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.
|Internalized TSH receptors en route to the TGN induce local Gs-protein signaling and gene transcription||2017||Godbole, A., Lyga, S., Lohse, M. J., and Calebiro, D.||Nat Comm||More|
|Single-molecule imaging reveals receptor-G protein interactions at cell surface hot spots||2017||Sungkaworn, T., Jobin, M.-L., Burnecki, K., Weron, A., Lohse, M. J., and Calebiro, D.||Nature||More|
|cAMP Signals in Drosophila Motor Neurons Are Confined to Single Synaptic Boutons||2016||Maiellaro, I., Lohse, M. J., Kittel, R. J. and Calebiro, D.||Cell Rep||More|
|Constitutive activation of PKA catalytic subunit in adrenal Cushing’s syndrome||2014||Beuschlein, F., Fassnacht, M., Assié, G., Calebiro, D., Stratakis, C. A., Osswald, A., Ronchi, C. L., Wieland, T., Sbiera, S., Faucz, F. R., Schaak, K., Schmittfull, A., Schwarzmayr, T., Barreau, O., Vezzosi, D., Rizk-Rabin, M., Zabel, U., Szarek, E., Salpea, P., Forlino, A., Vetro, A., Zuffardi, O., Kisker, C., Diener, S., Meitinger, T., Lohse, M. J., Reincke, M., Bertherat, J., Strom, T. M., and Allolio, B.||N Engl J Med||More|
|High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy||2014||Sungkaworn, T., Rieken, F., Lohse, M. J., and Calebiro, D.||J Vis Exp||More|
|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|
|Frequent TSH receptor genetic alterations with variable signaling impairment in a large series of children with nonautoimmune isolated hyperthyrotropinemia||2012||Calebiro, D., Gelmini, G., Cordella, D., Bonomi, M., Winkler, F., Biebermann, H., de Marco, A., Marelli, F., Libri, D. V., Antonica, F., Vigone, M. C., Cappa, M., Mian, C., Sartorio, A., Beck-Peccoz, P., Radetti, G., Weber, G., and Persani, L.||J Clin Endocrinol Metab||More|
|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|
|Real-time monitoring of somatostatin receptor-cAMP signaling in live pituitary||2010||Jacobs, S., Calebiro, D., Nikolaev, V. O., Lohse, M. J., and Schulz, S.||Endocrinology||More|
|Persistent cAMP-signals triggered by internalized G-protein-coupled receptors||2009||Calebiro, D., Nikolaev, V. O., Gagliani, M. C., de Filippis, T., Dees, C., Tacchetti, C., Persani, L., and Lohse, M. J.||PLoS Biol||More|
|Selective modulation of protein kinase A I and II reveals distinct roles in thyroid cell gene expression and growth||2006||Calebiro, D., de Filippis, T., Lucchi, S., Martinez, F., Porazzi, P., Trivellato, R., Locati, M., Beck-Peccoz, P., and Persani, L.||Mol Endocrinol||More|
|Intracellular entrapment of wild-type TSH receptor by oligomerization with mutants linked to dominant TSH resistance||2005||Calebiro, D., de Filippis, T., Lucchi, S., Covino, C., Panigone, S., Beck-Peccoz, P., Dunlap, D., and Persani, L.||Hum Mol Genet||More|