A universal bioluminescence resonance energy transfer sensor design enables high-sensitivity screening of GPCR activation dynamics

G-protein-coupled receptors (GPCRs) represent one of the most important classes of drug targets. The discovery of new GCPR therapeutics would greatly benefit from the development of a generalizable high-throughput assay to directly monitor their activation or de-activation. Here we screened a variety of labels inserted into the third intracellular loop and the C-terminus of the α-adrenergic receptor and used fluorescence (FRET) and bioluminescence resonance energy transfer (BRET) to monitor ligand-binding and activation dynamics. We then developed a universal intramolecular BRET receptor sensor design to quantify efficacy and potency of GPCR ligands in intact cells and real time. We demonstrate the transferability of the sensor design by cloning β-adrenergic and PTH1-receptor BRET sensors and monitored their efficacy and potency. For all biosensors, the Z factors were well above 0.5 showing the suitability of such design for microtiter plate assays. This technology will aid the identification of novel types of GPCR ligands

Printed organic TFTs on flexible substrate for complementary circuits

Organic Thin film Transistors (OTFT) have been widely investigated in these last years as potential candidate for the development of low cost, flexible and lightweight active-matrix backplanes for display applications. Indeed the organic semiconductors provide both promising electrical performances tunable by chemistry and the ability to be processed at low temperature with innovative printing technics on various large scale substrates. Thanks to the recent developments on both n-type and p-type solution-processed organic semiconductors, we have developed a printable organic complementary technology compatible with flexible PEN substrates. By combining state of the art materials exhibiting mobility in the range of 1 cm 2 /V.s and silicon inspired compact modeling and simulation approach, we were able to design and fabricate circuit's building blocks that provide the switching, digital and analog functions required for the fabrication of printed systems on foil

Bioluminescence in G protein-coupled receptors drug screening using nanoluciferase and halo-tag technology

Here we describe the stepwise application of bioluminescence resonance energy transfer (BRET)-based conformational receptor biosensors to study GPCR activation in intact cells. This technology can be easily adopted to various plate reader devices and microtiter plate formats. Due to the high sensitivity of these BRET-based receptor biosensors and their ability to quantify simultaneously receptor activation/de-activation kinetics as well as compound efficacy and potency, these optical tools provide the most direct and unbiased approach to monitor GPCR activity in a high-throughput-compatible assay format, representing a novel promising tool for the discovery of potential GPCR therapeutics

Calcium-sensing receptor signaling: it's all about multiplicity

The Calcium-Sensing Receptor (CaSR), a G-protein-coupled receptor mainly known for its role in the homeostatic regulation of Ca2+ levels in the extracellular fluid, is also expressed in a multiplicity of tissues where it regulates a variety of physiological and pathological processes. The main features of CaSR are its capacity to activate multiple downstream signaling pathways and its ability to itself be activated by a variety of ligands. Recent data have demonstrated that these features are actually connected by the concept of biased signaling. The recent availability of crystal structures of CaSR extracellular domain, and the functional characterization of clinically relevant mutations, have catalyzed a great step forward in the field of CaSR signaling. In the past two years, CaSR signaling characteristics have been shown to be even more complicated than expected: heterodimerization, phosphate-sensing, and compartment bias are only a fraction of the exciting developments. This review will focus on some of these topics, and on the debated case of CaSR signaling in cardiomyocytes

Termination and activation of store-operated cyclic AMP production.

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1-AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca(2+) ] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this 'store-operated' pathway requires the ER Ca(2+) sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET-based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store-operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca(2+) entry via the plasma membrane Ca(2+) channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca(2+) /cAMP crosstalk system