CAMP is a universal second messenger of many G-protein-coupled receptors and regulates a wide variety of cellular events. cAMP exerts its effects via cAMP-dependent protein kinase (PKA), cAMP-gated ion channels, and two isoforms of exchange protein directly activated by cAMP (Epac). Here we report the development of novel fluorescent indicators for cAMP based on the cAMP-binding domains of Epac and PKA. Fluorescence resonance energy transfer between variants of green fluorescent protein (enhanced cyan fluorescent protein and enhanced yellow fluorescent protein) fused directly to the cAMP-binding domains was used to analyze spatial and temporal aspects of cAMP-signaling in different cells. In contrast to previously developed PKA-based indicators, these probes are comprised of only a single binding site lacking cooperativity, catalytic properties, and interactions with other proteins and thereby allow us to easily image free intracellular cAMP and rapid signaling events. Rapid β-adrenergic receptor-induced cAMP signals were observed to travel with high speed (≈40 μm/s) throughout the entire cell body of hippocampal neurons and peritoneal macrophages. The developed indicators could be ubiquitously applied to studying cAMP, its physiological role and spatio-temporal regulation.

G-protein–coupled receptors (GPCRs) are generally thought to signal to second messengers like cyclic AMP (cAMP) from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK) activation. Here, we show that a GPCR continues to stimulate cAMP production in a sustained manner after internalization. We generated transgenic mice with ubiquitous expression of a fluorescent sensor for cAMP and studied cAMP responses to thyroid-stimulating hormone (TSH) in native, 3-D thyroid follicles isolated from these mice. TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein α_s-subunits and adenylyl cyclase III. Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors. These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell. Cells respond to many environmental cues through the activity of cell surface receptor proteins, which sense these cues and convey that information to signaling molecules inside the cell. G-protein–coupled receptors (GPCRs) form the largest eukaryotic family of plasma membrane receptors. They convert the information provided by extracellular stimuli into intracellular second messengers, like cyclic AMP (cAMP). After prolonged stimulation, they are internalized inside cells, an event that to date has been thought to terminate the production of second messengers. Though many of the key steps of GPCR signaling are known in detail, precisely how signaling and termination actually occur in time and space (i.e., in subcellular compartments or microdomains) is still largely unexplored. To observe GPCR signaling in living cells, we generated mice expressing a fluorescent sensor that allows monitoring the intracellular levels of cAMP with a microscope. We utilized this system to study, directly in native thyroid follicles, the signal sent by the receptor for thyroid-stimulating hormone (TSH). Our findings indicate that TSH receptors are internalized rapidly after activation but continue to stimulate cAMP production inside cells and that this sustained, cAMP production is apparently required for localized activation of downstream components. These data challenge the current model of the GPCR-cAMP pathway by suggesting the existence of previously unrecognized intracellular site(s) for cAMP generation and of differential signaling outcomes as a result of intracellular GPCR signaling. Such intracellular site(s) may provide specialized signaling platforms, thus contributing to the spatiotemporal regulation of cAMP production and to signaling specificity within the GPCR family.

Heart Cell Signaling in 3D: A healthy heart relies on the proper transduction of cellular signals through the β₁- and β₂-adrenergic receptors (βARs), which are located on the surface of the heart's muscle cells (cardiomyocytes). The surface of these cells resembles a highly organized series of hills and valleys and it has been unclear whether this topography plays a role in the βAR signaling events that are critical to cell function.Nikolaevet al.(p.1653, published online 25 February; see Perspective byDorn) monitored the cyclic adenosine monophosphate (cAMP) signals generated by the βARs in living cardiomyocytes. In cells from healthy rats and from rats with heart failure, the β₁ARs were localized across the entire cell surface. In contrast, the spatial localization of the β₂ARs differed in healthy and failing cells. In healthy cardiomyocytes, the β₂ARs resided exclusively within surface invaginations called transverse tubules, thereby producing spatially confined cAMP signals, whereas in failing cardiomyocytes, the β₂ARs redistributed to other cell surface areas, thereby producing diffuse cAMP signals. Thus, changes in the spatial localization of β₂AR-induced cAMP signaling may contribute to heart failure.

Mammalian oocytes are arrested in meiotic prophase by an inhibitory signal from the surrounding somatic cells in the ovarian follicle. In response to luteinizing hormone (LH), which binds to receptors on the somatic cells, the oocyte proceeds to second metaphase, where it can be fertilized. Here we investigate how the somatic cells regulate the prophase-to-metaphase transition in the oocyte, and show that the inhibitory signal from the somatic cells is cGMP. Using FRET-based cyclic nucleotide sensors in follicle-enclosed mouse oocytes, we find that cGMP passes through gap junctions into the oocyte,where it inhibits the hydrolysis of cAMP by the phosphodiesterase PDE3A. This inhibition maintains a high concentration of cAMP and thus blocks meiotic progression. LH reverses the inhibitory signal by lowering cGMP levels in the somatic cells (from ∼2 μM to ∼80 nM at 1 hour after LH stimulation)and by closing gap junctions between the somatic cells. The resulting decrease in oocyte cGMP (from ∼1 μM to ∼40 nM) relieves the inhibition of PDE3A, increasing its activity by ∼5-fold. This causes a decrease in oocyte cAMP (from ∼700 nM to ∼140 nM), leading to the resumption of meiosis.

β₁- and β₂-adrenergic receptors (βARs) are known to differentially regulate cardiomyocyte contraction and growth. We tested the hypothesis that these differences are attributable to spatial compartmentation of the second messenger cAMP. Using a fluorescent resonance energy transfer (FRET)-based approach, we directly monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes. We developed a new cAMP-FRET sensor (termed HCN2-camps) based on a single cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated potassium channel 2 (HCN2). Its cytosolic distribution, high dynamic range, and sensitivity make HCN2-camps particularly well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated HCN2-camps transgenic mice and performed single-cell FRET imaging on freshly isolated cardiomyocytes. Whole-cell superfusion with isoproterenol showed a moderate elevation of cAMP. Application of various phosphodiesterase (PDE) inhibitors revealed stringent control of cAMP through PDE4>PDE2>PDE3. The β₁AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas β₂AR-mediated cAMP was under control of multiple PDE isoforms. β₁AR subtype–specific stimulation yielded ≈2-fold greater cAMP responses compared with selective β₂-subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (ΔFRET, 17.3±1.3% [β₁AR] versus 8.8±0.4% [β₂AR]). Treatment with pertussis toxin to inactivate G_idid not affect cAMP production. Localized β₁AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local β₂AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, β₁ARs induce far-reaching cAMP signals, whereas β₂AR-induced cAMP remains locally confined.