Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2014) 35 MTE14 | DOI: 10.1530/endoabs.35.MTE14

ECE2014 Meet the Expert Sessions (1) (17 abstracts)

Real-time monitoring of GPCR signalling in living cells

Davide Calebiro


University of Wuerzburg, Wuerzburg, Germany.


G-protein coupled receptors (GPCRs) constitute the largest family of receptors and mediated the effects of many hormones and neurotransmitters. Whereas GPCRs and their signalling cascades have been intensively studied at the molecular and, more recently, structural level, many important aspects of GPCR signalling remain elusive. A major reason for this resides in the limited spatial and temporal resolution of standard biochemical methods. To overcome these limitations, we and other groups are developing methods based on fluorescence microscopy that can be used to monitor GPCR signalling directly in living cells and tissues with high spatiotemporal resolution. A first approach takes advantage of genetically encoded sensors based on the principle of fluorescence resonance energy transfer (FRET). These sensors allow monitoring all the major steps of GPCR signalling, ranging from ligand/receptor interactions to the production of soluble second messengers and the activation of downstream kinases. In order to study GPCR signalling under highly physiological conditions, transgenic animals expressing these sensors can also be generated. Such an approach was instrumental for demonstrating that GPCRs can continue signalling via classical G-protein pathways and cyclic AMP after internalization into the endosomal compartment (1,2). A more recent approach is based on single-molecule microscopy methods, which allow investigating the localization of receptors in nanodomains and their dynamic behaviour to an unprecedented level (3). The rapid advancement in this field will likely play a crucial role in the ongoing quest to unravel the intimate nature of GPCRs and their signalling cascades.

References

1. Calebiro D et al. 2009 Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol 7 e1000172.

2. Calebiro D et al. 2010 Signaling by internalized G-protein-coupled receptors. Trends Pharmacol Sci 31 221–8.

3. Calebiro D et al. 2013 Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization. PNAS 110 743–8.

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