scholarly journals GPCR signaling is highly compartmentalized in human cardiomyocytes and severely remodeled in atrial fibrillation

2021 ◽  
Vol 154 (9) ◽  
Author(s):  
Kira Beneke ◽  
Nefeli Grammatika Pavlidou ◽  
Andreas Schäfer ◽  
Viacheslav O. Nikolaev ◽  
Cristina E. Molina
Keyword(s):  
Atrial Fibrillation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Human Cardiomyocytes ◽  
Specific Effects ◽  
And Control ◽  
G Protein Coupled ◽  
Camp Levels ◽  
A2ar Agonist

Atrial fibrillation (AF) has been linked to the remodeling of membrane receptors and alterations in downstream cAMP-dependent regulation. However, to date, no study has elucidated how the increase on cAMP upon different G-protein-coupled receptors (GPCRs) can lead to different physiological compartmentalized responses. The aim of this study was to investigate the compartmentally specific effects of GPCRs on cAMP levels in human atrial myocytes (HAMs) from patients with AF and control patients without AF (Ctl), and how these compartmentalized effects are altered in AF. HAMs were isolated from 60 AF and 76 Ctl patient tissues. Cells were transduced with adenoviruses (Epac1-camps, pm-Epac1-camps and Epac1-JNC) and cultured for 48 hours to express the FRET-based cAMP sensor in the cytosolic, membrane, and RYR2 nanodomains. Förster-resonance energy transfer (FRET) was used to measure cAMP levels in 525 HAMs stimulated with isoprenaline (100 µM), serotonin (100 µM), or the A2AR agonist CGS (200 nM). A desensitization to β-adrenergic receptor stimulation was exclusively found in the cytosol of AF myocytes, while no difference was seen in the RYR2 or LTCC compartment. Similar effects were observed upon serotonin stimulation with a significant desensitization in the cytosol, and no difference in the RYR2 compartment. In response to A2ARs stimulation AF myocytes displayed a significantly higher cytosolic increase in cAMP levels. However, no response was seen in the LTCC compartment in response to serotonin or A2AR stimulation. Collectively, our data show that cAMP levels are highly compartmentalized and differentially regulated by GPCRs. Furthermore, these results provide a mechanistic insight for the previously reported functional effects seen upon stimulation of these three receptors.

P2869Role of PDE8 in cAMP dynamics in human atrial fibrillation

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
N Grammatika Pavlidou ◽  
S Pecha ◽  
H Reichenspurner ◽  
T Christ ◽  
V O Nikolaev ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Intracellular Camp ◽  
Atrial Myocytes ◽  
Human Atrial Myocytes ◽  
Intracellular Compartments ◽  
Camp Levels

Abstract Background Cardiac arrhythmias, such as atrial fibrillation (AF), are often related to remodeling of membrane receptors and alterations in cAMP-dependent regulation of Ca2+ handling mechanisms. For instance, decreased L-type calcium current (ICa,L) density but upregulated RyR2 are major hallmarks of AF. These inhomogeneous AF-associated changes of protein phosphorylation point to a local regulation of PKA activity within these intracellular compartments. Local cAMP compartmentation and the role of phosphodiesterase (PDEs) have ben extensively studied in ventricular myocytes from animals. However, only a few studies have evaluated the contribution of PDEs to the pathophysiology of AF and the reason for the persistent AF-associated hypophosphorylation of the L-type calcium channel (LTCC) is currently unknown. The aim of this study was to investigate whether a change in the expression level of PDE8 in human atrium may affects cAMP nearby LTCC promoting the reduction of the ICa,L observed in persistent AF. Methods Atrial myocytes were isolated from tissue of 47 patients in sinus rhythm (SR) and with AF. Cells were then transfect with an adenovirus (Epac1-camps or pm-Epac1-camps) in order to express the (cytosolic or membrane, respectively) FRET-based cAMP sensor and cultured during 48 hours. Föster-resonance energy transfer (FRET) was used to measure cAMP in 232 isolated human atrial myocytes. Ro-20-1724 (10 μM), Cilostamide (1 μM) and PF-04957325 (30 nM) and IBMX (100 μM) were used as PDE4, PDE3, PDE8 and non-selective phosphodiesterases (PDEs) inhibitor respectively. Results Effects of PDE4 and especially PDE3 inhibition on cytosolic [cAMP] are reduced in AF. Pharmacological PDE8 inhibition induces only a small increase in basal intracellular [cAMP] in AF but it showed a big synergic effect when PDE4 was inhibit at the same time. By contrast, PDE8 inhibition dramatically increased basal [cAMP] in the subsarcolemmal compartment in AF while PDE3 or PDE4 inhibition had a smaller effect that didn't change between SR and AF. Conclusions PDE8 controls basal cytosolic cAMP levels in human atrial myocytes from patients with persistent AF while PDE3 effects tends to be reduced in these patients. Furthermore, PDE8 is the main PDE in controlling cAMP levels at the membrane in persistent AF. Thus, our study may provide a clue for the reported reduction of the ICa,L in persistent AF.

scholarly journals Heterodimerization Between the Lutropin and Follitropin Receptors is Associated With an Attenuation of Hormone-Dependent Signaling

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3925-3930 ◽  
Author(s):  
Xiuyan Feng ◽  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Fsh Receptor ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Lh Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

scholarly journals Minireview: GPCR and G Proteins: Drug Efficacy and Activation in Live Cells

2009 ◽  
Vol 23 (5) ◽  
pp. 590-599 ◽  
Author(s):  
Jean-Pierre Vilardaga ◽  
Moritz Bünemann ◽  
Timothy N. Feinstein ◽  
Nevin Lambert ◽  
Viacheslav O. Nikolaev ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Live Cells ◽  
Mechanical Stimuli ◽  
G Protein Coupled

Abstract Many biochemical pathways are driven by G protein-coupled receptors, cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of Förster resonance energy transfer and bioluminescence resonance energy transfer-based methods has now permitted the kinetic analysis of initial steps involved in G protein-coupled receptor-mediated signaling in live cells and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by Förster resonance energy transfer is also now possible and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

scholarly journals Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

2012 ◽  
Vol 198 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Enora Moutin ◽  
Fabrice Raynaud ◽  
Jonathan Roger ◽  
Emilie Pellegrino ◽  
Vincent Homburger ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Physical Interaction ◽  
Scaffolding Proteins ◽  
Cellular Functions ◽  
Electrophysiological Recordings ◽  
Living Neurons ◽  
Activity Dependent

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

scholarly journals The luminescent HiBiT peptide enables selective quantitation of G protein–coupled receptor ligand engagement and internalization in living cells

2020 ◽  
Vol 295 (15) ◽  
pp. 5124-5135 ◽  
Author(s):  
Michelle E. Boursier ◽  
Sergiy Levin ◽  
Kris Zimmerman ◽  
Thomas Machleidt ◽  
Robin Hurst ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Cell Surface ◽  
G Protein ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
Cellular Interactions ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

scholarly journals Bioluminescence Resonance Energy Transfer Based G Protein-Activation Assay to Probe Duration of Antagonism at the Histamine H3 Receptor

2019 ◽  
Vol 20 (15) ◽  
pp. 3724 ◽  
Author(s):  
Tamara A. M. Mocking ◽  
Maurice C. M. L. Buzink ◽  
Rob Leurs ◽  
Henry F. Vischer
Keyword(s):  
G Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Short Residence Time ◽  
Protein Activation ◽  
G Protein Activation ◽  
Activation Assay ◽  
G Protein Coupled ◽  
Histamine H3

Duration of receptor antagonism, measured as the recovery of agonist responsiveness, is gaining attention as a method to evaluate the ‘effective’ target-residence for antagonists. These functional assays might be a good alternative for kinetic binding assays in competition with radiolabeled or fluorescent ligands, as they are performed on intact cells and better reflect consequences of dynamic cellular processes on duration of receptor antagonism. Here, we used a bioluminescence resonance energy transfer (BRET)-based assay that monitors heterotrimeric G protein activation via scavenging of released Venus-Gβ1γ2 by NanoLuc (Nluc)-tagged membrane-associated-C-terminal fragment of G protein-coupled receptor kinase 3 (masGRK3ct-Nluc) as a tool to probe duration of G protein-coupled receptor (GPCR) antagonism. The Gαi-coupled histamine H3 receptor (H3R) was used in this study as prolonged antagonism is associated with adverse events (e.g., insomnia) and consequently, short-residence time ligands might be preferred. Due to its fast and prolonged response, this assay can be used to determine the duration of functional antagonism by measuring the recovery of agonist responsiveness upon washout of pre-bound antagonist, and to assess antagonist re-equilibration time via Schild-plot analysis. Re-equilibration of pre-incubated antagonist with agonist and receptor could be followed in time to monitor the transition from insurmountable to surmountable antagonism. The BRET-based G protein activation assay can detect differences in the recovery of H3R responsiveness and re-equilibration of pre-bound antagonists between the tested H3R antagonists. Fast dissociation kinetics were observed for marketed drug pitolisant (Wakix®) in this assay, which suggests that short residence time might be beneficial for therapeutic targeting of the H3R.

scholarly journals Illuminating the allosteric modulation of the calcium-sensing receptor

2020 ◽  
Vol 117 (35) ◽  
pp. 21711-21722
Author(s):  
Hongkang Liu ◽  
Ping Yi ◽  
Wenjing Zhao ◽  
Yuling Wu ◽  
Francine Acher ◽  
...  
Keyword(s):  
Amino Acids ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Allosteric Modulators ◽  
Active Conformation ◽  
Calcium Sensing Receptor ◽  
Single Receptor ◽  
Calcium Sensing

Many membrane receptors are regulated by nutrients. However, how these nutrients control a single receptor remains unknown, even in the case of the well-studied calcium-sensing receptor CaSR, which is regulated by multiple factors, including ions and amino acids. Here, we developed an innovative cell-free Förster resonance energy transfer (FRET)-based conformational CaSR biosensor to clarify the main conformational changes associated with activation. By allowing a perfect control of ambient nutrients, this assay revealed that Ca2+alone fully stabilizes the active conformation, while amino acids behave as pure positive allosteric modulators. Based on the identification of Ca2+activation sites, we propose a molecular basis for how these different ligands cooperate to control CaSR activation. Our results provide important information on CaSR function and improve our understanding of the effects of genetic mutations responsible for human diseases. They also provide insights into how a receptor can integrate signals from various nutrients to better adapt to the cell response.

scholarly journals G-protein-coupled receptor 30 interacts with receptor activity-modifying protein 3 and confers sex-dependent cardioprotection

2013 ◽  
Vol 51 (1) ◽  
pp. 191-202 ◽  
Author(s):  
Patricia M Lenhart ◽  
Stefan Broselid ◽  
Cordelia J Barrick ◽  
L M Fredrik Leeb-Lundberg ◽  
Kathleen M Caron
Keyword(s):  
G Protein ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cardiac Cells ◽  
Receptor Activity ◽  
Hek293 Cells ◽  
G Protein Coupled

Receptor activity-modifying protein 3 (RAMP3) is a single-pass transmembrane protein known to interact with and affect the trafficking of several G-protein-coupled receptors (GPCRs). We sought to determine whether RAMP3 interacts with GPR30, also known as G-protein-coupled estrogen receptor 1. GPR30 is a GPCR that binds estradiol and has important roles in cardiovascular and endocrine physiology. Using bioluminescence resonance energy transfer titration studies, co-immunoprecipitation, and confocal microscopy, we show that GPR30 and RAMP3 interact. Furthermore, the presence of GPR30 leads to increased expression of RAMP3 at the plasma membrane in HEK293 cells. In vivo, there are marked sex differences in the subcellular localization of GPR30 in cardiac cells, and the hearts of Ramp3−/− mice also show signs of GPR30 mislocalization. To determine whether this interaction might play a role in cardiovascular disease, we treated Ramp3+/+ and Ramp3−/− mice on a heart disease-prone genetic background with G-1, a specific agonist for GPR30. Importantly, this in vivo activation of GPR30 resulted in a significant reduction in cardiac hypertrophy and perivascular fibrosis that is both RAMP3 and sex dependent. Our results demonstrate that GPR30–RAMP3 interaction has functional consequences on the localization of these proteins both in vitro and in vivo and that RAMP3 is required for GPR30-mediated cardioprotection.

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