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.

Studying multidimensional signaling of G protein-coupled receptors (GPCRs) in search of new and better treatments requires flexible, reliable and sensitive assays in high throughput screening (HTS) formats. Today, more than half of the detection techniques used in HTS are based on fluorescence, because of the high sensitivity and rich signal, but quenching, optical interferences and light scattering are serious drawbacks. In the 1990s the HTRF® (Cisbio Bioassays, Codolet, France) technology based on Förster resonance energy transfer (FRET) in a time-resolved homogeneous format was developed. This improved technology diminished the traditional drawbacks. The optimized protocol described here based on HTRF® technology was used to study the activation and signaling pathways of the calcium-sensing receptor, CaSR, a GPCR responsible for maintaining calcium homeostasis. Stimulation of the CaSR by agonists activated several pathways, which were detected by measuring accumulation of the second messengers d-myo-inositol 1-phosphate (IP1) and cyclic adenosine 3′,5′-monophosphate (cAMP), and by measuring the phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/2). Here we show how an optimized HTRF® platform with numerous advantages compared to previous assays provides a substantial and robust mode of investigating GPCR signaling. It is furthermore discussed how these assays can be optimized and miniaturized to meet HTS requirements and for screening compound libraries.

a b s t r a c tInvestigation of post-translational modifications of receptor proteins is important for our understanding of receptor pharmacology and disease physiology. However, our knowledge about posttranslational modifications of class C G protein-coupled receptors and how these modifications regulate expression and function is very limited. Herein, we show that the nutrient-sensing class C G protein-coupled receptor GPRC6A carries seven N-glycans and that one of these sites modulates surface expression whereas mutation of another site affects receptor function. GPRC6A has been speculated to form covalently linked dimers through cysteine disulfide linkage in the extracellular amino-terminal domain and here we show that GPRC6A indeed is a homodimer and that a disulfide bridge between the C131 residues is formed.

No abstract

The GABA receptor was the first G protein-coupled receptor identified as an obligate heterodimer. It is composed of two subunits, GABA containing the agonist binding site and GABA responsible for G protein activation. The GABA receptor was found to associate into larger complexes through GABA-GABA interactions, both in transfected cells and in brain membranes. Here we assessed the possible allosteric interactions between GABA heterodimers by analyzing the effect of mutations located at the putative interface between the extracellular binding domains. These mutations decrease, but do not suppress, the Förster resonance energy transfer (FRET) signal measured between GABA subunits. Further analysis of one of these mutations revealed an increase in G protein-coupling efficacy and in the maximal antagonist binding by approximately two-fold. Hypothesizing that a tetramer is an elementary unit within oligomers, additional FRET data using fluorescent ligands and tagged subunits suggest that adjacent binding sites within the GABA oligomers are not simultaneously occupied. Our data show a strong negative effect between GABA binding sites within GABA oligomers. Accordingly, GABA receptor assembly appears to limit receptor signaling to G proteins, a property that may offer novel regulatory mechanism for this important neuronal receptor. This article is part of the "Special Issue Dedicated to Norman G. Bowery".

G protein-coupled receptors (GPCRs) represent a biological target class of fundamental importance in drug therapy. The GPRC6A receptor is a newly deorphanized class C GPCR that we recently reported for the first allosteric antagonists based on the 2-arylindole privileged structure scaffold (e.g., 1-3). Herein, we present the first structure-activity relationship study for the 2-arylindole antagonist 3, comprising the design, synthesis, and pharmacological evaluation of a focused library of 3-substituted 2-arylindoles. In a FRET-based inositol monophosphate (IP1) assay we identified compounds 7, 13e, and 34b as antagonists at the GPRC6A receptor in the low micromolar range and show that 7 and 34b display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making 7 and 34b the most potent and selective antagonists for the GPRC6A receptor reported to date.