2-Adrenergic Receptor Signaling and Desensitization Elucidated by Quantitative Modeling of Real Time cAMP Dynamics

Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2008; 283(5):2949-61. DOI: 10.1074/jbc.M707009200
Source: PubMed


G protein-coupled receptor signaling is dynamically regulated by multiple feedback mechanisms, which rapidly attenuate signals elicited by ligand stimulation, causing desensitization. The individual contributions of these mechanisms, however, are poorly understood. Here, we use an improved fluorescent biosensor for cAMP to measure second messenger dynamics stimulated by endogenous beta(2)-adrenergic receptor (beta(2)AR) in living cells. beta(2)AR stimulation with isoproterenol results in a transient pulse of cAMP, reaching a maximal concentration of approximately 10 microm and persisting for less than 5 min. We investigated the contributions of cAMP-dependent kinase, G protein-coupled receptor kinases, and beta-arrestin to the regulation of beta(2)AR signal kinetics by using small molecule inhibitors, small interfering RNAs, and mouse embryonic fibroblasts. We found that the cAMP response is restricted in duration by two distinct mechanisms in HEK-293 cells: G protein-coupled receptor kinase (GRK6)-mediated receptor phosphorylation leading to beta-arrestin mediated receptor inactivation and cAMP-dependent kinase-mediated induction of cAMP metabolism by phosphodiesterases. A mathematical model of beta(2)AR signal kinetics, fit to these data, revealed that direct receptor inactivation by cAMP-dependent kinase is insignificant but that GRK6/beta-arrestin-mediated inactivation is rapid and profound, occurring with a half-time of 70 s. This quantitative system analysis represents an important advance toward quantifying mechanisms contributing to the physiological regulation of receptor signaling.

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    • "In contrast with quantitative analysis of b2AR-level desensitization , defining functional desensitization based on cAMP turnover by PDE or bronchodilation is more problematic. The role of PDE in isoproterenol stimulation of cAMP accumulation in HEK293 cells expressing only endogenous receptors has also been modeled and simulated (Violin et al., 2008; Xin et al., 2008). These findings highlighted the important role of PKA activation of PDE, which was nearly equivalent to the GRK pathway in terms of its effect on the rapid phase of functional desensitization (to be distinguished from b2AR desensitization). "
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    ABSTRACT: Salmeterol is a long acting β2-adrenergic receptor (β2AR) agonist widely used as a bronchodilator for the treatment of persistent asthma and COPD in conjunction with steroids. Previous studies demonstrated that salmeterol showed weak efficacy for activation of adenylyl cyclase; however, its efficacy in the complex desensitization of β2AR remains poorly understood. In the present work we provide insights into the roles played by GRK/arrestin and PKA in salmeterol-mediated desensitization through BRET studies of liganded-β2AR binding to arrestin, and kinetic studies of cAMP turnover. First, BRET demonstrated a much-reduced efficacy for salmeterol recruitment of arrestin to the β2AR relative to isoproterenol. The ratio of BRETISO/BRETSALM after 5 min stimulation was 20, decreasing to 5 after 35 min, reflecting a progressive decline in BRETISO, and a stable BRETSALM. Second, to assess salmeterol efficacy for functional desensitization, we examined the kinetics of salmeterol-induced cAMP accumulation (0-30 min) in human airway smooth muscle cells (HASM) in the presence and absence of PDE inhibition. Analysis of shaping of cAMP turnover for both agonists demonstrated significant salmeterol desensitization, although reduced relative to isoproterenol. Using an isoproterenol rescue protocol following either short- (10 min) or long-term (2 and 14 hr) salmeterol pretreatments, we found that salmeterol progressively depressed isoproterenol stimulation, but did not prevent subsequent rescue by isoproterenol and additional isoproterenol-mediated desensitization. Our findings reveal a complex efficacy for functional desensitization demonstrating that while salmeterol shows weak efficacy for adenylyl cyclase activation and GRK/arrestin-mediated desensitization, it acts as a strong agonist in highly amplified PKA-mediated events. The American Society for Pharmacology and Experimental Therapeutics.
    Molecular pharmacology 03/2015; 87(6). DOI:10.1124/mol.114.096800 · 4.13 Impact Factor
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    • "Like Epac1-camps and Epac2-camps, ICUE also responds to cAMP with a decrease in the yellow to cyan emission ratio. ICUE2, an improved version of ICUE1, has an EC 50 of ∼12.5 µM and contains a N-terminally truncated Epac1 protein (Epac1 149−881 ) (Violin et al., 2008). This biosensor showed improvement in localization over ICUE1 due to removal of a membrane and mitochondria targeting sequence located at the N-terminus. "
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    ABSTRACT: The second messengers cAMP and cGMP transduce many neuromodulatory signals from hormones and neurotransmitters into specific functional outputs. Their production, degradation and signaling are spatiotemporally regulated to achieve high specificity in signal transduction. The development of genetically encodable fluorescent biosensors has provided researchers with useful tools to study these versatile second messengers and their downstream effectors with unparalleled spatial and temporal resolution in cultured cells and living animals. In this review, we introduce the general design of these fluorescent biosensors and describe several of them in more detail. Then we discuss a few examples of using cyclic nucleotide fluorescent biosensors to study regulation of neuronal function and finish with a discussion of advances in the field. Although there has been significant progress made in understanding how the specific signaling of cyclic nucleotide second messengers is achieved, the mechanistic details in complex cell types like neurons are only just beginning to surface. Current and future fluorescent protein reporters will be essential to elucidate the role of cyclic nucleotide signaling dynamics in the functions of individual neurons and their networks.
    Frontiers in Cellular Neuroscience 11/2014; 8(395). DOI:10.3389/fncel.2014.00395 · 4.29 Impact Factor
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    • "In our proof of principle study we identified various previously known ȕ 2 AR interactors, such as ȕ 2 AR itself and GRK6 [28]. We also discovered novel interactors, which were confirmed in human cells by two independent orthogonal assays such as the high-throughput LUMIER assay and coimmunoprecipitation assays. "
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    ABSTRACT: G-protein Coupled Receptors (GPCRs) are involved in a variety of disease processes and comprise major drug targets. However, the complexity of integral membrane proteins such as GPCRs makes the identification of their interacting partners and subsequent drug development challenging. A comprehensive understanding of GPCR protein interaction networks is needed to design effective therapeutic strategies to inhibit these drug targets. Here, we developed a novel split-ubiquitin membrane yeast two-hybrid (MYTH) technology called CHIP-MYTH, which allows the unbiased characterization of interaction partners of full-length GPCRs in a drug-dependent manner. This was achieved by coupling DNA microarray technology to the MYTH approach, which allows a quantitative evaluation of interacting partners of a given integral membrane protein in the presence or absence of drug. As a proof of principle, we applied the CHIP-MYTH approach to the human ß2-adrenergic receptor (ß2AR), a target of interest in the treatment of asthma, chronic obstructive pulmonary disease (COPD), neurological disease, cardiovascular disease, and obesity. A CHIP-MYTH screen was performed in the presence or absence of salmeterol, a long-acting ß2AR-agonist. Our results suggest that ß2AR activation with salmeterol can induce the dissociation of heterotrimeric G-proteins, G, into G and G subunits, which in turn activates downstream signaling cascades. Using CHIP-MYTH, we confirmed previously known and identified novel ß2AR interactors involved in GPCR-mediated signaling cascades. Several of these interactions were confirmed in mammalian cells using LUMIER and co-immunoprecipitation assays. In summary, the CHIP-MYTH approach is ideal for conducting comprehensive PPI screenings of full-length GPCRs in the presence or absence of drugs, thus providing a valuable tool to further our understanding of GPCR-mediated signaling.
    Biochemical and Biophysical Research Communications 03/2014; 445(4). DOI:10.1016/j.bbrc.2014.02.033 · 2.30 Impact Factor
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