Solubilization and display of G protein-coupled receptors on beads for real-time fluorescence and flow cytometric analysis (original) (raw)
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Molecular pharmacology, 2003
G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gi␣2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein.
Real-time Analysis of G Protein-coupled Receptor Reconstitution in a Solubilized System
Journal of Biological Chemistry, 2001
Receptor based signaling mechanisms are the primary source of cellular regulation. The superfamily of G protein-coupled receptors is the largest and most ubiquitous of the receptor mediated processes. We describe here the analysis in real-time of the assembly and disassembly of soluble G protein-coupled receptor-G protein complexes. A fluorometric method was utilized to determine the dissociation of a fluorescent ligand from the receptor solubilized in detergent. The ligand dissociation rate differs between a receptor coupled to a G protein and the receptor alone. By observing the sensitivity of the dissociation of a fluorescent ligand to the presence of guanine nucleotide, we have shown a time-and concentration-dependent reconstitution of the N-formyl peptide receptor with endogenous G proteins. Furthermore, after the clearing of endogenous G proteins, purified G␣ subunits premixed with bovine brain G␥ subunits were also able to reconstitute with the solubilized receptors. The solubilized N-formyl peptide receptor and G␣ i3 protein interacted with an affinity of ϳ10 ؊6 M with other ␣ subunits exhibiting lower affinities (G␣ i3 > G␣ i2 > G␣ i1 > > G␣ o). The N-formyl peptide receptor-G protein interactions were inhibited by peptides corresponding to the G␣ i C-terminal regions, by G␣ i mAbs, and by a truncated form of arrestin-3. This system should prove useful for the analysis of the specificity of receptor-G protein interactions, as well as for the elucidation and characterization of receptor molecular assemblies and signal transduction complexes.
Fluorescent ligands, antibodies, and proteins for the study of receptors
Pharmacology & Therapeutics, 2003
Fluorescent molecules bound to receptors can show their location and, if binding is reversible, can provide pharmacological information such as affinity and proximity between interacting molecules. The spatial precision offered by visualisation transcends the diverse localisation and low molecular concentration of receptor molecules. Consequently, the relationships between receptor location and function and life cycles of receptors have become better understood as a result of fluorescent labeling. Each of these aspects contributes new insights to drug action and potential new targets. The relationships between spatial distribution of receptor and function are largely unknown. This is particularly apparent for native receptors expressed in their normal host tissues where communication between heterogeneous cell types influences receptor distribution and function. In cultured cell systems, particularly for G-protein-coupled receptors (GPCR), fluorescencebased methods have enabled the visualisation of the cycle of agonist-stimulated receptor clustering, endocytic internalisation to the perinuclear region, degradation of the receptor-ligand complex, and recycling back to the surface membrane. Using variant forms of green fluorescent protein (GFP), antibodies, or fluorescent ligands, it is possible to detect or visualise the formation of oligomeric receptor complexes. Careful selection of fluorescent molecules based on their spectral properties enables resonance energy transfer and multilabel visualisation with colocalisation studies. Fluorescent agonist and antagonist ligands are now being used in parallel with GFP to study receptor cycling in live cells. This review covers how labeling and visualisation technologies have been applied to the study of major pharmacologically important receptors and illustrates this by giving examples of recent techniques that have relied on GFP, antibodies, or fluorescent ligands alone or in combination for the purpose of studying GPCR. D
ChemBioChem, 2014
Classical fluorescence-based approaches to monitor ligandprotein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells.
Fluorescence techniques: shedding light on ligand–receptor interactions
Trends in Pharmacological Sciences, 2000
The ability of organisms, or individual cells, to react to external chemical signals, which are detected and transduced by cell-surface receptors, is crucial for their survival. These receptors are the targets of the majority of clinically used medicines. Combinatorial genetics can provide almost unlimited numbers of mutant receptor proteins and combinatorial chemistry can produce large libraries of potential therapeutic compounds that act on these membrane receptors. What is missing for the fundamental understanding of receptor function and for the discovery of new medicines are efficient procedures to screen both ligand-receptor interactions and the subsequent functional consequences. Ultrasensitive fluorescence spectroscopic approaches, in combination with efficient labelling protocols, offer enormous possibilities for highly parallel functional bioanalytics at the micro-and nanometer level.
Bioscience Reports, 2015
G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A 2A receptor (A 2A R)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A 2A R-SMALP , generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5 • C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A 2A R controls. The A 2A R-SMALP was also stable when stored for prolonged periods at 4 • C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR-SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([ 3 H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.
Fluorescence Analysis of Receptor−G Protein Interactions in Cell Membranes †
Biochemistry, 2002
The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluoresceinlabeled myristoylated GR i1 (F-R i1 ) was used as the ligand bound to G γ in competition binding studies with differently modified GR subunit isoforms. In detergent solutions, the binding affinity of GR i to γ was 2 orders of magnitude higher than for GR o and GR s (IC 50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in R 2A AR receptor expressing CHO cell membranes into which purified γ subunits and F-R i1 were reconstituted. These cell membrane studies led to the following observations: (1) binding of R subunit to the γ was not enhanced by receptor in the presence or absence of agonist, indicating that γ contributed essentially all of the binding energy for R i1 interaction with the membrane; (2) activation of the receptor facilitated GTPγS-stimulated detachment of F-R i1 from γ and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.
Fluorescent ligands to investigate GPCR binding properties and oligomerization
Biochemical Society Transactions, 2013
Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands tur...
Methods, 2002
Green fluorescent protein (GFP)-centered fluorescence resonance energy transfer (FRET) relies on a distance-dependent transfer of energy from a donor fluorophore to an acceptor fluorophore and can be used to examine protein interactions in living cells. Here we describe a method to monitor the association and disassociation of heterotrimeric GTP-binding (G-proteins) from one another before and after stimulation of coupled receptors in living Dictyostelium discoideum cells. The Ga 2 and Gbc proteins were tagged with cyan and yellow fluorescent proteins and used to observe the state of the G-protein heterotrimer. Data from emission spectra were used to detect the FRET fluorescence and to determine kinetics and dose-response curves of bound ligand and analogs. Extending G-protein FRET to mammalian G-proteins should enable direct in situ mechanistic studies and applications such as drug screening and identifying ligands of new G-protein-coupled receptors.
Analytical biochemistry, 2016
G protein-coupled receptors (GPCRs) mediate many important physiological functions and are considered as one of the most successful therapeutic target classes for a wide spectrum of diseases. Drug discovery projects generally benefit from a broad range of experimental approaches for screening compound libraries and for the characterization of binding modes of drug candidates. Owing to the difficulties in solubilizing and purifying GPCRs, assay formats have been so far mainly limited to cell-based functional assays and radioligand binding assays. In this study, we used fluorescence cross-correlation spectroscopy (FCCS) to analyze the interaction of detergent-solubilized receptors to various types of GPCR ligands: endogenous peptides, small molecules, and a large surrogate antagonist represented by a blocking monoclonal antibody. Our work demonstrates the suitability of the homogeneous and time-resolved FCCS assay format for a robust, high-throughput determination of receptor-ligand b...