No abstract

Fluorescence resonance energy transfer (FRET) from cyan to yellow fluorescent proteins (CFP/YFP) is a well-established method to monitor protein-protein interactions or conformational changes of individual proteins. But protein functions can be perturbed by fusion of large tags such as CFP and YFP. Here we use G protein-coupled receptor (GPCR) activation in living cells as a model system to compare YFP with the small, membrane-permeant fluorescein derivative with two arsen-(III) substituents (fluorescein arsenical hairpin binder; FlAsH) targeted to a short tetracysteine sequence. Insertion of CFP and YFP into human adenosine A(2A) receptors allowed us to use FRET to monitor receptor activation but eliminated coupling to adenylyl cyclase. The CFP/FlAsH-tetracysteine system gave fivefold greater agonist-induced FRET signals, similar kinetics (time constant of 66-88 ms) and perfectly normal downstream signaling. Similar results were obtained for the mouse alpha(2A)-adrenergic receptor. Thus, FRET from CFP to FlAsH reports GPCR activation in living cells without disturbing receptor function and shows that the small size of the tetracysteine-biarsenical tag can be decisively advantageous.

Hormones and neurotransmitters transduce signals through G protein-coupled receptors (GPCR). Despite their common signaling pathways, however, the responses they elicit have different temporal patterns. To reveal the molecular basis for these differences we have developed a generally applicable fluorescence-based technique for real-time monitoring of the activation switch of GPCRs in living cells. We used such direct measurements to investigate the activation of the alpha(2A)-adrenergic receptor (alpha(2A)AR; neurotransmitter) and the parathyroid hormone receptor (PTHR; hormone) and observed much faster kinetics than expected: approximately 40 ms for the alpha(2A)AR and approximately 1 s for the PTHR. The different switch times are in agreement with the different receptors' biological functions. Agonists and antagonists could rapidly switch the receptors on or off, whereas a partial agonist caused only a partial signal. This approach allows the comparison of agonist and partial agonist intrinsic activities at the receptor level and provides evidence for millisecond activation times of GPCRs.

G protein-coupled receptors transduce diverse extracellular signals, such as neurotransmitters, hormones, chemokines, and sensory stimuli, into intracellular responses through activation of heterotrimeric G proteins. G proteins play critical roles in determining specificity and kinetics of subsequent biological responses by modulation of effector proteins. We have developed a fluorescence resonance energy transfer (FRET)-based assay to directly measure mammalian G protein activation in intact cells and found that Gi proteins activate within 1-2 s, which is considerably slower than activation kinetics of the receptors themselves. More importantly, FRET measurements demonstrated that G␣i-and G␤␥-subunits do not dissociate during activation, as has been previously postulated. Based on FRET measurements between G␣i-yellow fluorescent protein and G␤␥-subunits that were fused to cyan fluorescent protein at various positions, we conclude that, instead, G protein subunits undergo a molecular rearrangement during activation. The detection of a persistent heterotrimeric composition during G protein activation will impact the understanding of how G proteins achieve subtype-selective coupling to effectors. This finding will be of particular interest for unraveling G␤␥-induced signaling pathways. Avariety of physiological signals such as neurotransmitters, hormones, and light are detected by members of the seven transmembrane domain receptor family. These G protein-coupled receptors (GPCRs) activate G proteins by promoting binding of GTP in exchange for GDP. Both, G␣ and G␤␥-subunits of activated G proteins can regulate downstream effectors such as adenylyl cyclases, phospholipases, or ion channels. Based on biochemical experiments and structural studies, it is known that conformational rearrangements in the ''switch regions'' of the ␣-subunits on GTP binding weaken the interaction with G␤␥-subunits (1-3). It is generally assumed that the reduced affinity of G␣-GTP for G␤␥ causes G proteins to dissociate into G␣-GTP and a G␤␥ complex. Reassociation is then assumed to occur on hydrolysis of the GTP bound to G␣, which can be accelerated by RGS proteins (4-6). However, this model fails to explain well established phenomena in G protein-mediated signaling. Most importantly, a major open question is how G␤␥ effectors, such as G proteinactivated inwardly rectifying K ϩ (GIRK) channel, are selectively regulated through specific G␣ subtypes despite the lack of subtype selectivity for G␤␥ subtypes (7-10). In attempts to answer this central question, it has been hypothesized that selectivity may be caused by scaffolding of G proteins and effectors, either by direct binding of G␣ to its effector (11), or by temporally and spatially restricting G protein activity (12, 13).The possibility to investigate protein-protein interactions in living cells by using fluorescence resonance energy transfer (FRET) between recombinant fluorescent proteins (14), has recently led to new insights in temporal signaling properties of G protein effectors (1...

Abstract-␤ 1 -and ␤ 2 -adrenergic receptors (␤ARs) 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 PDE4ϾPDE2ϾPDE3. The ␤ 1 AR-mediated cAMP signals were entirely dependent on PDE4 activity, whereas ␤ 2 AR-mediated cAMP was under control of multiple PDE isoforms. ␤ 1 AR subtype-specific stimulation yielded Ϸ2-fold greater cAMP responses compared with selective ␤ 2 -subtype stimulation, even on treatment with the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) (⌬FRET, 17.3Ϯ1.3% [␤ 1 AR] versus 8.8Ϯ0.4% [␤ 2 AR]). Treatment with pertussis toxin to inactivate G i did not affect cAMP production. Localized ␤ 1 AR stimulation generated a cAMP gradient propagating throughout the cell, whereas local ␤ 2 AR stimulation did not elicit marked cAMP diffusion. Our data reveal that in adult cardiac myocytes, ␤ 1 ARs induce far-reaching cAMP signals, whereas ␤ 2 AR-induced cAMP remains locally confined. by the endogenous agonists noradrenaline and adrenaline represents the strongest mechanism to increase cardiac chronotropy and inotropy. 1 The mammalian heart contains 3 ␤AR subtypes: the ␤ 1 -, the ␤ 2 -, and the ␤ 3 AR. The ␤ 1 -and ␤ 2 AR subtypes dominate the cardiac response to adrenergic stimulation. Both are expressed in cardiomyocytes, couple primarily to G s , and mediate cAMP formation, whereas coupling of the ␤ 2 AR to G i has been described in several animal species and in failing human cardiomyocytes. [2][3][4] The second messenger cAMP then leads to activation of protein kinase A (PKA), which phosphorylates key regulators of the cardiac excitation/contraction machinery, including the L-type Ca 2ϩ channel, phospholamban, the ryanodine receptor, and troponin T and I. However, selective stimulation of these 2 receptor subtypes elicits different physiological responses. ␤ 1 AR stimulation, but not ␤ 2 AR stimulation, seems to induce cardiomyocyte hypertrophy. 5 Transgenic mice with cardiomyocyte-specific overexpression of the ␤ 1 AR develop progressive cardiac hypertrophy and heart failure, whereas ␤ 2 AR transgenic mice do not show such abnormalities. 6,7 Isolated cardio...