Spatial organization and dynamics of GPCR signaling as revealed by single-molecule and super-resolution microscopy

Project Description

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.

Title Year Authors Journal Links
Internalization of G-protein-coupled receptors: Implication in receptor function, physiology and diseases 2018 Calebiro, D., and Godbole, A. Best Pract Res Clin Endocrinol Metab More
Single-Molecule Imaging of GPCR Interactions 2018 Calebiro, D. and Sungkaworn, T. TIPS More
Single-molecule microscopy reveals dynamic FLNA interactions governing SSTR2 clustering and internalization 2018 Treppiedi, D., Jobin, M. L., Peverelli, E., Giardino, E., Sungkaworn, T., Zabel, U., Arosio, M., Spada, A., Mantovani, G., and Calebiro, D. Endocrinology More
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 Commun 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
Persistent cAMP signaling by internalized LH receptors in ovarian follicles 2016 Lyga, S., Volpe, S., Werthmann, R. C., Götz, K., Sungkaworn, T., Lohse, M. J., and Calebiro, D. Endocrinology More
The activation mechanism of glycoprotein hormone receptors with implications in the cause and therapy of endocrine diseases 2016 Brüser, A., Schulz, A., Rothemund, S., Ricken, A., Calebiro, D., Kleinau, G., and Schöneberg T. J Biol Chem 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 internalized 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