G protein-coupled receptors (GPCRs) have key roles in cellcell communication. Recent data suggest that these receptors can form large complexes, a possibility expected to expand the complexity of this regulatory system. Among the brain GPCRs, the heterodimeric GABA B receptor is one of the most abundant, being distributed in most brain regions, on either pre-or post-synaptic elements. Here, using specific antibodies labelled with time-resolved FRET compatible fluorophores, we provide evidence that the heterodimeric GABA B receptor can form higher-ordered oligomers in the brain, as suggested by the close proximity of the GABA B1 subunits. Destabilizing the oligomers using a competitor or a GABA B1 mutant revealed different G protein coupling efficiencies depending on the oligomeric state of the receptor. By examining, in heterologous system, the G protein coupling properties of such GABA B receptor oligomers composed of a wild-type and a non-functional mutant heterodimer, we provide evidence for a negative functional cooperativity between the GABA B heterodimers.

The GPRC6A receptor is a recently "deorphanized" class C G protein-coupled receptor. We and others have shown that this receptor is coactivated by basic L-a-amino acids and divalent cations, whereas other groups have also suggested osteocalcin and testosterone to be agonists. Likewise, the GPRC6A receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort to establish fully the signaling properties of the receptor, we tested representatives of four previously reported GPRC6A agonist classes for activity in the G q , G s , G i , and extracellular-signal regulated kinase signaling pathways. Our results confirm that GPRC6A is activated by basic L-a-amino acids and divalent cations, and for the first time, we conclusively show that these responses are mediated through the G q pathway. We were not able to confirm previously published data demonstrating G i -and G s -mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of G q activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A is thus useful in highthroughput screening for novel pharmacological tool compounds, which are necessary to unravel the physiologic function of the receptor.