Spatial organization and dynamics of GPCR signaling as revealed by single-molecule and super-resolution microscopy
G protein-coupled receptors (GPCRs) constitute the largest family of receptors for hormones and neurotransmitters and represent major pharmacological targets. They signal through heterotrimeric G proteins, which activate a number of effectors, such as enzymes and ion channels. Increasing evidence suggest that GPCR signaling cascades are organized in dynamic micro- and nanodomains both on the cell surface and inside cells. However, direct observation of such domains has been hampered by the insufficient spatiotemporal resolution of conventional biochemical and optical methods.
The central hypothesis of this proposal is that the organization of receptor–G protein–effector cascades in micro-/nanodomains, likely through intervention of scaffold proteins and the cytoskeleton, is crucial for achieving efficient and specific signaling. We anticipate that such an organization provides an additional and important level of regulation. To verify this hypothesis, we plan to use high-end optical methods such as single-molecule microscopy and super-resolution imaging (dSTORM) to directly observe with few nanometer and millisecond precision the localization of receptors, G proteins and effectors and to investigate the role of the cytoskeleton in organizing GPCR signaling cascades.
Specifically, we will focus on three prototypical GPCRs, i.e. β2-adrenergic (β2-AR), α2A-adrenergic (α2A-AR) and metabotropic γ-aminobutyric acid (GABAB) receptors, which play fundamental roles in the regulation of neurotransmission and cardiovascular functions, are coupled to different G proteins and, as we have recently shown, possess distinct and peculiar spatial arrangements on the surface of living cells. These experiments will be performed on a simple cell model, which allows easy genetic manipulation and imaging, as well as in primary hippocampal neurons, where GABAB receptors have been suggested to form highly organized signaling complexes with Gi/o proteins and ion channels. We expect these experiments to lead to a deeper understanding of the basic mechanisms of GPCR signaling and to shed new light on signaling micro-/nanodomains, which might pave the way to innovative and more specific pharmacological therapies for a wide range of human diseases.
Davide Calebiro will coordinate and supervise the project together with other members of the team.
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|Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization||2013||Calebiro, D., Rieken, F., Wagner, J., Sungkaworn, T., Zabel, U., Borzi, A., Cocucci, E., Zurn, A., and Lohse, M. J.||Proc Natl Acad Sci USA||More|
|Persistent cAMP signaling by inter- nalized TSH receptors occurs in thyroid but not in HEK293 cells||2012||Werthmann, R. C., Volpe, S., Lohse, M. J., and Calebiro, D.||FASEB J||More|
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|FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells||2011||Börner, S., Schwede, F., Schlipp, A., Berisha, F., Calebiro, D., Lohse, M. J., and Nikolaev, V. O.||Nat Protoc||More|
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