N-glycosylation and disulfide bonding affects GPRC6A receptor expression, function, and dimerization

AbstractInvestigation of post-translational modifications of receptor proteins is important for our understanding of receptor pharmacology and disease physiology. However, our knowledge about post-translational 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

G protein-coupled receptor signaling analysis using homogenous time-resolved Förster resonance energy transfer (HTRF®) technology.

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 (IP(1)) 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

Allosteric interactions between GABA B1 subunits control orthosteric binding sites occupancy within GABA B oligomers

International audienceThe GABAB receptor was the first G protein-coupled receptor identified as an obligate heterodimer. It is composed of two subunits, GABAB1 containing the agonist binding site and GABAB2 responsible for G protein activation. The GABAB receptor was found to associate into larger complexes through GABAB1-GABAB1 interactions, both in transfected cells and in brain membranes. Here we assessed the possible allosteric interactions between GABAB 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 GABAB1 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 GABAB oligomers are not simultaneously occupied. Our data show a strong negative effect between GABAB1 binding sites within GABAB oligomers. Accordingly, GABAB 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"

Selective Allosteric Antagonists for the G Protein-Coupled Receptor GPRC6A Based on the 2‑Phenylindole Privileged Structure Scaffold

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., <b>1</b>–<b>3</b>). Herein, we present the first structure–activity relationship study for the 2-arylindole antagonist <b>3</b>, 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 <b>7</b>, <b>13e</b>, and <b>34b</b> as antagonists at the GPRC6A receptor in the low micromolar range and show that <b>7</b> and <b>34b</b> display >9-fold selectivity for the GPRC6A receptor over related GPCRs, making <b>7</b> and <b>34b</b> the most potent and selective antagonists for the GPRC6A receptor reported to date

The oligomeric state sets GABAB receptor signalling efficacy

This paper adds to the complexity of GPCR signalling, revealing hetero-tetramer formation for native GABAB receptors in brain tissue. An elegant mutational approach further reveals functionally relevant negative cooperativity between GABAB heterodimers