RESUMEN
Cells relay a plethora of extracellular signals to specific cellular responses by using only a few second messengers, such as cAMP. To explain signaling specificity, cAMP-degrading phosphodiesterases (PDEs) have been suggested to confine cAMP to distinct cellular compartments. However, measured rates of fast cAMP diffusion and slow PDE activity render cAMP compartmentalization essentially impossible. Using fluorescence spectroscopy, we show that, contrary to earlier data, cAMP at physiological concentrations is predominantly bound to cAMP binding sites and, thus, immobile. Binding and unbinding results in largely reduced cAMP dynamics, which we term "buffered diffusion." With a large fraction of cAMP being buffered, PDEs can create nanometer-size domains of low cAMP concentrations. Using FRET-cAMP nanorulers, we directly map cAMP gradients at the nanoscale around PDE molecules and the areas of resulting downstream activation of cAMP-dependent protein kinase (PKA). Our study reveals that spatiotemporal cAMP signaling is under precise control of nanometer-size domains shaped by PDEs that gate activation of downstream effectors.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Transducción de Señal , Análisis de la Célula Individual/métodos , Simulación por Computador , AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/química , Citoplasma/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HEK293 , Humanos , Modelos Moleculares , Hidrolasas Diéster Fosfóricas/química , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes , Análisis Espacio-Temporal , Espectrometría de FluorescenciaRESUMEN
In their role as second messengers, cyclic nucleotides such as cAMP have a variety of intracellular effects. These complex tasks demand a highly organized orchestration of spatially and temporally confined cAMP action which should be best achieved by compartmentalization of the latter. A great body of evidence suggests that cAMP compartments may be established and maintained by cAMP degrading enzymes, e.g. phosphodiesterases (PDEs). However, the molecular and biophysical details of how PDEs can orchestrate cAMP gradients are entirely unclear. In this paper, using fusion proteins of cAMP FRET-sensors and PDEs in living cells, we provide direct experimental evidence that the cAMP concentration in the vicinity of an individual PDE molecule is below the detection limit of our FRET sensors (<100nM). This cAMP gradient persists in crude cytosol preparations. We developed mathematical models based on diffusion-reaction equations which describe the creation of nanocompartments around a single PDE molecule and more complex spatial PDE arrangements. The analytically solvable equations derived here explicitly determine how the capability of a single PDE, or PDE complexes, to create a nanocompartment depend on the cAMP degradation rate, the diffusive mobility of cAMP, and geometrical and topological parameters. We apply these generic models to our experimental data and determine the diffusive mobility and degradation rate of cAMP. The results obtained for these parameters differ by far from data in literature for free soluble cAMP interacting with PDE. Hence, restricted cAMP diffusion in the vincinity of PDE is necessary to create cAMP nanocompartments in cells.
Asunto(s)
AMP Cíclico/metabolismo , Citosol/metabolismo , Modelos Biológicos , Hidrolasas Diéster Fosfóricas/metabolismo , Línea Celular , HumanosRESUMEN
We recently identified a high prevalence of mutations affecting the catalytic (Cα) subunit of protein kinase A (PKA) in cortisol-secreting adrenocortical adenomas. The two identified mutations (Leu206Arg and Leu199_Cys200insTrp) are associated with increased PKA catalytic activity, but the underlying mechanisms are highly controversial. Here we utilize a combination of biochemical and optical assays, including fluorescence resonance energy transfer in living cells, to analyze the consequences of the two mutations with respect to the formation of the PKA holoenzyme and its regulation by cAMP. Our results indicate that neither mutant can form a stable PKA complex, due to the location of the mutations at the interface between the catalytic and the regulatory subunits. We conclude that the two mutations cause high basal catalytic activity and lack of regulation by cAMP through interference of complex formation between the regulatory and the catalytic subunits of PKA.
Asunto(s)
Neoplasias de la Corteza Suprarrenal/enzimología , Adenoma Corticosuprarrenal/enzimología , Síndrome de Cushing/enzimología , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/metabolismo , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Adenoma Corticosuprarrenal/genética , Adenoma Corticosuprarrenal/patología , Dominio Catalítico , Línea Celular Tumoral , Síndrome de Cushing/genética , Síndrome de Cushing/patología , AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIIbeta de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/química , Subunidad RIalfa de la Proteína Quinasa Dependiente de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Mutación , Unión Proteica , Estabilidad ProteicaRESUMEN
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 beta-adrenergic receptor-induced cAMP signals were observed to travel with high speed ( approximately 40 microm/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.
Asunto(s)
AMP Cíclico/metabolismo , Transducción de Señal , Animales , Línea Celular , Cricetinae , Fluorescencia , Humanos , Sistemas de Mensajero SecundarioRESUMEN
Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a beta-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/fisiología , AMP Cíclico/metabolismo , Miocitos Cardíacos/enzimología , 3',5'-AMP Cíclico Fosfodiesterasas/análisis , 3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Agonistas alfa-Adrenérgicos/farmacología , Animales , Animales Recién Nacidos , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3 , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 4 , Transferencia Resonante de Energía de Fluorescencia , Norepinefrina/farmacología , Inhibidores de Fosfodiesterasa/farmacología , RatasRESUMEN
The beta(2)-adrenergic receptor has been shown to display significant constitutive activity (i.e., in the absence of agonist) in addition to agonist-induced activation. Various studies have suggested that a movement in transmembrane helix VI plays a role in activation of various G-protein-coupled receptors. Here we show that a mutation in this domain of the beta(2)-adrenergic receptor abolishes agonist activation but not constitutive activity. An Asn293Asp mutant of the human beta(2)-adrenergic receptor was expressed either transiently in COS-7 cells or stably in Chinese hamster ovary cells. The mutant receptors were unable to couple to G(s), as seen by the lack of high-affinity agonist binding as well as a reduction of the affinities of several agonists correlating with their intrinsic activities. The mutant receptors caused only minimal activation of adenylyl cyclase (2.5% of wild-type activity) and also failed to show agonist-induced phosphorylation by G-protein-coupled receptor kinase 2. In contrast, the mutant receptors were much less affected in their constitutive activity: transient transfection of wild-type and mutant receptors into COS-7 cells caused an increase in intracellular cAMP-levels that was dependent on the level of receptor expression and was maximally 5.4-fold for the mutant and 6.8-fold for the wild-type receptors (67% of wild-type activity). Introduction of the Asn293Asp mutation into a constitutively active mutant receptor did not affect the constitutive activity of this mutant. These results underscore the importance of transmembrane helix VI in controlling agonist-induced activation of the receptor and suggest that constitutive activity is different from agonist-induced activity. Furthermore, they indicate that Asn293 is a key residue in transferring conformational information from the agonist-binding site to the intracellular surface.