1,049 research outputs found
Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors
Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains of single living cells. However, relating these properties to molecular mechanisms can be challenging. We investigated the influence of β-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluorescence complementation (BiFC) system also identified Y1 receptor-β-arrestin complexes. Diffusion co-efficients (D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15×10−9 cm2 s−1 respectively. At a concentrationwhich promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFPmotility
(D 1.48×10−9 cm2 s−1), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced
changes in Y1 receptor motility were inhibited by mutations (6A) which prevented β-arrestin recruitment and
internalisation; conversely they became apparent in a Y2 receptor mutant with increased β-arrestin affinity.
NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering,
and which were abolished by the 6A mutation. The importance of β-arrestin recruitment for these effects was
illustrated by reduced lateral mobility (D 1.20–1.33×10−9 cm2 s−1) of Y1 receptor-β-arrestin BiFC complexes.
Thus NPY-induced changes in Y receptormotility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect
the underlying receptor-β-arrestin signalling complex
Fluorescence correlation spectroscopy, combined with bimolecular fluorescence complementation, reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors
Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains of single living cells. However, relating these properties to molecular mechanisms can be challenging. We investigated the influence of β-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluorescence complementation (BiFC) system also identified Y1 receptor-β-arrestin complexes. Diffusion co-efficients (D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15×10−9 cm2 s−1 respectively. At a concentrationwhich promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFPmotility
(D 1.48×10−9 cm2 s−1), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced
changes in Y1 receptor motility were inhibited by mutations (6A) which prevented β-arrestin recruitment and
internalisation; conversely they became apparent in a Y2 receptor mutant with increased β-arrestin affinity.
NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering,
and which were abolished by the 6A mutation. The importance of β-arrestin recruitment for these effects was
illustrated by reduced lateral mobility (D 1.20–1.33×10−9 cm2 s−1) of Y1 receptor-β-arrestin BiFC complexes.
Thus NPY-induced changes in Y receptormotility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect
the underlying receptor-β-arrestin signalling complex
Geographic variation in the response of Culex pipiens life history traits to temperature
BackgroundClimate change is predicted to alter the transmission of many vector-borne pathogens. The quantitative impact of climate change is usually estimated by measuring the temperature-performance relationships for a single population of vectors, and then mapping this relationship across a range of temperatures or locations. However, life history traits of different populations often differ significantly. Specifically, performance across a range of temperatures is likely to vary due to local adaptation to temperature and other factors. This variation can cause spatial variation in pathogen transmission and will influence the impact of climate change on the transmission of vector-borne pathogens.MethodsWe quantified variation in life history traits for four populations of Culex pipiens (Linnaeus) mosquitoes. The populations were distributed along altitudinal and latitudinal gradients in the eastern United States that spanned ~3 °C in mean summer temperature, which is similar to the magnitude of global warming expected in the next 3-5 decades. We measured larval and adult survival, development rate, and biting rate at six temperatures between 16 and 35 °C, in a common garden experiment.ResultsTemperature had strong and consistent non-linear effects on all four life history traits for all four populations. Adult female development time decreased monotonically with increasing temperature, with the largest decrease at cold temperatures. Daily juvenile and adult female survival also decreased with increasing temperature, but the largest decrease occurred at higher temperatures. There was significant among-population variation in the thermal response curves for the four life history traits across the four populations, with larval survival, adult survival, and development rate varying up to 45, 79, and 84 % among populations, respectively. However, variation was not correlated with local temperatures and thus did not support the local thermal adaptation hypothesis.ConclusionThese results suggest that the impact of climate change on vector-borne disease will be more variable than previous predictions, and our data provide an estimate of this uncertainty. In addition, the variation among populations that we observed will shape the response of vectors to changing climates
The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors
The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties
Kinetic analysis of fluorescent ligand binding to cell surface receptors: Insights into conformational changes and allosterism in living cells
Equilibrium binding assays are one of the mainstays of current drug discovery efforts to evaluate the interaction of drugs with receptors in membranes and intact cells. However, in recent years there has been increased focus on the kinetics of the drug-receptor interaction to gain insight into the lifetime of drug-receptor complexes and the rate of association of a ligand with its receptor. Furthermore, drugs that act on topically distinct sites (allosteric) from those occupied by the endogenous ligand (orthosteric site) can induce conformational changes in the orthosteric binding site leading to changes in the association and/or dissociation rate constants of orthosteric ligands. Conformational changes in the orthosteric ligand binding site can also be induced through interaction with neighbouring accessory proteins and receptor homo- and hetero-dimerization. In this review, we provide an overview of the use of fluorescent ligand technologies to interrogate ligand-receptor kinetics in living cells and the novel insights that they can provide into the conformational changes induced by drugs acting on a variety of cell surface receptors including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and cytokine receptors
Transactivation of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs): Recent insights using luminescence and fluorescence technologies
© 2020 The Authors Alterations in signalling due to bidirectional transactivation of G protein-coupled receptor (GPCRs) and receptor tyrosine kinases (RTKs) are well established. Transactivation significantly diversifies signalling networks within a cell and has been implicated in promoting both advantageous and disadvantageous physiological and pathophysiological outcomes, making the GPCR/RTK interactions attractive new targets for drug discovery programmes. Transactivation has been observed for a plethora of receptor pairings in multiple cell types; however, the precise molecular mechanisms and signalling effectors involved can vary with receptor pairings and cell type. This short review will discuss the recent applications of proximity-based assays, such as resonance energy transfer and fluorescence-based imaging in investigating the dynamics of GPCR/RTK complex formation, subsequent effector protein recruitment and the cellular locations of complexes in living cells
Kinetic analysis of antagonist-occupied adenosine-A3 receptors within membrane microdomains of individual cells provides evidence of receptor dimerization and allosterism
In our previous work, using a fluorescent adenosine-A3 receptor (A3AR) agonist and fluorescence correlation spectroscopy (FCS), we demonstrated high-affinity labeling of the active receptor (R*) conformation. In the current study, we used a fluorescent A3AR antagonist (CA200645) to study the binding characteristics of antagonist-occupied inactive receptor (R) conformations in membrane microdomains of individual cells. FCS analysis of CA200645-occupied A3ARs revealed 2 species, τD2 and τD3, that diffused at 2.29 ± 0.35 and 0.09 ± 0.03 μm2/s, respectively. FCS analysis of a green fluorescent protein (GFP)-tagged A3AR exhibited a single diffusing species (0.105 μm2/s). The binding of CA200645 to τD3 was antagonized by nanomolar concentrations of the A3 antagonist MRS 1220, but not by the agonist NECA (up to 300 nM), consistent with labeling of R. CA200645 normally dissociated slowly from the A3AR, but inclusion of xanthine amine congener (XAC) or VUF 5455 during washout markedly accelerated the reduction in the number of particles exhibiting τD3 characteristics. It is notable that this effect was accompanied by a significant increase in the number of particles with τD2 diffusion. These data show that FCS analysis of ligand-occupied receptors provides a unique means of monitoring ligand A3AR residence times that are significantly reduced as a consequence of allosteric interaction across the dimer interfac
A G protein-coupled receptor dimer imaging assay reveals selectively modified pharmacology of neuropeptide Y Y1/Y5 receptor heterodimers
The ability of G protein-coupled receptors (GPCRs) to form dimers, and particularly heterodimers, offers potential for targeted therapeutics with improved selectivity. However, studying dimer pharmacology is challenging, because of signaling cross-talk or because dimerization may often be transient in nature. Here we develop a system to isolate the pharmacology of precisely defined GPCR dimers, trapped by bimolecular fluorescence complementation (BiFC). Specific effects of agonist activation on such dimers are quantified using automated imaging and analysis of their internalization, controlled for by simultaneous assessment of endocytosis of one coexpressed protomer population. We applied this BiFC system to study example neuropeptide Y (NPY) Y1 receptor dimers. Incorporation of binding-site or phosphorylation-site mutations into just one protomer of a Y1/Y1 BiFC homodimer had no impact on efficient NPY-stimulated endocytosis, demonstrating that single-site agonist occupancy, and one phosphorylated monomer within this dimer, was sufficient. For two Y1 receptor heterodimer combinations (with the Y4 receptor or β2-adrenoceptor), agonist and antagonist pharmacology was explained by independent actions on the respective orthosteric binding sites. However, Y1/Y5 receptor BiFC dimers, compared with the constituent subtypes, were characterized by reduced potency and efficacy of Y5-selective peptide agonists, the inactivity of Y1-selective antagonists, and a change from surmountable to nonsurmountable antagonism for three unrelated Y5 antagonists. Thus, allosteric interactions between Y1 and Y5 receptors modify the pharmacology of the heterodimer, with implications for potential antiobesity agents that target centrally coexpressed Y1 and Y5 receptors to suppress appetite
Characterisation of tyrosine kinase inhibitor-receptor interactions at VEGFR2 using sunitinib-red and nanoBRET
Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis, proliferation and migration of vascular endothelial cells. It is well known that cardiovascular safety liability for a wide range of small molecule tyrosine kinase inhibitors (TKIs) can result from interference with the VEGFR2 signalling system. In this study we have developed a ligand-binding assay using a fluorescent analogue of sunitinib (sunitinib-red) and full length VEGFR2 tagged on its C-terminus with the bioluminescent protein nanoluciferase to monitor ligand-binding to VEGFR2 using bioluminescence resonance energy transfer (BRET). This NanoBRET assay is a proximity-based assay (requiring the fluorescent and bioluminescent components to be within 10nm of each other) that can monitor the binding of ligands to the kinase domain of VEGFR2. Sunitinib-red was not membrane permeable but was able to monitor the binding affinity and kinetics of a range of TKIs in cell lysates. Kinetic studies showed that sunitinib-red bound rapidly to VEGFR2 at 25 °C and that cediranib had slower binding kinetics with an average residence time of 112 min. Comparison between the log Ki values for inhibition of binding of sunitinib-red and log IC50 values for attenuation of VEGF165a-stimulated NFAT responses showed very similar values for compounds that inhibited sunitinib-red binding. However, two compounds that failed to inhibit sunitinib-red binding (dasatinib and entospletinib) were still able to attenuate VEGFR2-mediated NFAT signalling through inhibition of downstream signalling events. These results suggest that these compounds may still exhibit cardiovascular liabilities as a result of interference with downstream VEGFR2 signalling
A nanoluciferase biosensor to investigate endogenous chemokine secretion and receptor binding
© 2020 The Author(s) Secreted chemokines are critical mediators of cellular communication that elicit intracellular signaling by binding membrane-bound receptors. Here we demonstrate the development and use of a sensitive real-time approach to quantify secretion and receptor binding of native chemokines in live cells to better understand their molecular interactions and function. CRISPR/Cas9 genome editing was used to tag the chemokine CXCL12 with the nanoluciferase fragment HiBiT. CXCL12 secretion was subsequently monitored and quantified by luminescence output. Binding of tagged CXCL12 to either chemokine receptors or membrane glycosaminoglycans could be monitored due to the steric constraints of nanoluciferase complementation. Furthermore, binding of native CXCL12-HiBiT to AlexaFluor488-tagged CXCR4 chemokine receptors could also be distinguished from glycosaminoglycan binding and pharmacologically analyzed using BRET. These live cell approaches combine the sensitivity of nanoluciferase with CRISPR/Cas9 genome editing to detect, quantify, and monitor binding of low levels of native secreted proteins in real time
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