Signaltransduktion durch internalisierte G-Protein-gekoppelte Rezeptoren: Molekulare Mechanismen und physiologische Relevanz
Zusammenfassung der Projektergebnisse
G protein-coupled receptors (GPCRs) mediate the effects of several hormones and neurotransmitters and are major pharmacological targets. These receptors were long believed to signal via classical pathways, such as the Gs protein-stimulated production of cyclic AMP (cAMP), only when located at the cell surface. Thus, the ligand induced internalization of these receptors was thought to be associated with termination of G protein-dependent signaling. More recently, findings by our and other groups challenged this view by showing that internalized receptors can induce cAMP signaling at intracellular sites. However, the mechanisms involved in this phenomenon and its functional consequences remained largely unknown. Aim of this project was to investigate the functional roles of intracellular G protein-dependent signaling in two physiologically relevant contexts: TSH receptor signaling in the thyroid gland and LH receptor signaling in the ovary. To investigate GPCR signaling under physiological conditions, we utilized a combination of optical and biochemical methods in primary cells and tissues. In particular, cAMP levels and protein kinase A (PKA) activity were monitored directly in living thyroid cells and ovarian follicles by FRET microscopy. In addition, we evaluated the involvement of receptor signaling at intracellular sites on downstream signaling events as well as on final biological effects. Our data indicate that GPCR signaling at intracellular sites induces a second, persistent phase of cAMP signaling, which is distinct from the transient one generated by receptors at the cell surface and is required for the effects of TSH and LH. In particular, we show that in thyroid cells this second phase is generated on membranes of the trans-Golgi network, where internalized receptors meet a resident pool of G proteins and adenylyl cyclases. Moreover, we demonstrate that this second phase is required to induce CREB phosphorylation and gene transcription in response to TSH. Finally, we show that a similar phenomenon is taking place in ovarian follicles, where it is required for inducing meiosis resumption in the oocyte, a key biological effect of LH. Thus, our results provide strong evidence that GPCR signaling at intracellular sites is distinct from the one occurring at the cell surface and is physiologically highly relevant. These findings might have important pathophysiological implications and might constitute the basis for the development of new drugs capable of modulating receptor signaling by controlling internalization or signaling at intracellular sites.
Projektbezogene Publikationen (Auswahl)
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(2012) Persistent cAMP signaling by internalized TSH receptors occurs in thyroid but not in HEK293 cells. FASEB J 26, 2043-2048
Werthmann RC, Volpe S, Lohse MJ, Calebiro D
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(2013) Cell biology: Receptor signals come in waves. Nature 495, 457-458
Lohse MJ, Calebiro D
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(2014) cAMP signaling microdomains and their observation by optical methods. Frontiers in cellular neuroscience 8, 350
Calebiro D, Maiellaro I
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(2014) Constitutive Activation of PKA Catalytic Subunit in Adrenal Cushing's Syndrome. New England Journal of Medicine 370, 1019-1028
Beuschlein F, Fassnacht M, Assié G, Calebiro D, Stratakis CA, Osswald A, Ronchi CL, Wieland T, Sbiera S, Faucz FR, 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 MJ, Reincke M, Bertherat J, Strom TM, Allolio B
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(2014) High-resolution spatiotemporal analysis of receptor dynamics by single-molecule fluorescence microscopy. J Vis Exp, e51784
Sungkaworn T, Rieken F, Lohse MJ, Calebiro D
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(2014) PKA catalytic subunit mutations in adrenocortical Cushing’s adenoma impair association with the regulatory subunit. Nature communications 5, 5680
Calebiro D, Hannawacker A, Lyga S, Bathon K, Zabel U, Ronchi C, Beuschlein F, Reincke M, Lorenz K, Allolio B, Kisker C, Fassnacht M, Lohse MJ
