Imaging of persistent cAMP signaling by internalized G protein-coupled receptors
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Figure 1
Structure of a single-chain cAMP sensor (Epac1-camps) and generation of a cAMP reporter mouse. (A) The Epac1-camps sensor reports intracellular levels of cAMP based on FRET between CFP and YFP. It consists of CFP and YFP fused to a single cAMP-binding domain derived from the Epac1 protein. In the absence of cAMP, the two fluorophores are in close proximity, and a strong FRET signal is observed. Upon cAMP binding to the sensor, the distance between the two fluorophores increases, leading to a decrease in FRET. (B) Generation of transgenic mice expressing Epac1-camps. To obtain ubiquitous expression, the Epac1-camps sensor was put under the control of a strong constitutive promoter, i.e. the hybrid CMV enhancer/chicken β-actin (CAG) promoter. Pronuclear injection of this construct gave rise to transgenic mice that express high levels of the sensor in virtually all tissues. Bottom, representative fluorescence image (YFP channel) of a living transgenic mouse showing diffuse strong fluorescence.
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Figure 2
Schematic structure and function of the thyroid follicle. The thyroid follicle constitutes both the anatomical and functional unit of thyroid tissue. It consists of a monolayer of epithelial cells that define an inner cavity, where thyroid hormones are synthesized and stored in the form of iodinated thyroglobulin (Tg-I). Upon Tg-I reuptake from the follicle lumen, thyroid hormones (T4 and T3) are released by an enzymatic digestion occurring in lysosomes (Lys). The proteins involved in thyroid hormone secretion and thyroid cell proliferation are under strict transcriptional and post-transcriptional regulation by the TSH/TSHR signaling pathway. TSH binding to the TSHR triggers the Gs-dependent activation of adenylyl cyclase (AC), leading to a stimulation of cAMP production. As a result, PKA is activated and phosphorylates a number of targets that are ultimately responsible for the effects of TSH.
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Figure 3
cAMP imaging in living cells by FRET. (A) Typical microscopic setup for real-time FRET imaging, consisting of an inverted fluorescent microscope equipped with a light source for CFP excitation (436 nm). Cells attached to glass-bottom dishes are superfused with various ligands from the tip of a perfusion pipette. CFP (480 nm) and YFP (535 nm) fluorescence is split into two channels by a beam splitter device, and the two images (CFP and YFP) are detected using a CCD camera. (B) Representative FRET imaging experiment, in which a thyroid follicle freshly isolated from an Epac1-camps transgenic mouse is stimulated with TSH for 10 min. Hormone application leads to a rapid increase of cAMP levels detected as a change in FRET between CFP and YFP (from red to green in false-color FRET ratio images; note that the FRET ratio is inverted, i.e. calculated as CFP emission/YFP emission, so that FRET ratio values are positively related to intracellular cAMP concentrations). Upon washout of TSH, only an incomplete reversal of the FRET signal is observed, which suggests that the TSHR continues signaling to cAMP after internalization.
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Figure 4
TSHR signaling to cAMP from intracellular compartments. TSH binding to the TSHR activates the cAMP signaling cascade close to the cell surface. Upon prolonged stimulation, both TSH and TSHR are transferred into endosomes. Recent findings suggest that internalized TSH–TSHR complexes continue to stimulate cAMP production at endosomes. This results in persistent signaling and may lead to specific outcomes. For example, persistent receptor signaling from endosomes is apparently involved in the cAMP-mediated effects of TSH on actin depolymerization, an event involved in thyroid hormone release.
- © 2010 Society for Endocrinology
