Single-cell imaging of ERK and Akt activation dynamics and heterogeneity induced by G-protein-coupled receptors.

Kinases play key roles in signaling networks that are activated by G-protein-coupled receptors (GPCRs). Kinase activities are generally inferred from cell lysates, hiding cell-to-cell variability. To study the dynamics and heterogeneity of ERK and Akt proteins, we employed high-content biosensor imaging with kinase translocation reporters.

Imaging of Genetically Encoded FRET-Based Biosensors to Detect GPCR Activity.

A wealth of assays for screening GPCR activity have been developed. Biosensors that employ Förster Resonance Energy transfer (FRET) are specific and enable dynamic measurements. Moreover, FRET biosensors are ideally suited for the analysis of single living cells.

Sensing Extracellular Calcium - An Insight into the Structure and Function of the Calcium-Sensing Receptor (CaSR).

The calcium-sensing receptor (CaSR) is a G protein-coupled receptor that plays a key role in calcium homeostasis, by sensing free calcium levels in blood and regulating parathyroid hormone secretion in response. The CaSR is highly expressed in parathyroid gland and kidney where its role is well characterised, but also in other tissues where its function remains to be determined. The CaSR can be activated by a variety of endogenous ligands, as well as by synthetic modulators such as Cinacalcet, used in the clinic to treat secondary hyperparathyroidism in patients with chronic kidney disease.

Mutational Analysis and Modeling of Negative Allosteric Modulator Binding Sites in AMPA Receptors.

The -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) constitute a subclass of the ionotropic glutamate receptor superfamily, which functions as glutamate-gated cation channels to mediate the majority of excitatory neurotransmission in the central nervous system. AMPARs are therapeutic targets in a range of brain disorders associated with abnormal glutamate hyperactivity. Multiple classes of AMPAR inhibitors have been developed during the past decades, including competitive antagonists, ion channel blockers, and negative allosteric modulators (NAMs).

Physical biology of GPCR signalling dynamics inferred from fluorescence spectroscopy and imaging.

The physical biology of G protein-coupled receptor (GPCR) signalling can be inferred from imaging of single molecules and single living cells. In this opinion paper, we highlight recent developments in technologies to study GPCR signalling in vitro and in cyto. We start from mobility and localisation characteristics of single receptors in membranes.