR4b revealed respective affinities (Kd) of 480 and 360 nM and maximal binding densities (Bmax) of 4.2 and 0.8 pmol/mg protein. The ligand selectivity of mGluR4a and mGluR4b was similar: L-AP4 > L-serine-O-phosphate > L-glutamate > L-2-amino 2-methyl-4-phosphonobutyrate > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate > or = quisqualate. A decrease in the affinity of [3H]-L-AP4 was observed in the presence of 0.1 mM guanosine 5'-O-(3-thio)trisphosphate-gamma-S, indicating that mGluR4a and mGluR4b were functionally coupled to G-proteins in Sf-9 cells. Agonists of mGluR4 caused a minor decrease in forskolin-induced cAMP formation in Sf-9 cells expressing either mGluR4a or mGluR4b, suggesting that both receptors are coupled to adenylate cyclase in an inhibitory manner. Thus, mGluR4a and mGluR4b share a common signal transduction pathway and pharmacology when expressed in Sf-9 insect cells.