- Open Access
Illuminating the life of GPCRs
© Böhme and Beck-Sickinger; licensee BioMed Central Ltd. 2009
- Received: 08 May 2009
- Accepted: 14 July 2009
- Published: 14 July 2009
The investigation of biological systems highly depends on the possibilities that allow scientists to visualize and quantify biomolecules and their related activities in real-time and non-invasively. G-protein coupled receptors represent a family of very dynamic and highly regulated transmembrane proteins that are involved in various important physiological processes. Since their localization is not confined to the cell surface they have been a very attractive "moving target" and the understanding of their intracellular pathways as well as the identified protein-protein-interactions has had implications for therapeutic interventions. Recent and ongoing advances in both the establishment of a variety of labeling methods and the improvement of measuring and analyzing instrumentation, have made fluorescence techniques to an indispensable tool for GPCR imaging. The illumination of their complex life cycle, which includes receptor biosynthesis, membrane targeting, ligand binding, signaling, internalization, recycling and degradation, will provide new insights into the relationship between spatial receptor distribution and function. This review covers the existing technologies to track GPCRs in living cells. Fluorescent ligands, antibodies, auto-fluorescent proteins as well as the evolving technologies for chemical labeling with peptide- and protein-tags are described and their major applications concerning the GPCR life cycle are presented.
- Green Fluorescent Protein
- Fluorescence Correlation Spectroscopy
- Affibody Molecule
- Enhanced Yellow Fluorescent Protein
- Chemical Label
G-protein coupled receptors (GPCRs) are integral membrane proteins, consisting of a single polypeptide chain with seven transmembrane domains (TMD). They control and influence a diversity of physiological functions by mediating the signal of a wide variety of stimuli such as peptide hormones, neurotransmitters, neuropeptides, autocrine factors and even photons. Thereby the ligand transmits its activity to an intracellular signal through activation of a heterotrimeric guanosine triphosphate-binding protein (G-protein) by the receptor. As a result, a broad range of downstream intracellular signals are activated, leading to both short-term effects (e.g. changes in intracellular calcium levels) and long-term effects (e.g. gene transcription). Representing the largest family of transmembrane signaling molecules in the human genome, GPCRs are a very important class of therapeutic targets for the pharmaceutical industry and nearly half of the drugs currently in use act on these biomolecules.
In addition to the binding of ligands and G-proteins, GPCRs interact with a broad range of other proteins with potential roles specifically in receptor biosynthesis, distribution, signaling, desensitization, clustering, internalization, trafficking and degradation. These include other GPCRs, GPCR kinases (GRKs), second-messenger-dependent kinases, arrestin molecules, molecular chaperones, receptor-activity-modifying proteins (RAMPs) and PDZ-domain-containing proteins . For an excellent review that summarizes these activities including schematic figures see . The relationship between agonist-induced activation of receptors, receptor translocation and cell function has previously been shown to be difficult to investigate because it is a dynamic process and localization of receptors by standard biochemical methods offers insufficient high-resolution spatial information. In addition, the expression levels of GPCRs are generally low in native systems, which make the detection even more difficult. But elucidating these interactions will help to understand their cellular functions in order to develop new and improved pharmaceuticals. Since there is evidence that several peptide hormone receptors are over-expressed in various human cancer cells it has been a challenge to develop regulatory, receptor-binding peptides as agents for tumor diagnosis and therapy. However receptor-mediated internalization is a prerequisite for this type of study . Therefore novel methods to study receptor localization and function are needed as well as the extension of techniques to visualize and quantify involved biomolecules and processes with a spatiotemporal high-resolution and sensitivity .
Classically, receptors have been studied using radioactive isotopes, enzyme-linked immunosorbent assays (ELISAs) or functional responses in isolated tissue or organ preparations. The disadvantages of these methods, such as radioactive hazards and the limitations of studying the molecular dynamics of receptor activation have hindered advancements in receptor research. Biochemical methods for the investigation of protein-protein-interactions, such as co-immunoprecipitation assays, Western-blot analysis, "pulldown" approaches or yeast two-hybrid experiments have several drawbacks, e.g. artifacts owing to harsh techniques that are required to isolate membrane proteins, failure to identify components of a protein complex or false-positive as well as false-negative results. Therefore, non-invasive, real-time imaging methods applied to living cells have become very important in cell biology.
Fluorescence techniques that allow imaging of reporter gene expression, protein trafficking and monitoring of many dynamic biochemical signals have become feasible through the development of novel fluorophores as well as through the improvement of fluorescence instrumentation and advanced data analysis methods . They are considered superior over other existing molecular detection technologies because of their enhanced sensitivity, minimal perturbation, multiplicity of measurable parameters and suitable time scales. This allows the analysis of several biologically relevant molecular processes . Fluorescence is the most sensitive spectroscopic method. Reproducible signals from samples containing less than 1 nM concentrations of some fluorophores can be quantified. The signal can be analyzed by different methods, including its intensity, lifetime, energy (wavelength) and rotational freedom (polarization or anisotropy), to reveal different aspects of a structure, interaction, mechanism or process [7, 8]. Furthermore, fluorescence is a non-destructive phenomenon, so any signal change can be monitored as a function of time to determine its kinetics. Of course it has to be considered that some of the fluorophores might be toxic in certain systems, either themselves or by generating free radicals.
Techniques such as scanning confocal microscopy (SCM) and fluorescence correlation spectroscopy (FCS) have offered the establishment of assays at the single cell  and the single molecule level . Confocal and multiphoton microscopes coupled with sophisticated image analysis software packages are becoming affordable. The development of very high resolution, high sensitivity cameras and 3D deconvolution methods advances the area of quantitative 4D imaging . Microscopy methods such as total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) can be used for selective imaging of the plasma membrane of cells, e.g. to study exo- and endocytic pathways or plasma membrane dynamics during internalization [12–14].
Fluorescent labeling reagents are an essential component of a huge industry built on sensitive fluorescence detection and reagents with close to maximum theoretical brightness are available in many different colors. Hundreds of small organic dyes for covalent labeling of macromolecules have been developed and industrially optimized in their wavelength range, brightness, photo-stability and reduced in self-quenching. Strategies have included extension of double-bond conjugation, rigidification through extra rings and decoration with electron-withdrawing or obligatorily charged substituents such as fluorines or sulfonates [15, 16]. Labeling of proteins with fluorescent probes or affinity reagents has facilitated in vitro studies of protein structure, dynamics and protein-protein-interactions. However, traditional methods of protein labeling are often inadequate for in vivo studies, because they require purification of the protein, chemical labeling, re-purification and re-introduction into cells by invasive methods such as micro-injection or electroporation. These limitations have spawned efforts to label proteins in living cells or tissues non-invasively.
Appropriate methods for the study of receptor trafficking and regulation in native systems have not been available up to now. The limited axial (z) resolution of fluorescence and confocal-based microscopy impedes the imaging of receptors in individual cells deep within living tissues. The recent development and availability of fluorescent antibodies, fluorescent ligands and recombinant DNA technologies to label GPCRs in living cells provide new insights into their "real life" and "fate". Studies with GPCRs in living dissociated hepatocytes and vascular smooth muscle cells revealed quantitative data on receptor localization and translocation, which highly correlated with results obtained with fluorescent ligands or heterologous expression systems .
Subcellular distribution patterns have become an essential component of GPCR characterization that might have multiple regulatory consequences. For example, intracellular receptor pools that are rapidly transported to the cell membrane upon activation have been suggested to reduce desensitization and/or potentiate signaling. Many receptor subtypes have been shown to differ in their subcellular localization within the same cell type and particular receptors might localize differently depending on the cell type in which they are expressed. Unraveling these trafficking pathways and heterologous interactions by live imaging methods is strongly supported by the existence of various markers for intracellular compartments and pathways, as well as by inclusion of inhibitors for these processes, to fully understand the complex network .
Methods to label and visualize GPCRs
Immunohistochemistry (IHC) is based on an antigen-antibody reaction. In the case of GPCR labeling the antigen is the receptor protein or a certain epitope tag and the antibody is a glycoprotein targeting a particular recognition sequence. In most cases the protein of interest (POI) is labeled with a primary antibody followed by amplification with a secondary antibody that is conjugated to a small organic dye or an enzyme. Alternatively, primary antibodies can also be directly conjugated to fluorophores or enzymes [18, 19]. This is especially useful when antibodies are injected into living cells or when the increase of spectral diversity is required to analyze multiple proteins.
The availability of specific and potent antibody reagents is essential to obtain reliable and interpretable results in IHC studies. Accordingly, it has to be paid attention whether antibodies recognize the naturally folded protein as well. Antibodies against receptors can be generated in animals, e.g. rabbits, chickens or mice, through immunization with cells bearing receptors at their surface, injection of affinity-purified receptor or immunization with synthetic peptides derived from the nucleotide sequence of receptor genes . In contrast to polyclonal antibodies (PAbs), monoclonal antibodies (MAbs) are directed against a single epitope of an antigen, which makes them extremely selective. The established phage display technology provides a more effective tool for their generation compared to hybridoma technology or immunization .
Important epitope tags used on GPCRs for IF studies
peptide from human influenza hemagglutinin protein
major capsid protein of the T7 phage
human c-myc gene protein
His6 or His10
vesicular stomatitis virus glycoprotein
Auto-fluorescent proteins (AFPs)
The discovery and isolation of the green fluorescent protein (GFP) from the light-emitting organ of the jellyfish Aequorea victoria in 1962 and the gene cloning of the complementary DNA (cDNA) initiated the broad use of fluorescence imaging in cell biology [44, 45]. GFP is a 238 amino acid protein that consists of 11 anti-parallel β-strands surrounding a central α-helix to form a barrel-such as β-can structure . Its natural function is to convert the blue chemiluminescence of the Ca2+-sensitive photo-protein aequorin into green light . The tripeptide chromophore is localized in the centre of the protein and therefore well protected from the environment. The main reason for the success of GFP is its own nature: it is auto-fluorescent and the chromophore is auto-catalytically generated. Thus GFP does not require any additional substrates or co-factors and the fluorescence is not species specific.
The heterologous expression of GFP allows its application as a reporter molecule or the genetic labeling of biomolecules and therefore their direct visualization in vivo . Standard genetic-engineering allows the covalent labeling of proteins, subcellular compartments, cells of interest and specific tissue regions by using the protein expression system of the cell. Transfection and transgenic techniques enable the delivery of exogenous DNA more easily than the delivery of dyes to cells and even to whole organisms . However, early variants of GFP were frequently misfolded and led to the aggregation of the fusions. In addition, GFP is a full-sized protein and therefore its fusion may interfere with the expression, function and activity of the protein of interest [50, 51]. Creating a successful fusion protein requires the maintenance of the fluorescence of GFP, the functionality of the protein of interest (POI) and the integrity of the chimeric protein. This can be highly dependent on the length and sequence of the linker between GFP and the POI and should be taken into account and optimized for each specific application. To avoid difficulties in protein folding, mostly N- and C-terminal fusions are generated, but the cDNA of the fluorophore can also be integrated into the DNA sequence of many biomolecules . However, GFP turned out to be a rather inert molecule which in most cases did not affect the functional integrity of its fusion partner, which might be explained by its compact molecular structure.
Most important monomeric GFP variants and their spectral properties
[% of EGFP]
[t1/2 in s]
Blue fluorescent proteins
Cyan fluorescent proteins
Green fluorescent proteins
BD Biosciences Clontech
Yellow fluorescent proteins
Orange and red fluorescent proteins
, MBL Intl.
BD Biosciences Clontech
The development of advanced auto-fluorescent proteins, such as photo-activatable, photo-switchable and photo-convertible AFPs , fluorescent sensors , timer [71, 72] and split AFPs  provides novel applications for fluorescent labeling in vivo for studying the biosynthesis, expression, localization, movement, activity and turn-over of proteins as well as the direct measurement of cellular parameters and organelle functions at the single cell and even down to the single molecule level .
With the development of resonance energy transfer (RET) techniques such as FRET [75, 76], bioluminescence resonance energy transfer (BRET)  and the bimolecular fluorescence complementation (BiFC) [78, 79] approach the opportunity arose to gain insights into protein-protein-interactions in living cells by using the appropriate pairs of autofluorescence proteins.
Peptide- and protein-tags
Important peptide- and protein-tags for fluorescently chemical labeling applied on living cells
tetracysteine motif: CCXXCC
size: 6–20 aa
biarsenical dyes, e.g.:
non-fluorescent until binding
oligo-histidine sequence: (H)n, n = 6
size: 6–10 aa
nickel-nitrilotriacetic acid (NTA-Ni2+) dyes, e.g.:
mutated human O6-alkylguanine alkyltransferase (hAGT)
size: 182 aa
fluorescent benzylguanine (BG) derivatives, e.g.:
cell-permeable or cell-impermeable
acyl carrier/peptide carrier protein tag labeled via phosphopatheine transferase (PPTase)
size: 80 aa
fluorescent coenzyme A conjugates, e.g.:
mutated prokaryotic dehalogenase
size: 297 aa
fluorescent haloalkane ligand, e.g.:
HaloTag 488 ligand (green)
HaloTag TMR ligand (red)
cell-permeable or cell-impermeable
biotin acceptor peptide labeled via E. coli biotin ligase BirA
size: 15 aa
fluorescent streptavidin, e.g.:
Concerning the binding mechanism most labeling techniques can be classified into two major categories: affinity labeling and enzymatic labeling. Affinity labeling is based on a non-covalent chelation and provides a simple and highly selective labeling procedure that is applicable to various sites within the POI. On the other hand, irreversible covalent labelling by enzyme-catalyzed labeling methods still is more suited for the clear analysis of the POI, since these methods provide a higher stability. Therefore non-enzymatic covalent labeling is highly attractive since there is no need for a large enzyme or a protein domain and many labeling reagents as well as reaction conditions are well suited to this strategy.
In most cases self-labeling tags are smaller in size than AFPs and can be post-translationally labeled with a variety of synthetic fluorescent probes. These can provide alternative spectroscopic properties compared to AFPs . They can be advantageous in applications, if the size of the tag is important  or if the conditions are not suited for AFPs, such as anaerobic environments . Self-labeling tags are also a promising addition to the tools available for the immobilization and purification of proteins via affinity chromatography and can be useful in microarrays or on beads for pulldown assays.
The opportunity to control the localization and the time point of labeling enables scientists to study protein function in time and space inside a living cell at the molecular level. The choice of using different dyes at various time points during the experiment in cells, when protein translation is not inhibited, will lead to distinct populations of otherwise identical proteins whose discriminating features are determined through the time point of the respective labeling of each population. Such pulse-chase-experiments will reveal further insights into protein function and localization.
Fluorescent dyes to label receptor ligands
cover green wavelengths of the visible spectrum
contra: photo-bleaching, pH sensitivity
cover red wavelenghts of the visible spectrum
pro: pH insensitivity, photo-stability
contra: water solubility, non-specific binding, quenching
cover full spectrum from UV to IR
pro: water solubility, reduced quenching, diverse reactive groups
cover visible range
pro: sharp excitation and emission, high and insensitive quantum yields, pH insensitivity
BODIPY 558/568 pirenzepine
Alexa fluor dyes:
cover spectrum from visible range to IR
pro: pH insensitivity, photo-stability, increased brightness, diverse reactive groups
Alexa 532 adrenaline
Alexa 647 CXCL11
pro: broad absorption, narrow and tune-able emission, photo-stability, strong luminescence, long luminescent lifetimes, multivalent surface
contra: large size
There is a remarkable trend to develop and use labeling reagents that fluoresce at longer wavelengths, which allow the measurement of still more parameters within multi-color experiments and are suited for fluorescence imaging in vivo . The most significant advantage of these dyes is the reduced background fluorescence from cells, cell debris, buffer components and plastic materials. However, infrared fluorescent labels have a lower chemical and photo-stability. Semiconductor nanocrystals, called quantum dots (QDs), provide a great alternative to traditional dyes . They fluoresce throughout the visible and near-infrared spectrum and can be excited very efficiently with one excitation source. Since they possess narrow emission bands, up to 20 QD reagents could be detected separately with narrow band-pass filters. Therefore they will also promote the applications of fluorescently labeled peptide ligands in the future [107, 108].
Besides its pharmacology, the fluorescent ligand has chemical properties that will determine its behavior on and in a cell. Most drugs can be placed within a spectrum of lipophilicity to hydrophilicity. It is important to determine these properties and if they are altered by the addition of a distinct fluorescent molecule dependent on its position . Therefore preference should be given to fluorophores with high stability and fluorescent yield as well as resistance to photo-bleaching and which preserve the pharmacological properties of the ligand.
Many fluorescent ligands are peptides. The synthesis of fluorescent small-molecule ligands of GPCRs is not a trivial process. A potential site for fluorophore conjugation is in much closer proximity to the pharmacophore for small molecules and as a consequence, much more likely to affect ligand affinity and efficacy. A common approach includes the separation of the ligand and the fluorophore by some form of linker or spacer, which may vary in length and chemical nature as requested by the biological activity. Well established high-affinity ligands with excisting structure-activity relationship data can suggest whether a modification of a chemical site in the molecule might be tolerated. Positional scanning peptide combinatorial libraries can also be used to identify new fluorescent ligands  and procedures such as the Macro-model's large-scale low mode (LLMOD) enable the conformational profiling of fluorophore-modified peptides .
Fluorescence applications to study GPCRs
The described methodologies to label GPCRs provide the possibility to monitor the expression and cellular localization of these biomolecules. But visualization represents only the first step of a variety of colorful applications for unraveling biological processes. The investigation of GPCR functionality, clustering, trafficking, biosynthesis and degradation as well as the identification and visualization of important protein-protein-interactions, such as receptor oligomerization, ligand binding, G-protein coupling and arrestin recruitment in living cells has become feasible. Recent advances in fluorescence instrumentation not only allow qualitative but also quantitative data analysis and led to the ongoing development of high-throughput applications.
Expression and localization
Fluorescent antibodies and ligands as well as auto-fluorescent proteins and self-labeling tags are suited for monitoring receptor expression and cellular distribution. However, some considerations due to the difference in the labeling procedures have to be taken into account to receive reliable results.
Immunohistochemistry is a valuable method to localize GPCR expression and provides important information for defining receptor function and disease association. Fluorescent antibodies can be used to visualize the tissue and cellular distribution of receptors with a far greater lateral and axial resolution than offered by autoradiography [114, 115]. The fluorescence can be quantified by fluorescence activated cell sorting (FACS) analysis or by special microscopic software. A limitation of the immunofluorescence method is the low expression level of wild-type receptors in normal cells and native tissues. One early method, which was developed to overcome this problem, is the tyramide signal amplification (TSA) method . This is a sensitive immunodetection technique based on the peroxidase (POD) catalyzed deposition of labeled tyramide molecules. TSA in both direct (fluorophore conjugated) and indirect (biotin or dinitrophenol conjugated) variants has been used to amplify signals. It was furthermore successfully applied in double and triple labeling immunofluorescence confocal-based studies  and allows the use of low antibody concentrations to reduce background and non-specific binding.
Since receptors are highly mobile within the cell and the membrane, IHC generally requires fixation of cells and tissues. Therefore, this approach can be used to detect receptor localization at fixed time points however chemical fixation may negatively translocate them on the cellular level. It is very important to choose a fixative with relatively mild shrinkage effects on cells, as well as non-fluorescent properties to avoid cross-talk of signals between the chemical and the applied fluorophore(s). The fixation of cells could also negatively affect the conformation of the receptor antigen and thus the antibody binding. The conditions of fixation (temperature, time, pH) are furthermore critical for the accessibility of the antigenic epitope . Since the antibodies are not able to pass the cell membrane, IF is usually restricted to the extracellular receptor site. Only cell permeabilization enables the receptor detection at the intracellular receptor site or in subcellular compartments.
However, labeled antibodies provide the possibility to quantify the surface expression of receptors, because the antigenic epitope can also be detected via a cell surface ELISA . In this approach the secondary antibody is labeled with an enzyme, e.g. peroxidase (POD) or alkaline phosphatase (AP) that catalyzes a reaction, which results in a chromogene product that is easily measurable with a plate reader. This application is widely used to compare the surface expression of different receptor subtype mutants . Using permeabilized cells in a parallel approach the ratio between total and surface expressed receptors can be determined. Additionally, both populations can be visualized e.g. with species-specific primary and different fluorescent secondary antibodies .
Fluorophore-tagged GPCR ligands allow the direct non-radioactive visualization of their receptor target at the single-cell level, provided that the affinity and selectivity of the modified ligand is known not only at recombinant cells over-expressing a particular receptor but can also lead to the endogenous receptor. With respect to improved stability, detection of different receptor subtypes in cells and tissues and prevention of receptor internalization small non-peptidic fluorescent antagonists should be applied. It should be noted, that the choice of the fluorophore cannot only variably influence the host pharmacophore but also can lead to different fluorescence distribution patterns in receptor expressing cells . Since it is impossible to predict exactly how a fluorescent drug analogue will perform or distribute within a living cell careful pharmacological, chemical and biological validation of the fluo-ligand are required. Compounds that only show fluorescence when they are bound to the receptor offer low background fluorescence in the aqueous phase and thus a high signal to noise ratio. Novel methodologies for studying receptors in native cell tissue or recombinant receptors in cell culture were developed and validated  and used for the identification and localization of receptors in primary cultures of native cells [9, 124] and also in intact blood vessels [125, 126].
Sufficient washing steps or quenching procedures are required to receive clear images of GPCR expression and localization and to specifically detect the GPCR signal. This can lead to complications, such as influencing cell viability or to promote ligand dissociation. The commonly first and simplest method to monitor GPCR expression and localization is the genetic generation of GFP receptor fusions. Because GFP is covalently attached to the protein of interest potential problems with non-specific fluorescence can be avoided. GPCR-GFP fusions can also overcome problems of relatively low levels of receptor expression, the stoichiometry of the receptor and the fluorescent protein is well defined. These fusions offer enhanced sensitivity and resolution in comparison to standard antibody staining techniques and there is no distortion or alteration of membrane compartments, since there is no need for cell fixation, cell permeabilization or additional labeling steps . However, cells can be fixed for convenience, because GFP is chemically resistant. Additionally, GPCR-GFP fusions are more resistant to photo-bleaching than antibodies or ligands. They exhibit low background fluorescence and permit kinetic studies of protein localization and trafficking. This allows investigators to visualize proteins for a longer duration of time in an intact cellular environment than currently possible with the use of other extrinsic fluorescent probes. The real time expression in living cells can be easily detected and quantified by fluorescence microscopy, fluorescence spectroscopy, FACS analysis or fluorometric assays. Recent advances in instrumentation and image analysis have opened the door to high-throughput in vivo studies that can provide the morphological and temporal context for the biochemical pathways regulating cell function .
As mentioned above it is very important to check, whether the fluorophore-tagged receptor retains its natural properties, because it can be important to either label the receptor at its N- or C-terminus. Labeling can negatively influence the ligand binding pocket or the coupling of the G-protein in the signal transduction process. Thus, careful experimental comparisons to untagged receptors should be made whenever possible. For successful fluorescence detection enhanced and optimized monomeric GFP variants should be applied, since the formation of GFP oligomers can result in aggregation of the chimera and disturbance of the target protein function and localization.
Being fused to a GPCR the GFP fluorescence is expected to be well located within the cell membrane but possibly also to certain amounts in subcellular compartments, such as the endoplasmic reticulum (ER) or the Golgi apparatus (GA), because the chimeric protein in living cells reflects the normal turn-over of the GPCR. Thus, the surface expression of a given GPCR can be easily monitored but this might also depend on the cell system used, because the protein repertoire of the cell line might have an influence on the expression, localization and distribution of the GPCR-GFP chimera . Therefore, it is important to always combine visualization with functional studies, in order to identify the possible role of intracellularly located receptors and their relationship to surface membrane-located proteins. Appropriately membrane-located GPCRs can also be used to screen for mutants to identify regions within the receptor that are important for subcellular targeting .
Despite the rapid progress in visualization of GPCRs in living cells caused by auto-fluorescent proteins these illuminators have limitations. They are relatively bulky, therefore interference with protein structure, localization and function may occur. Genetically encoded peptide- and protein-tags for chemical labeling provide an alternative method to label and visualize receptors in living cells, thereby reducing the size of the receptor-fluorophore complex and expanding the spectral range to the near-infrared region.
However, there is great variation among these methods in terms of labeling specificity, velocity, stability, size of the tag, toxicity and versatility to probe structure and cell type and no single method yet succeeds in all these respects. Unspecific labeling within the cells has to be avoided for a specific detection. Therefore washing steps are indispensable to reduce possible background fluorescence. Systems providing dyes that are non-fluorescent before binding, e.g. the Lumio™-tag technology, or that perform a spectral change while binding to the tag positively influence the signal to noise ratio. Another important consideration for chemical labeling in living cells is the potential of the probe or targeting sequence to affect the cellular system via toxicity or more complicated interactions, which have to be ruled out by suitable controls and improved labeling protocols. The stability of the fluorescent complex is influenced by the possible dissociation of the fluorescent probe as well as the degradation of the complex. The labeling timescale will define the number of biological processes that can be studied, the faster the labeling, the more processes may be addressed. However, these self-labeling tags can be used to differentiate easily between intracellular and extracellular pools of the membrane localized proteins, e.g. GPCRs. For this application the tag has to be introduced at the N-terminal of the receptor site and a tag system has to be applied that provides both cell-permeable as well as cell-impermeable labeling dyes.
Cell surface dynamics and mobility
Increasing evidence favors the concept of membranes being organized into domains with defined lipid and protein compositions. These domains are believed to serve as platforms for trafficking, sorting, signalling and pathogen entry by concentrating certain selected lipids (e.g. cholesterol and sphingolipids) and proteins [131–133]. Resistance to solubilization by mild non-ionic detergents at low temperature represents an extensively used biochemical criterion to identify, isolate and characterize those membrane domains . Detection of proteins in detergent-resistant membranes is usually performed either by immunoblotting or ligand binding. However, these methods are not suitable in cases in which ligand binding is compromised in the presence of the detergent and/or is limited by the availability of antibodies with high specificity. GFP tagged membrane proteins are an alternative to directly determine detergent insolubility of GPCRs based on the fluorescence quantification of the membrane protein before and after detergent treatment . Specific lipid (DiIC16 and FAST DiI) and protein (transferrin receptor) markers have been utilized to validate this fluorescence approach. The method of analysis of detergent insolubility can be useful in exploring localization and organization of GPCRs in membranes and has the potential to be used in large-scale screening as well.
The activation of GPCRs leads to the recruitment and activation of heterotrimeric G-proteins and occurs at the plasma-membrane. The lateral mobility of the activated receptor on the cell surface represents an important prerequisite for the interaction with G-proteins and has a significant impact on the overall efficiency of the signal transduction process [136, 137]. To examine the cell surface dynamics of a GPCR in the plasma membrane the fluorescence recovery after photo-bleaching (FRAP) technique can be applied . This method involves the generation of a concentration gradient of fluorescent molecules by irreversibly photo-bleaching a fraction of fluorophores with a high intensity laser in a small area of the cell membrane. The recovery of fluorescence into the bleached region is used to measure the membrane diffusion characteristics of the GPCR, but also their changes in terms of ligand binding, activation as well as receptor oligomerization [139–141].
The application of confocal microscopy and multiple fluorophore-tagged ligands can provide the basis for time course studies of receptor cluster formation . The receptor mobility can be visualized by photo-dissociation of a fluorescent ligand – a process distinct from photo-bleaching. The ability of intense focused light to remove a fluorescent ligand from one site and hence allow the binding of a ligand molecule of another color enables the visualization of the movements of spatially restricted subpopulations of cell surface receptors .
Comparing FRAP and FCS, both methods can be used for different applications. Whereas FCS can only be used to monitor mobile receptors, FRAP also allows to follow immobile molecules. In contrast, only FCS can be used to provide information on a single molecule level.
Trafficking – internalization and recycling
In response to agonists GPCRs desensitize, aggregate on the cell surface and move from the plasma membrane into intracellular vesicles at different rates and to varying extents. Then, GPCRs are either recycled to the cell surface or degraded in lysosomes [1, 2].
Classically, receptor internalization after ligand exposure has been measured by radio-ligand binding and laborious physical fractionation techniques. This process can also be visualized and quantified by immunofluorescence studies. One approach is the measurement of the loss of surface receptors from the cell membrane as a result of internalization. In this case the receptors are not accessible to antibodies from outside the cell anymore. Receptor sequestration is then defined as the fraction of total cell surface receptors that, after ligand treatment, are removed from the cell surface . Otherwise it is also possible to detect the internalized receptors inside the cells in comparison to un-stimulated examples. Accordingly, the receptors have to be labeled with the antibodies prior or after ligand exposure. Since cell fixation and permeabilization are a prerequisite for receptor detection after ligand treatment, the labeling with antibodies within this application can not be performed in living cells. The receptor labeling before the stimulation provides the detection of receptors available on the cell surface at a certain time point but requires the ability of the ligand to bind to the bulky antibody-tagged receptor compared to the un-tagged one. The bound antibodies should not interfere with the ligand binding process and for that reason fluorescently labeled primary antibodies may facilitate this approach by a smaller antibody-receptor-complex. If there is no fluorophore-labeled primary antibody available, it will be a compromise to perform the receptor stimulation after labeling with the un-labeled antibody and thus visualize it with the labeled receptor after fixation and permeabilization.
Labeling of cell surface receptors with fluorescent antibodies at a specific time and the subsequent ligand exposure provides the basis for receptor recycling experiments in living cells by applying either fluorescence microscopy, FACS or ELISA. If the labeled GPCR recycles back to the cell surface after the removal of the ligand then these defined GPCRs will be detected and quantified in the cell membrane again, in contrast to only stimulated or degraded receptors . Receptor recycling can then be defined as the fraction of total cell surface receptors, after ligand treatment and clearance of stimulus, that are back at the cell surface and therefore accessible for antibodies [144–146]. But without receptor pre-labeling, also un-stimulated and membrane-destined, recruited and recycled receptors can be co-detected with the stimulated and recycled receptors as a separate second population.
N-terminally located self-labeling tags and their corresponding membrane-impermeable dyes can also serve as a tool for studying GPCR internalization and recycling, since only the labeled receptors are visualized prior to the ligand stimulation. In contrast to antibodies, the size of the GPCR-fluorophore complex is significantly reduced. However this approach also requires ligand binding to the pre-labeled receptor that is not interfered with by the label.
The use of GFP-tagged receptors enables a more simple and rapid assessment of GPCR trafficking, which is not possible by other techniques. The ligand can be applied to living cells and the movement of GPCRs can be visualized in real-time under varying conditions concerning incubation time, temperature as well as ligand concentration and specificity. The internalized GFP-labeled receptors are visualized as numerous punctuated spots in the perinuclear region of the cell. Algorithms have been developed that identify and collect information about these spots, allowing the quantification of the internalization process and the screening of ligand-induced receptor dynamics in whole cells. The obvious advantage to pharmacologists, in using GFP fusion proteins over antibodies is the ability to promote receptor sequestration by using both, agonists and inverse agonists . With the help of fluorescent markers for subcellular compartments, e.g. for endosomes (transferrin) or for lysosomes (dextran, LysoTracker), the GPCR can be directly located within the cell and its recycling back to the cell membrane and/or degradation can be easily detected. GFPs that are more sensitive to lower pH levels, such as the "ecliptic pHluorin" or the enhanced yellow fluorescent protein (EYFP), can be used a non-invasive pH indicator for intracellular organelles and cytoplasm, and for the quantification of GPCR trafficking, because the fluorescence is quenched as these protein chimeras enter lysosomes [148, 149].
Since only agonist ligands seem to promote receptor internalization, also fluorescent agonistic ligands are a valuable tool for the investigation of receptor trafficking, not only in living cell culture studies but also in living neuronal cells. The accumulation of fluorescent ligands at the perinuclear region appears to be a common feature of many GPCR-ligand complexes . To study the cellular receptor distribution after agonist exposure it is necessary to remove unspecifically bound molecules as completely as possible. Frequently applied methods include hypertonic acid stripping that removes surface-bound ligand while leaving the intracellularly bound sites for analysis . The fluorescent yield of some dyes was found to be pH sensitive, an effect that possibly is potentially helpful in internalization studies by indicating the presence of GPCRs in endosomal compartments with relatively high pH. The fate of both, receptor and ligand can be simultaneously determined and visualized when a fluorescent ligand and a fluorophore labeled receptor are used together [97, 129]. Studies have examined the trafficking of the ligand as well as of the receptor, which provides further important insights into the fate of the receptor-ligand complex as well as into cellular mechanisms such as the regulation of the GPCR signal via ligand degradation .
Of course the value of the fluorescent methods has critically to be compared with classical biochemical methods, e. g. co-immunoprecipitation and fractionation. A clear advantage of the fluorescent methods is their application on whole cells as well as the possibility to monitor the effects in a time-dependant manner. Limits, however, are due to cross-talk, channel bleed-through and limited resolution because membrane microdomains usually are below light resolution.
GPCR biosynthesis, folding and assembly take place in the endoplasmic reticulum (ER). When the receptors are correctly folded, they are packaged into ER-derived vesicles and migrate from the ER to the ER-Golgi intermediate complex (ERGIC), the Golgi apparatus and the trans-Golgi-network (TGN). During this transport process receptors undergo post-translational modifications (e.g. glycosylation) to ensure final migration to the cell surface . The export from the ER and the membrane targeting are highly regulated processes and the detailed mechanisms are not explicitly understood. These investigations will need further information on the age, expression, lifetime and movement of GPCRs also for the comparison of ligand-stimulated and un-stimulated GPCR-expressing cells. Therefore, it is indispensible to apply methodologies which can distinguish between old and newly synthesized receptors in living cells.
In order to discriminate between different populations of membrane located GPCRs pulse-chase studies can be performed with different fluorescent variants of the used antibody. This application on living cells can provide new insights into the receptor turnover, but is dependent on the applicability of primary fluorescent antibodies. In terms of investigating receptor biosynthesis after ligand stimulation again the appropriate ligand binding to the antibody-receptor complex is necessary. Intracellular receptors have not yet been delivered to the plasma membrane and receptors present at the plasma membrane have their first N-terminal epitope irreversibly cleaved by the enzyme .
Receptor-specific fluorescent ligands with high binding affinity and low off-rates have also been found to be suited for receptor pulse labeling . The low off-rate of the ligands ensures the stability of the ligand-receptor complex during the whole period of the experiment. Different chromophores attached at the same pharmacophore enable the consecutive application of spectroscopically distinguishable ligands to visualize the surface appearance and turnover of GPCRs.
The development of fluorescent protein variants opens up the possibility of performing "pulse-chase" experiments in living cells, by visualizing a distinct pool of the protein of interest in a defined region of the cell and following its transport and turn-over in real time. Important advances include the development of GFP-variants that can be activated by ultra violet (UV-) light, such as photo-activatable GFP (PA-GFP) , or those that can change their color from cyan to green , or from green to red  upon intense illumination with violet or UV-light. However, the complex photochemical processes underlying the phenomena of photo-activation and photo-conversion can also cause problems with normal fluorescent proteins leading to potential artifacts under certain circumstances. Intense illumination of EYFP with 514 nm laser light can lead to photo-conversion to a protein with ECFP-like fluorescence properties, which can be a problem in experiments that are based on bleaching of EYFP . Photo-bleaching that leads to photo-toxicity can be significantly reduced by specialized forms of confocal microscopy such as spinning-disk microscopy, in which the excitation light is guided through a series of small pinholes  or by excitation technologies that are applied in two-photon or multi-photon laser scanning microscopy .
However, the recently established self-labeling tags are and will be suited for pulse-chase studies more efficiently than all other fluorescence technologies. The free choice of specific time points for pulse labeling of the receptor and the sequential labeling of receptor subpopulations with spectrally distinguishable fluorophores provide a promising tool for the imaging of consecutively expressed GPCRs and their spatiotemporal organization .
Functionality – ligand binding and signal transduction
Since GPCRs are associated with diseases they are a very important target for the pharmaceutical industry. The ability to measure and quantify the binding of ligands to these receptors and the obtained responses has been, and remains, a key element of the drug discovery process. The most common way to study this include radioactively labeled drug molecules to label receptors directly on the cell surface or in membrane fragments from cells over-expressing the receptor of interest.
Because of high-throughput and high-content drug discovery assays with improved detection efficiencies, increased health, safety and disposal issues associated with the application of radio-ligands, there is a need to develop more robust fluorescence-based techniques and receptor-specific ligands with fluorescent properties. Fluorescent ligands have several advantages over traditional radio-ligand binding techniques  and studies with different neuropeptides comparing radio-ligand with fluorescent ligand binding revealed a higher resolution with fluo-peptides . The interest and use of fluorescent ligands is growing not only to reveal novel information on the life cycle of the receptors but also to develop receptor binding peptides, e.g. small molecular weight antagonists, for diagnosis as well as for therapy .
The application of flow cytometry can be used to characterize the specificity of fluorescent ligands . Within this method the applied laser beam is precisely directed towards the surfaces of the receptor-expressing cells, which are centered in the core of a thin sample stream. Therefore it is mainly cell-associated fluorescence that is detected and the signal is hardly impaired by free fluorescent ligand in solution thus allowing measurements of ligand binding under equilibrium conditions . Several innovative approaches in flow cytometry to investigate GPCRs have been described. The determination of binding and/or functional data with intact cells as well as the potential of flow cytometric techniques in high-throughput screening will further advance and accelerate the drug discovery process through experimental setups to gain equilibrium binding, selectivity data and the functional activity of GPCR ligands in one single step .
Cell-based FCS measurements with fluorescent ligands can be applied to determine the properties of ligand-receptor complexes within small areas of the cell membrane by measuring the fluctuations of fluorescence intensities and employing mathematical correlation algorithms . Since FCS can determine the diffusion rate of a tagged receptor directly and yield quantitative information about its membrane environment it can provide important insights into subcellular quantitative GPCR pharmacology .
Instead of the direct measurement of the fluorescence of the bound or free fluorescent ligand after separation, fluorescence polarization has been identified as a useful method to follow receptor bound ligands even in high throughput assays. As some of the organic fluorescent molecules are sensitive to their surrounding the measurement of the anisotropy or polarization may depend on whether the ligand is bound to the receptor or free. This method has been used to investigate ligand binding of different GPCRs, including vasopressin, melanocortin, neurotensin and opioid receptors .
Ligands carrying two fluorophores with spectral characteristics, that are well-suited for FRET measurements, can provide further insights into the bioactive conformation versus the conformation in solution of the ligand by changes in intramolecular FRET due to distance change between the fluorescently labeled residues .
Because GPCRs constitute excellent putative therapeutic targets, identification of their endogenous ligands has a great potential for drug discovery. The expression of GFP-tagged GPCRs followed by incubation of the transfected cells with fractions purified from tissue extracts and imaging of ligand-induced receptor internalization has become very important in functional characterization of orphan receptors. The GFP-based internalization assay provides a highly specific quantitative cytosensor technique with sensitivity in the nanomolar range to identify natural and synthetic ligands for GPCRs. Additionally, further improvements in GPCR antibody technology led to conformation state-sensitive antibodies that can also be useful for the identification of molecules with therapeutic interest .
The activation of GPCRs is traditionally measured either by membrane-based biochemical assays or by monitoring downstream physiological events. Fluorescence spectrophotometers are widely used for the quantification of photometric and fluorometric cell-based assays, e.g. for the investigation of second messenger systems as an indirect way of assessing receptor function.
Since these methods are not suited for detailed kinetic or spatial analysis of receptor activation and signaling, several optical techniques have been developed to monitor receptor activation continuously and in real-time. These provide new insights in both the mechanistic basis of the signaling process and the kinetic and spatial properties of GPCR-mediated signals . Fluorescent or luminescent labeled ligands, receptors and G-protein subunits, in combination with the development of FRET and BRET approaches, has allowed the determination of kinetic parameters for many steps of the signaling process, including ligand binding [171, 172], receptor activation [173, 174], receptor-G-protein interaction [175, 176], G-protein activation  and effector activation . Moreover, sensors have also been developed and further optimized to measure second messenger molecules such as phosphatidylinositol-3,4,5-trisphosphate (PIP3) , cyclic adenosine monophosphate (cAMP) [180, 181] or cyclic guanosine monophosphate (cGMP) [182, 183]. FRET sensors are usually fusion proteins of ECFP and EYFP or EGFP and a monomeric red fluorescent protein linked by a sensory domain. This domain is responsive to changes in distinct cellular parameters by a conformational change, leading to a change of the FRET signal. A variety of enzymatic or biological activities can be determined by the appropriate choice of the sensory domain and are also relevant for studying intracellular processes, as well as processes at the cell surface. Despite the development of FRET based sensors, which measure changes between two GFP variants to assess second messengers, sensors have also been developed which measure changes in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP), e.g. for Ca2+ sensing .
When GPCRs bind agonists, they are thought to change into an active conformation, which in turn binds to and activates G-proteins. To study this receptor activation by FRET fluorescent probes can be inserted in the third intracellular loop and the C-terminus, respectively. In addition to the most common ECFP/EYFP receptor sensors an ECFP/FlAsH sensor was recently developed . Although labeling with FlAsH requires an extra step and the resultant receptor sensor bleaches more rapidly, similar results have been obtained and the ECFP/FlAsH sensor has the advantage of leaving the receptor more intact with respect to its ability to signal to G-proteins.
Further alternatives to assess GPCR activation are approaches based on protein complementation . In this field enzyme fragment complementation (EFC) assays notably have the advantage that the signal is generated catalytically, and thus the assay can exhibit high sensitivity. Enzyme reporter proteins such as β-galactosidase, dihydrofolate reductase (DHFR) or lactamase have been utilized, all of which can turn over chromogenic or fluorogenic substrates. Luminescent signals can be generated, either with luciferase or β-galactosidase as an enzyme reporter. However, as split enzyme-based reporters require substrate incubation, these assays often need optimization with respect to concentration and incubation time to exclude the background signal caused by the substrate. Alternatively, complementation assays with split fluorescent proteins with direct read-outs, such as microscopy or scanning spectroscopy instruments, were developed for the detection of rapid interactions without interfering background signals .
An important aim in cell biology has been to identify and to observe dynamic interactions between protein molecules, as they execute the reactions of a particular biochemical pathway. Concerning GPCRs, despite elucidating ligand binding and signaling, the knowledge and investigation of conformational changes and further occurring protein-protein-interactions as well as their meaning for the life cycle of GPCRs and changes in cellular responses is of utmost interest. Frequently applied biochemical methods for the investigation of protein-protein-interactions are immunoprecipitation, photo-affinity labeling, cross-linking, size-separation chromatography and Western blot analysis. All these methods include cell lysis and are not able to follow protein-protein interaction in a living cell. The development of fluorophores and imaging techniques, such as FRET, BRET and BiFC, provide experimental alternatives to these denaturing techniques (e. g. immunoprecipitation). Fluorescence methods allow the localization of specific biomolecules in a time dependant manner, and accordingly also the protein-protein interaction processes. Accordingly, these techniques will help to find further answers to controversially discussed issues such as the role of interacting proteins in signal transduction cascades and their temporarily or permanent contacts.
Different FRET techniques and fusions with different spectral characteristics have been described and used for the study of protein interactions. Many modified versions of FRET were developed to suit individual needs, e.g. for large-scale quantitative analysis by flow cytometry [186, 187]. FRET can be measured in different ways. The sensitized emission method is a spectroscopic approach in which the sample of interest is excited at the wavelength of the donor and thus the increase of the acceptor fluorescence is quantified. Applying this technique requires additional measurements of samples that only contain the donor or acceptor, respectively, for correct data evaluation. Acceptor photo-bleaching FRET using confocal microscopy has become widely employed and has the advantage to localize occurring FRET events at the subcellular level . In this approach the reduced fluorescence intensity of the donor in the presence of the acceptor within a FRET is focused. To demonstrate interactions images before and after complete photo-bleaching of the acceptor are obtained. When the donor image is brighter following the acceptor photo-bleaching, then FRET can be assumed to have taken place. Within BRET approaches microscopy is rarely applied, the majority of studies utilize plate-reading instrumentation . Besides the possibility to perform single cell BRET assays , there are many technical limitations because of the reduced intensity of light released when luciferase oxidizes its substrate. Thus the taking place, but not the locations of energy transfer can be investigated. However, advantages of the BRET technique include the independence of a light source to initiate the energy transfer and the lack of photo-bleaching. In contrast, the BiFC approach allows spectroscopic as well as microscopic examinations. The complementary parts of the different GFP variants, when brought in close vicinity, produce BiFC with unique spectral properties. Thus the technique allows detection of multiple as well as competing interactions in vivo. By combining BiFC with either FRET or BRET, it is theoretically possible to demonstrate the simultaneous interaction of three or more interacting partners. Recently, it was demonstrated that Renilla luciferase (RLuc) [191, 192] can be used as a split enzyme. Accordingly BRET experiments also might be possible now to study the assembly of multiple proteins into a complex.
Comparing the three methods, which one is the most powerful? In fact, all three have advantages and disadvantages and specific applications they could be used for best. FRET and BRET are powerful techniques for real time experiments, with reversible energy transfer but limited dynamic range and the requirement of a complex multiwave lengths analysis that is prone to artefacts. In contrast, BiFC studies are no real time experiments, very sensitive and easy to measure, but represent the end-point as the fluorscence formation is irreversible. Localisation of the interaction is best studied with BiFC, whereas multiprotein dynamics are possible for competitive (multicolour BiFC) and cooperative (BiFC/BRET) interactions.
Through the application of these fluorescence based methods it is now widely accepted that the formation of homo- and/or heterodimers or higher order complexes is a universal aspect of GPCR biology  and could have important functional roles, e.g. in receptor maturation, function and trafficking [194, 195]. A lot of GPCR systems have already been examined by these non-invasive methods  and these investigations have supported our understanding of the functional significance of homo- and heterodimerization of GPCRs .
Despite the wide-spread application of GFP and its variants, the recent development of self-labeling tags provides further alternatives of donor and acceptor receptor fusions within FRET experiments. Recently the SNAP™-tag technology was successfully applied in time-resolved FRET measurements . The acyl carrier protein (ACP) labeling technique was used to simultaneously label the N-terminus of the neurokinin-1 receptor (NK1R) with Cy3 and Cy5 at different, but well-defined ratios and thus allowed FRET studies with high signal-to-noise ratios . Also fluorescent antibodies were successfully used to detect receptor oligomerization. The various existing epitopes allow not only co-expression and co-visualization of two differently tagged GPCRs in one cell, but also provide information about possible protein-protein-interactions. Because of the existence of different fluorescent dye pairs, e.g. fluorescein isothiocyanate (FITC, donor) and rhodamine (acceptor), which have an overlap in the donor emission and the acceptor excitation spectra, labeled antibodies have already been applied in FRET studies to investigate GPCR oligomerization . Fluorescent ligands are also used to study receptor subtype oligomerization by FRET. GPCR-dimers as GFP-fusions can be determined even when they still accumulate in the endosomal compartments. As receptor oligomerization is discussed in the context of receptor trafficking this is of major interest .
Receptor-mediated activation of a G-protein is an early event in signal transduction and is thought to be a result of a transient interaction between an agonist occupied receptor and its G-protein. However, also data from FRET and BRET studies provided evidence that GPCRs and G-proteins can form stable complexes [202, 203]. G-protein β and γ subunits bind to each other with high affinity, forming a heterodimeric complex, which is essential for the stability of these peptides. The stability of the heterotrimeric complex formed by the association of Gα and Gβγ is a controversial. The hypothesis that Gβ dissociates from Gβγ when heterotrimeric G-proteins are activated in vivo is generally accepted, however some experiments suggest that they do not. Both hypotheses have been based largely on data from in vitro experiments  and therefore FRET has been used to probe this interaction during signal transduction in vivo. However, the construction of a functional fluorescently tagged Gα subunit was not as simple as producing their tagged and functional counterparts. Although functional Gα fusion proteins have been prepared by inserting GFP variants into their α-helical domains [204, 205], FRET experiments in intact cells also led to controversial results [177, 178]. A decrease in FRET signal can be due to subunit dissociation, however a conformational change within the heterotrimeric complex can also produce the same result. The interpretation of changes in FRET can be even more complicated, assuming that each receptor monomer forms a complex with a G-protein. The resulting proximity of G-proteins could produce FRET between subunits of different G-proteins as well as between subunits within the same G-protein and changes in FRET may be due to conformational changes within an individual G-protein, between different G-proteins or a combination of both.
Prolonged agonist activation of a GPCR is followed by desensitization that occurs when G-protein coupled receptor kinases (GRKs) phosphorylate the agonist-occupied receptor and prevent further stimulation. This process facilitates the interaction of the receptor with the protein arrestin, resulting in internalization of the desensitized GPCR, further leading either to receptor down regulation or to resensitization and receptor recycling to the plasma membrane . BRET is widely used to detect the interaction of arrestin with a GPCR [207–209] and FRET is applied to follow the time course of interaction [173, 210]. BRET studies additionally probing the interaction between GPCRs and GRKs revealed that the time course for the interaction with arrestin lagged behind the interaction with GRK, which is consistent with the requirement, that GRK-catalyzed phosphorylation must precede arrestin binding .
It is proven that fluorescence techniques are powerful tools for investigation of the very dynamic family of GPCRs to understand their subcellular localisation and to further elucidate key elements in GPCR trafficking and interaction with other signal pathways.
However, to obtain physiologically relevant results, some considerations have to be made. First, it is of utmost importance that the investigated GPCR, ligand or interacting protein is not influenced in its functionality by the fluorescent modification. Therefore careful characterizations are needed and to exclude interferences it might be helpful to apply different labels at different sites of the protein for data evaluation . Additionally, the label should be as small as possible, the recent and ongoing development and optimization of self-labeling tags will be advantageous in this field. Using unnatural amino acid mutagenesis the site-specific incorporation of reactive keto groups, such as p-benzoyl-L-phenylalanine (Bzp) or p-acetyl-L-phenylalanine (Acp), into functional GPCRs and their ability to react with a variety of spectroscopic and other probes was previously described . Because of their excellent fluorescent properties quantum dots are very attractive for labeling, however the full potential of QDs for cellular imaging has not yet been realized because of problems with large QD size, QD multivalency and the difficulty of delivering QDs into the cytosol. Recently, monovalent and reduced-size quantum dots were generated and successfully applied for receptor imaging in living cells .
Fluorescent antibodies provide a powerful tool for examining the cellular distribution of GPCRs. However, quantification is highly depended on the accessibility of – in most cases – the small epitope by the large antibody. The challenge is to develop even more high-affinity fluorophore- or enzyme-conjugated primary antibodies for one-step labeling assays on living cells. The generation of bright and stable dyes as well as pH sensitive ones, such as CypHer 5 , will lead to further insights into the life of GPCRs and will enable high-throughput screening applications. A new group of molecules, called affibody molecules, is especially interesting for imaging applications because of their small size (7–15 kDa) compared to antibodies. These proteins are composed of a three-helix bundle of 58 amino acids and are derived from the scaffold of one of the IgG-binding domains of the staphylococcal protein A . The binding site is equivalent to an antibody with respect to the surface area. The size, the simple structure, the specific target recognition, the ease of production and the high stability give affibody molecules significant advantages over antibodies. These molecules can be labeled with fluorophores but also with radionuclides which make them promising candidates for GPCRs associated tumor diagnosis and therapy .
Recombinant DNA technologies have highly advanced fluorescence labeling as well as transfection and transgenic techniques that enable simple DNA delivery to cells that results in covalent labeling by using the protein expression system of the cell. However, expression levels in cell cultures may significantly differ from those in natural systems. Concerning the signaling and trafficking behavior of GPCRs the relationship between the occupation of the receptor by physiological levels of agonists and the initiation of translocation is an important issue. The general use of very high concentrations of agonist leaves open the possibility that the investigated processes are more pharmacological than physiological.
A major criticism of FRET/BRET studies used to investigate protein-protein-interactions, is that the required protein overexpression can result in RET attributed to a high incidence of random collisions, rather than direct protein-protein-interactions. If low expression levels can not be obtained by varying DNA amounts within transient cell transfections, stable cell transfections will provide an alternative, since there is a homogenous population of cells expressing the protein of interest at the same level. Another possibility is the baculovirus expression system which enables protein expression levels to be controlled more closely than with transient transfection, because protein expression can be titrated by adjusting the multiplicity of viral infection .
Since protein co-localization is the first prerequisite for interactions, this should be proven by fluorescence microscopy, and by using parallel labeling strategies to locate subcellularly the interaction of interest. For correct evaluation of FRET and BRET data appropriate controls have to be used to demonstrate the specificity of the interactions and to establish levels of RET considered to be background in any given experiment. The additional application of a biochemical approach might support the results. To validate the physiological role of the detected interaction studies in other, more natural cell systems, e.g. cell lines endogenously expressing one protein of interest, as well as investigations on tissues and animals will be indispensable in proving the relevance of the interactions in the future. For example, the in vivo co-expression of GPCRs has to be demonstrated in the same tissue, and ideally in the same cell for establishing the physiological relevance of receptor oligomerization. Functional cross-talk between the receptor signaling pathways as well as novel pharmacological and/or functional properties will provide evidence for the mechanism by which receptor-receptor-interactions modulate cellular activity .
An exciting application of GPCR-GFP chimeras involves their use in genetic screens in genetically tractable organisms such as yeast, e.g. to identify mutant yeast strains in which the receptor is mis-localized. Such strategies contribute greatly to the identification of new components involved in GPCR targeting and trafficking in additional model organisms . New approaches using whole organisms, in which the GFP-chimera can be expressed under the control of the endogenous promoter, e.g. invertebrates as C. elegans or mouse models, allow cell biological, molecular and biochemical results to be interpreted in a physiologically relevant context and to be compared to those observed in cultured cells [220, 221]. GFP and its variants as reporters represent the next step in mouse genome engineering technology by opening up the possibility of combinatorial non-invasive reporter usage within a single animal, e.g. for gene-expression, as well as for co-visualization and FRET assays .
In summary, many issues concerning the life of GPCRs can be addressed by fluorescence techniques, however many remain challenging. Further rapid advances in labeling and imaging technology can be expected and their parallel as well as their combined application will provide novel insights that will also broaden the range of new therapeutic interventions.
The financial contribution of the Deutsche Forschungsgemeinschaft (Grants BE1264/5-2 and SFB 610 TP A1) is kindly acknowledged.
- Brady AE, Limbird LE: G protein-coupled receptor interacting proteins: emerging roles in localization and signal transduction. Cell Signal. 2002, 14: 297-309.PubMedGoogle Scholar
- Luttrell LM, Lefkowitz RJ: The role of beta-arrestins in the termination and transduction of G-protein-coupled receptor signals. J Cell Sci. 2002, 115: 455-465.PubMedGoogle Scholar
- Reubi JC: Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev. 2003, 24: 389-427.PubMedGoogle Scholar
- Zhang J, Campbell RE, Ting AY, Tsien RY: Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol. 2002, 3: 906-918.PubMedGoogle Scholar
- Giepmans BN, Adams SR, Ellisman MH, Tsien RY: The fluorescent toolbox for assessing protein location and function. Science. 2006, 312: 217-224.PubMedGoogle Scholar
- Chattopadhyay A: Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach. Chem Phys Lipids. 2003, 122: 3-17.PubMedGoogle Scholar
- Ramachandran R, Tweten RK, Johnson AE: Membrane-dependent conformational changes initiate cholesterol-dependent cytolysin oligomerization and intersubunit beta-strand alignment. Nat Struct Mol Biol. 2004, 11: 697-705.PubMedGoogle Scholar
- Woolhead CA, McCormick PJ, Johnson AE: Nascent membrane and secretory proteins differ in FRET-detected folding far inside the ribosome and in their exposure to ribosomal proteins. Cell. 2004, 116: 725-736.PubMedGoogle Scholar
- Mackenzie JF, Daly CJ, Pediani JD, McGrath JC: Quantitative imaging in live human cells reveals intracellular alpha(1)-adrenoceptor ligand-binding sites. J Pharmacol Exp Ther. 2000, 294: 434-443.PubMedGoogle Scholar
- Haustein E, Schwille P: Single-molecule spectroscopic methods. Curr Opin Struct Biol. 2004, 14: 531-540.PubMedGoogle Scholar
- Jaiswal JK, Simon SM: Imaging single events at the cell membrane. Nat Chem Biol. 2007, 3: 92-98.PubMedGoogle Scholar
- Axelrod D: Total internal reflection fluorescence microscopy. Methods Cell Biol. 1989, 30: 245-270.PubMedGoogle Scholar
- Llobet A, Beaumont V, Lagnado L: Real-time measurement of exocytosis and endocytosis using interference of light. Neuron. 2003, 40: 1075-1086.PubMedGoogle Scholar
- Toomre D, Manstein DJ: Lighting up the cell surface with evanescent wave microscopy. Trends Cell Biol. 2001, 11: 298-303.PubMedGoogle Scholar
- Haugland RP: The Handbook – A Guide to Fluorescent Probes and Labeling Technologies. 2005, Eugene: Molecular ProbesGoogle Scholar
- Waggoner A: Fluorescent labels for proteomics and genomics. Curr Opin Chem Biol. 2006, 10: 62-66.PubMedGoogle Scholar
- Watson P, Jones AT, Stephens DJ: Intracellular trafficking pathways and drug delivery: fluorescence imaging of living and fixed cells. Adv Drug Deliv Rev. 2005, 57: 43-61.PubMedGoogle Scholar
- Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG: Molecular determinants underlying the formation of stable intracellular G protein-coupled receptor-beta-arrestin complexes after receptor endocytosis*. J Biol Chem. 2001, 276: 19452-19460.PubMedGoogle Scholar
- Kalatskaya I, Schussler S, Blaukat A, Muller-Esterl W, Jochum M, Proud D, Faussner A: Mutation of tyrosine in the conserved NPXXY sequence leads to constitutive phosphorylation and internalization, but not signaling, of the human B2 bradykinin receptor. J Biol Chem. 2004, 279: 31268-31276.PubMedGoogle Scholar
- Eckard CP, Beck-Sickinger AG: Characterisation of G-protein-coupled receptors by antibodies. Curr Med Chem. 2000, 7: 897-910.PubMedGoogle Scholar
- Brissette R, Goldstein NI: The use of phage display peptide libraries for basic and translational research. Methods Mol Biol. 2007, 383: 203-213.PubMedGoogle Scholar
- Marion S, Oakley RH, Kim KM, Caron MG, Barak LS: A beta-arrestin binding determinant common to the second intracellular loops of rhodopsin family G protein-coupled receptors. J Biol Chem. 2006, 281: 2932-2938.PubMedGoogle Scholar
- Kelley-Hickie LP, Kinsella BT: Homologous desensitization of signalling by the beta (beta) isoform of the human thromboxane A2 receptor. Biochim Biophys Acta. 2006, 1761: 1114-1131.PubMedGoogle Scholar
- Conner AC, Simms J, Conner MT, Wootten DL, Wheatley M, Poyner DR: Diverse functional motifs within the three intracellular loops of the CGRP1 receptor. Biochemistry. 2006, 45: 12976-12985.PubMedGoogle Scholar
- Holliday ND, Lam CW, Tough IR, Cox HM: Role of the C terminus in neuropeptide Y Y1 receptor desensitization and internalization. Mol Pharmacol. 2005, 67: 655-664.PubMedGoogle Scholar
- Matharu AL, Mundell SJ, Benovic JL, Kelly E: Rapid agonist-induced desensitization and internalization of the A(2B) adenosine receptor is mediated by a serine residue close to the COOH terminus. J Biol Chem. 2001, 276: 30199-30207.PubMedGoogle Scholar
- Holliday ND, Holst B, Rodionova EA, Schwartz TW, Cox HM: Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor. Mol Endocrinol. 2007, 21: 3100-3112.PubMedGoogle Scholar
- Estall JL, Yusta B, Drucker DJ: Lipid raft-dependent glucagon-like peptide-2 receptor trafficking occurs independently of agonist-induced desensitization. Mol Biol Cell. 2004, 15: 3673-3687.PubMed CentralPubMedGoogle Scholar
- Kim KM, Caron MG: Complementary roles of the DRY motif and C-terminus tail of GPCRS for G protein coupling and beta-arrestin interaction. Biochem Biophys Res Commun. 2008, 366: 42-47.PubMedGoogle Scholar
- Cescato R, Schulz S, Waser B, Eltschinger V, Rivier JE, Wester HJ, Culler M, Ginj M, Liu Q, Schonbrunn A, Reubi JC: Internalization of sst2, sst3, and sst5 receptors: effects of somatostatin agonists and antagonists. J Nucl Med. 2006, 47: 502-511.PubMedGoogle Scholar
- Johnson EA, Oldfield S, Braksator E, Gonzalez-Cuello A, Couch D, Hall KJ, Mundell SJ, Bailey CP, Kelly E, Henderson G: Agonist-selective mechanisms of mu-opioid receptor desensitization in human embryonic kidney 293 cells. Mol Pharmacol. 2006, 70: 676-685.PubMedGoogle Scholar
- Hamdan FF, Rochdi MD, Breton B, Fessart D, Michaud DE, Charest PG, Laporte SA, Bouvier M: Unraveling G protein-coupled receptor endocytosis pathways using real-time monitoring of agonist-promoted interaction between beta-arrestins and AP-2. J Biol Chem. 2007, 282: 29089-29100.PubMedGoogle Scholar
- Sklar LA, Vilven J, Lynam E, Neldon D, Bennett TA, Prossnitz E: Solubilization and display of G protein-coupled receptors on beads for real-time fluorescence and flow cytometric analysis. BioTechniques. 2000, 28: 976-980.PubMedGoogle Scholar
- Adie EJ, Francis MJ, Davies J, Smith L, Marenghi A, Hather C, Hadingham K, Michael NP, Milligan G, Game S: CypHer 5: a generic approach for measuring the activation and trafficking of G protein-coupled receptors in live cells. Assay Drug Dev Technol. 2003, 1: 251-259.PubMedGoogle Scholar
- Liu Q, Dewi DA, Liu W, Bee MS, Schonbrunn A: Distinct phosphorylation sites in the SST2A somatostatin receptor control internalization, desensitization, and arrestin binding. Mol Pharmacol. 2008, 73: 292-304.PubMedGoogle Scholar
- Attal H, Cohen-Hillel E, Meshel T, Wang JM, Gong W, Ben-Baruch A: Intracellular cross-talk between the GPCR CXCR1 and CXCR2: role of carboxyl terminus phosphorylation sites. Exp Cell Res. 2008, 314: 352-365.PubMedGoogle Scholar
- Nasser MW, Raghuwanshi SK, Malloy KM, Gangavarapu P, Shim JY, Rajarathnam K, Richardson RM: CXCR1 and CXCR2 activation and regulation. Role of aspartate 199 of the second extracellular loop of CXCR2 in CXCL8-mediated rapid receptor internalization. J Biol Chem. 2007, 282: 6906-6915.PubMedGoogle Scholar
- Huttenrauch F, Nitzki A, Lin FT, Honing S, Oppermann M: Beta-arrestin binding to CC chemokine receptor 5 requires multiple C-terminal receptor phosphorylation sites and involves a conserved Asp-Arg-Tyr sequence motif. J Biol Chem. 2002, 277: 30769-30777.PubMedGoogle Scholar
- Langer I, Langlet C, Robberecht P: Effect of inactivating mutations on phosphorylation and internalization of the human VPAC2 receptor. J Mol Endocrinol. 2005, 34: 405-414.PubMedGoogle Scholar
- Langlet C, Langer I, Vertongen P, Gaspard N, Vanderwinden JM, Robberecht P: Contribution of the carboxyl terminus of the VPAC1 receptor to agonist-induced receptor phosphorylation, internalization, and recycling. J Biol Chem. 2005, 280: 28034-28043.PubMedGoogle Scholar
- Pheng LH, Dumont Y, Fournier A, Chabot JG, Beaudet A, Quirion R: Agonist- and antagonist-induced sequestration/internalization of neuropeptide Y Y1 receptors in HEK293 cells. Br J Pharmacol. 2003, 139: 695-704.PubMed CentralPubMedGoogle Scholar
- Voisin T, Goumain M, Lorinet AM, Maoret JJ, Laburthe M: Functional and molecular properties of the human recombinant Y4 receptor: resistance to agonist-promoted desensitization. J Pharmacol Exp Ther. 2000, 292: 638-646.PubMedGoogle Scholar
- Hein L, Meinel L, Pratt RE, Dzau VJ, Kobilka BK: Intracellular trafficking of angiotensin II and its AT1 and AT2 receptors: evidence for selective sorting of receptor and ligand. Mol Endocrinol. 1997, 11: 1266-1277.PubMedGoogle Scholar
- Shimomura O, Johnson FH, Saiga Y: Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol. 1962, 59: 223-239.PubMedGoogle Scholar
- Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ: Primary structure of the Aequorea victoria green-fluorescent protein. Gene. 1992, 111: 229-233.PubMedGoogle Scholar
- Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC: Green fluorescent protein as a marker for gene expression. Science. 1994, 263: 802-805.PubMedGoogle Scholar
- Shimomura O: The discovery of aequorin and green fluorescent protein. J Microsc. 2005, 217: 1-15.PubMedGoogle Scholar
- Tsien RY: The green fluorescent protein. Annu Rev Biochem. 1998, 67: 509-544.PubMedGoogle Scholar
- Stewart CN Jr: Go with the glow: fluorescent proteins to light transgenic organisms. Trends Biotechnol. 2006, 24: 155-162.PubMedGoogle Scholar
- Meyer E, Fromherz P: Ca2+ activation of hSlo K+ channel is suppressed by N-terminal GFP tag. Eur J Neurosci. 1999, 11: 1105-1108.PubMedGoogle Scholar
- Lalonde S, Weise A, Walsh RP, Ward JM, Frommer WB: Fusion to GFP blocks intercellular trafficking of the sucrose transporter SUT1 leading to accumulation in companion cells. BMC Plant Biol. 2003, 3: 8-PubMed CentralPubMedGoogle Scholar
- Zimmer M: Green fluorescent protein (GFP): applications, structure, and related photophysical behavior. Chem Rev. 2002, 102: 759-781.PubMedGoogle Scholar
- Zapata-Hommer O, Griesbeck O: Efficiently folding and circularly permuted variants of the Sapphire mutant of GFP. BMC Biotechnol. 2003, 3: 5-PubMed CentralPubMedGoogle Scholar
- Zacharias DA, Violin JD, Newton AC, Tsien RY: Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science. 2002, 296: 913-916.PubMedGoogle Scholar
- Rizzo MA, Springer GH, Granada B, Piston DW: An improved cyan fluorescent protein variant useful for FRET. Nat Biotechnol. 2004, 22: 445-449.PubMedGoogle Scholar
- Nguyen AW, Daugherty PS: Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol. 2005, 23: 355-360.PubMedGoogle Scholar
- Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A: A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol. 2002, 20: 87-90.PubMedGoogle Scholar
- Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY: Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J Biol Chem. 2001, 276: 29188-29194.PubMedGoogle Scholar
- Karasawa S, Araki T, Nagai T, Mizuno H, Miyawaki A: Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer. Biochem J. 2004, 381: 307-312.PubMed CentralPubMedGoogle Scholar
- Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY: Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004, 22: 1567-1572.PubMedGoogle Scholar
- Wang L, Jackson WC, Steinbach PA, Tsien RY: Evolution of new nonantibody proteins via iterative somatic hypermutation. Proc Natl Acad Sci USA. 2004, 101: 16745-16749.PubMed CentralPubMedGoogle Scholar
- Arun KH, Kaul CL, Ramarao P: Green fluorescent proteins in receptor research: an emerging tool for drug discovery. J Pharmacol Toxicol Methods. 2005, 51: 1-23.PubMedGoogle Scholar
- Chudakov DM, Lukyanov S, Lukyanov KA: Fluorescent proteins as a toolkit for in vivo imaging. Trends Biotechnol. 2005, 23: 605-613.PubMedGoogle Scholar
- Matz MV, Labas YA, Ugalde J: Evolution of function and color in GFP-like proteins. Methods Biochem Anal. 2006, 47: 139-161.PubMedGoogle Scholar
- Pedelacq JD, Cabantous S, Tran T, Terwilliger TC, Waldo GS: Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol. 2006, 24: 79-88.PubMedGoogle Scholar
- Shaner NC, Steinbach PA, Tsien RY: A guide to choosing fluorescent proteins. Nat Methods. 2005, 2: 905-909.PubMedGoogle Scholar
- Hutter H: Five-colour in vivo imaging of neurons in Caenorhabditis elegans. J Microsc. 2004, 215: 213-218.PubMedGoogle Scholar
- Anderson KI, Sanderson J, Gerwig S, Peychl J: A new configuration of the Zeiss LSM 510 for simultaneous optical separation of green and red fluorescent protein pairs. Cytometry A. 2006, 69: 920-929.PubMedGoogle Scholar
- Muller-Taubenberger A, Anderson KI: Recent advances using green and red fluorescent protein variants. Appl Microbiol Biotechnol. 2007, 77: 1-12.PubMedGoogle Scholar
- Griesbeck O: Fluorescent proteins as sensors for cellular functions. Curr Opin Neurobiol. 2004, 14: 636-641.PubMedGoogle Scholar
- Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao X, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P: "Fluorescent timer": protein that changes color with time. Science. 2000, 290: 1585-1588.PubMedGoogle Scholar
- Wiegand UK, Duncan RR, Greaves J, Chow RH, Shipston MJ, Apps DK: Red, yellow, green go! – A novel tool for microscopic segregation of secretory vesicle pools according to their age. Biochem Soc Trans. 2003, 31: 851-856.PubMedGoogle Scholar
- Hu CD, Kerppola TK: Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis. Nat Biotechnol. 2003, 21: 539-545.PubMed CentralPubMedGoogle Scholar
- Willig KI, Kellner RR, Medda R, Hein B, Jakobs S, Hell SW: Nanoscale resolution in GFP-based microscopy. Nat Methods. 2006, 3: 721-723.PubMedGoogle Scholar
- Jares-Erijman EA, Jovin TM: FRET imaging. Nat Biotechnol. 2003, 21: 1387-1395.PubMedGoogle Scholar
- Wallrabe H, Periasamy A: Imaging protein molecules using FRET and FLIM microscopy. Curr Opin Biotechnol. 2005, 16: 19-27.PubMedGoogle Scholar
- Boute N, Jockers R, Issad T: The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol Sci. 2002, 23: 351-354.PubMedGoogle Scholar
- Kerppola TK: Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu Rev Biophys. 2008, 37: 465-487.PubMed CentralPubMedGoogle Scholar
- Kerppola TK: Visualization of molecular interactions by fluorescence complementation. Nat Rev Mol Cell Biol. 2006, 7: 449-456.PubMed CentralPubMedGoogle Scholar
- Bohme I, Morl K, Bamming D, Meyer C, Beck-Sickinger AG: Tracking of human Y receptors in living cells – a fluorescence approach. Peptides. 2007, 28: 226-234.PubMedGoogle Scholar
- Griffin BA, Adams SR, Tsien RY: Specific covalent labeling of recombinant protein molecules inside live cells. Science. 1998, 281: 269-272.PubMedGoogle Scholar
- Kapanidis AN, Ebright YW, Ebright RH: Site-specific incorporation of fluorescent probes into protein: hexahistidine-tag-mediated fluorescent labeling with (Ni(2+):nitrilotriacetic Acid (n)-fluorochrome conjugates. J Am Chem Soc. 2001, 123: 12123-12125.PubMedGoogle Scholar
- Guignet EG, Hovius R, Vogel H: Reversible site-selective labeling of membrane proteins in live cells. Nat Biotechnol. 2004, 22: 440-444.PubMedGoogle Scholar
- Keppler A, Gendreizig S, Gronemeyer T, Pick H, Vogel H, Johnsson K: A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat Biotechnol. 2003, 21: 86-89.PubMedGoogle Scholar
- Keppler A, Pick H, Arrivoli C, Vogel H, Johnsson K: Labeling of fusion proteins with synthetic fluorophores in live cells. Proc Natl Acad Sci USA. 2004, 101: 9955-9959.PubMed CentralPubMedGoogle Scholar
- George N, Pick H, Vogel H, Johnsson N, Johnsson K: Specific labeling of cell surface proteins with chemically diverse compounds. J Am Chem Soc. 2004, 126: 8896-8897.PubMedGoogle Scholar
- Jacquier V, Prummer M, Segura JM, Pick H, Vogel H: Visualizing odorant receptor trafficking in living cells down to the single-molecule level. Proc Natl Acad Sci USA. 2006, 103: 14325-14330.PubMed CentralPubMedGoogle Scholar
- Los GV, Wood K: The HaloTag: a novel technology for cell imaging and protein analysis. Methods Mol Biol. 2007, 356: 195-208.PubMedGoogle Scholar
- Svendsen S, Zimprich C, McDougall MG, Klaubert DH, Los GV: Spatial separation and bidirectional trafficking of proteins using a multi-functional reporter. BMC Cell Biol. 2008, 9: 17-PubMed CentralPubMedGoogle Scholar
- Chen I, Howarth M, Lin W, Ting AY: Site-specific labeling of cell surface proteins with biophysical probes using biotin ligase. Nat Methods. 2005, 2: 99-104.PubMedGoogle Scholar
- Chen I, Ting AY: Site-specific labeling of proteins with small molecules in live cells. Curr Opin Biotechnol. 2005, 16: 35-40.PubMedGoogle Scholar
- Keppler A, Arrivoli C, Sironi L, Ellenberg J: Fluorophores for live cell imaging of AGT fusion proteins across the visible spectrum. BioTechniques. 2006, 41: 167-170.PubMedGoogle Scholar
- Hoffmann C, Gaietta G, Bunemann M, Adams SR, Oberdorff-Maass S, Behr B, Vilardaga JP, Tsien RY, Ellisman MH, Lohse MJ: A FlAsH-based FRET approach to determine G protein-coupled receptor activation in living cells. Nat Methods. 2005, 2: 171-176.PubMedGoogle Scholar
- Regoes A, Hehl AB: SNAP-tag mediated live cell labeling as an alternative to GFP in anaerobic organisms. BioTechniques. 2005, 39: 809-810.PubMedGoogle Scholar
- Madsen BW, Beglan CL, Spivak CE: Fluorescein-labeled naloxone binding to mu opioid receptors on live Chinese hamster ovary cells using confocal fluorescent microscopy. J Neurosci Methods. 2000, 97: 123-131.PubMedGoogle Scholar
- Fabry M, Cabrele C, Hocker H, Beck-Sickinger AG: Differently labeled peptide ligands for rapid investigation of receptor expression on a new human glioblastoma cell line. Peptides. 2000, 21: 1885-1893.PubMedGoogle Scholar
- Hunyady L, Baukal AJ, Gaborik Z, Olivares-Reyes JA, Bor M, Szaszak M, Lodge R, Catt KJ, Balla T: Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor. J Cell Biol. 2002, 157: 1211-1222.PubMed CentralPubMedGoogle Scholar
- Chevessier F, Faraut B, Ravel-Chapuis A, Richard P, Gaudon K, Bauche S, Prioleau C, Herbst R, Goillot E, Ioos C, Azulay JP, Attarian S, Leroy JP, Fournier E, Legay C, Schaeffer L, Koenig J, Fardeau M, Eymard B, Pouget J, Hantai D: MUSK, a new target for mutations causing congenital myasthenic syndrome. Hum Mol Genet. 2004, 13: 3229-3240.PubMedGoogle Scholar
- Sako Y, Minoghchi S, Yanagida T: Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol. 2000, 2: 168-172.PubMedGoogle Scholar
- Pick H, Preuss AK, Mayer M, Wohland T, Hovius R, Vogel H: Monitoring expression and clustering of the ionotropic 5HT3 receptor in plasma membranes of live biological cells. Biochemistry. 2003, 42: 877-884.PubMedGoogle Scholar
- Dumont Y, Gaudreau P, Mazzuferi M, Langlois D, Chabot JG, Fournier A, Simonato M, Quirion R: BODIPY-conjugated neuropeptide Y ligands: new fluorescent tools to tag Y1, Y2, Y4 and Y5 receptor subtypes. Br J Pharmacol. 2005, 146: 1069-1081.PubMed CentralPubMedGoogle Scholar
- Tahtaoui C, Parrot I, Klotz P, Guillier F, Galzi JL, Hibert M, Ilien B: Fluorescent pirenzepine derivatives as potential bitopic ligands of the human M1 muscarinic receptor. J Med Chem. 2004, 47: 4300-4315.PubMedGoogle Scholar
- Hegener O, Prenner L, Runkel F, Baader SL, Kappler J, Haberlein H: Dynamics of beta2-adrenergic receptor-ligand complexes on living cells. Biochemistry. 2004, 43: 6190-6199.PubMedGoogle Scholar
- Strong AE, Thierry AC, Cousin P, Moulon C, Demotz S: Synthetic chemokines directly labeled with a fluorescent dye as tools for studying chemokine and chemokine receptor interactions. Eur Cytokine Netw. 2006, 17: 49-59.PubMedGoogle Scholar
- Rao J, Dragulescu-Andrasi A, Yao H: Fluorescence imaging in vivo: recent advances. Curr Opin Biotechnol. 2007, 18: 17-25.PubMedGoogle Scholar
- Zhou M, Ghosh I: Quantum dots and peptides: a bright future together. Biopolymers. 2007, 88: 325-339.PubMedGoogle Scholar
- Zhou M, Nakatani E, Gronenberg LS, Tokimoto T, Wirth MJ, Hruby VJ, Roberts A, Lynch RM, Ghosh I: Peptide-labeled quantum dots for imaging GPCRs in whole cells and as single molecules. Bioconjug Chem. 2007, 18: 323-332.PubMedGoogle Scholar
- Clarke SJ, Hollmann CA, Zhang Z, Suffern D, Bradforth SE, Dimitrijevic NM, Minarik WG, Nadeau JL: Photophysics of dopamine-modified quantum dots and effects on biological systems. Nat Mater. 2006, 5: 409-417.PubMedGoogle Scholar
- Sugawara T, Hirasawa A, Hashimoto K, Tsujimoto G: Differences in the subcellular localization of alpha1-adrenoceptor subtypes can affect the subtype selectivity of drugs in a study with the fluorescent ligand BODIPY FL-prazosin. Life Sci. 2002, 70: 2113-2124.PubMedGoogle Scholar
- Houghten RA, Dooley CT, Appel JR: De novo identification of highly active fluorescent kappa opioid ligands from a rhodamine labeled tetrapeptide positional scanning library. Bioorg Med Chem Lett. 2004, 14: 1947-1951.PubMedGoogle Scholar
- Kolossváry I, Keserü GM: Hessian-free low-mode conformational search for large-scale protein loop optimization: application to c-jun N-terminal kinase JNK3. J Comput Chem. 2001, 22: 21-30.Google Scholar
- Schneider E, Keller M, Brennauer A, Hoefelschweiger BK, Gross D, Wolfbeis OS, Bernhardt G, Buschauer A: Synthesis and characterization of the first fluorescent nonpeptide NPY Y1 receptor antagonist. Chembiochem. 2007, 8: 1981-1988.PubMedGoogle Scholar
- Hazum E, Chang KJ, Cuatrecasas P: Cluster formation of opiate (enkephalin) receptors in neuroblastoma cells: differences between agonists and antagonists and possible relationships to biological functions. Proc Natl Acad Sci USA. 1980, 77: 3038-3041.PubMed CentralPubMedGoogle Scholar
- Gustafsson MG: Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J Microsc. 2000, 198: 82-87.PubMedGoogle Scholar
- Gustafsson MG, Agard DA, Sedat JW: I5M: 3D widefield light microscopy with better than 100 nm axial resolution. J Microsc. 1999, 195: 10-16.PubMedGoogle Scholar
- Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ: Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods. 1989, 125: 279-285.PubMedGoogle Scholar
- Wang G, Achim CL, Hamilton RL, Wiley CA, Soontornniyomkij V: Tyramide signal amplification method in multiple-label immunofluorescence confocal microscopy. Methods. 1999, 18: 459-464.PubMedGoogle Scholar
- Montero C: The antigen-antibody reaction in immunohistochemistry. J Histochem Cytochem. 2003, 51: 1-4.PubMedGoogle Scholar
- Schoneberg T, Liu J, Wess J: Plasma membrane localization and functional rescue of truncated forms of a G protein-coupled receptor. J Biol Chem. 1995, 270: 18000-18006.PubMedGoogle Scholar
- Merten N, Lindner D, Rabe N, Rompler H, Morl K, Schoneberg T, Beck-Sickinger AG: Receptor subtype-specific docking of Asp6.59 with C-terminal arginine residues in Y receptor ligands. J Biol Chem. 2007, 282: 7543-7551.PubMedGoogle Scholar
- Guthrie CR, Murray AT, Franklin AA, Hamblin MW: Differential agonist-mediated internalization of the human 5-hydroxytryptamine 7 receptor isoforms. J Pharmacol Exp Ther. 2005, 313: 1003-1010.PubMedGoogle Scholar
- Arttamangkul S, Alvarez-Maubecin V, Thomas G, Williams JT, Grandy DK: Binding and internalization of fluorescent opioid peptide conjugates in living cells. Mol Pharmacol. 2000, 58: 1570-1580.PubMedGoogle Scholar
- Daly CJ, Milligan CM, Milligan G, Mackenzie JF, McGrath JC: Cellular localization and pharmacological characterization of functioning alpha-1 adrenoceptors by fluorescent ligand binding and image analysis reveals identical binding properties of clustered and diffuse populations of receptors. J Pharmacol Exp Ther. 1998, 286: 984-990.PubMedGoogle Scholar
- McGrath JC, Naghadeh MA, Pediani JD, Mackenzie JF, Daly CJ: Importance of agonists in alpha-adrenoceptor classification and localisation of alpha1-adrenoceptors in human prostate. Eur Urol. 1999, 36 (Suppl 1): 80-88.PubMedGoogle Scholar
- Brahmadevara N, MacDonald A, McGrath JC, Daly CJ: Measurement of cellular and tissue distribution of beta-adrenoceptors in rat thoracic aorta using BODIPY-CGP 12177. Br J Pharmacol. 2002, 137: 93P-Google Scholar
- Daly CJ, Pediani JD, Mackenzie JF, McGrath JC: Visualising agonist and antagonist interactions; The value of fluorescent ligands. The Pharmacologist. 2002, 44:Google Scholar
- Wang S, Hazelrigg T: Implications for bcd mRNA localization from spatial distribution of exu protein in Drosophila oogenesis. Nature. 1994, 369: 400-403.PubMedGoogle Scholar
- Fernandez-Gonzalez R, Munoz-Barrutia A, Barcellos-Hoff MH, Ortiz-de-Solorzano C: Quantitative in vivo microscopy: the return from the 'omics'. Curr Opin Biotechnol. 2006, 17: 501-510.PubMedGoogle Scholar
- Tarasova NI, Stauber RH, Choi JK, Hudson EA, Czerwinski G, Miller JL, Pavlakis GN, Michejda CJ, Wank SA: Visualization of G protein-coupled receptor trafficking with the aid of the green fluorescent protein. Endocytosis and recycling of cholecystokinin receptor type A. J Biol Chem. 1997, 272: 14817-14824.PubMedGoogle Scholar
- Schulein R, Lorenz D, Oksche A, Wiesner B, Hermosilla R, Ebert J, Rosenthal W: Polarized cell surface expression of the green fluorescent protein-tagged vasopressin V2 receptor in Madin Darby canine kidney cells. FEBS Lett. 1998, 441: 170-176.PubMedGoogle Scholar
- Pike LJ: Lipid rafts: heterogeneity on the high seas. Biochem J. 2004, 378: 281-292.PubMed CentralPubMedGoogle Scholar
- Edidin M: Shrinking patches and slippery rafts: scales of domains in the plasma membrane. Trends Cell Biol. 2001, 11: 492-496.PubMedGoogle Scholar
- Mukherjee S, Maxfield FR: Membrane domains. Annu Rev Cell Dev Biol. 2004, 20: 839-866.PubMedGoogle Scholar
- Chamberlain LH: Detergents as tools for the purification and classification of lipid rafts. FEBS Lett. 2004, 559: 1-5.PubMedGoogle Scholar
- Kalipatnapu S, Chattopadhyay A: A GFP fluorescence-based approach to determine detergent insolubility of the human serotonin1A receptor. FEBS Lett. 2004, 576: 455-460.PubMedGoogle Scholar
- Ostrom RS, Insel PA: The evolving role of lipid rafts and caveolae in G protein-coupled receptor signaling: implications for molecular pharmacology. Br J Pharmacol. 2004, 143: 235-245.PubMed CentralPubMedGoogle Scholar
- Hur EM, Kim KT: G protein-coupled receptor signalling and cross-talk: achieving rapidity and specificity. Cell Signal. 2002, 14: 397-405.PubMedGoogle Scholar
- Klonis N, Rug M, Harper I, Wickham M, Cowman A, Tilley L: Fluorescence photobleaching analysis for the study of cellular dynamics. Eur Biophys J. 2002, 31: 36-51.PubMedGoogle Scholar
- Barak LS, Ferguson SS, Zhang J, Martenson C, Meyer T, Caron MG: Internal trafficking and surface mobility of a functionally intact beta2-adrenergic receptor-green fluorescent protein conjugate. Mol Pharmacol. 1997, 51: 177-184.PubMedGoogle Scholar
- Nelson S, Horvat RD, Malvey J, Roess DA, Barisas BG, Clay CM: Characterization of an intrinsically fluorescent gonadotropin-releasing hormone receptor and effects of ligand binding on receptor lateral diffusion. Endocrinology. 1999, 140: 950-957.PubMedGoogle Scholar
- Hebert TE, Bouvier M: Structural and functional aspects of G protein-coupled receptor oligomerization. Biochem Cell Biol. 1998, 76: 1-11.PubMedGoogle Scholar
- Akaaboune M, Grady RM, Turney S, Sanes JR, Lichtman JW: Neurotransmitter receptor dynamics studied in vivo by reversible photo-unbinding of fluorescent ligands. Neuron. 2002, 34: 865-876.PubMedGoogle Scholar
- Daigle TL, Kwok ML, Mackie K: Regulation of CB1 cannabinoid receptor internalization by a promiscuous phosphorylation-dependent mechanism. J Neurochem. 2008, 106: 70-82.PubMed CentralPubMedGoogle Scholar
- Anborgh PH, Seachrist JL, Dale LB, Ferguson SS: Receptor/beta-arrestin complex formation and the differential trafficking and resensitization of beta2-adrenergic and angiotensin II type 1A receptors. Mol Endocrinol. 2000, 14: 2040-2053.PubMedGoogle Scholar
- Estall JL, Koehler JA, Yusta B, Drucker DJ: The glucagon-like peptide-2 receptor C terminus modulates beta-arrestin-2 association but is dispensable for ligand-induced desensitization, endocytosis, and G-protein-dependent effector activation. J Biol Chem. 2005, 280: 22124-22134.PubMedGoogle Scholar
- Oakley RH, Laporte SA, Holt JA, Barak LS, Caron MG: Association of beta-arrestin with G protein-coupled receptors during clathrin-mediated endocytosis dictates the profile of receptor resensitization. J Biol Chem. 1999, 274: 32248-32257.PubMedGoogle Scholar
- Milligan G: Exploring the dynamics of regulation of G protein-coupled receptors using green fluorescent protein. Br J Pharmacol. 1999, 128: 501-510.PubMed CentralPubMedGoogle Scholar
- Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY: Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA. 1998, 95: 6803-6808.PubMed CentralPubMedGoogle Scholar
- Miesenbock G, De Angelis DA, Rothman JE: Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature. 1998, 394: 192-195.PubMedGoogle Scholar
- Beaudet A, Nouel D, Stroh T, Vandenbulcke F, Dal-Farra C, Vincent JP: Fluorescent ligands for studying neuropeptide receptors by confocal microscopy. Braz J Med Biol Res. 1998, 31: 1479-1489.PubMedGoogle Scholar
- Sarret P, Gendron L, Kilian P, Nguyen HM, Gallo-Payet N, Payet MD, Beaudet A: Pharmacology and functional properties of NTS2 neurotensin receptors in cerebellar granule cells. J Biol Chem. 2002, 277: 36233-36243.PubMedGoogle Scholar
- Duvernay MT, Filipeanu CM, Wu G: The regulatory mechanisms of export trafficking of G protein-coupled receptors. Cell Signal. 2005, 17: 1457-1465.PubMedGoogle Scholar
- Daunt DA, Hurt C, Hein L, Kallio J, Feng F, Kobilka BK: Subtype-specific intracellular trafficking of alpha2-adrenergic receptors. Mol Pharmacol. 1997, 51: 711-720.PubMedGoogle Scholar
- Patterson GH, Lippincott-Schwartz J: A photoactivatable GFP for selective photolabeling of proteins and cells. Science. 2002, 297: 1873-1877.PubMedGoogle Scholar
- Chudakov DM, Verkhusha VV, Staroverov DB, Souslova EA, Lukyanov S, Lukyanov KA: Photoswitchable cyan fluorescent protein for protein tracking. Nat Biotechnol. 2004, 22: 1435-1439.PubMedGoogle Scholar
- Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, Salih A, Spindler KD, Nienhaus GU: EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sci USA. 2004, 101: 15905-15910.PubMed CentralPubMedGoogle Scholar
- Valentin G, Verheggen C, Piolot T, Neel H, Coppey-Moisan M, Bertrand E: Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments. Nat Methods. 2005, 2: 801-PubMedGoogle Scholar
- Maddox PS, Moree B, Canman JC, Salmon ED: Spinning disk confocal microscope system for rapid high-resolution, multimode, fluorescence speckle microscopy and green fluorescent protein imaging in living cells. Meth Enzymol. 2003, 360: 597-617.PubMedGoogle Scholar
- White N, Errington R: Multi-photon microscopy: seeing more by imaging less. BioTechniques. 2002, 33: 298-300. 302, 304-295PubMedGoogle Scholar
- Pick H, Jankevics H, Vogel H: Distribution plasticity of the human estrogen receptor alpha in live cells: distinct imaging of consecutively expressed receptors. J Mol Biol. 2007, 374: 1213-1223.PubMedGoogle Scholar
- McGrath JC, Arribas S, Daly CJ: Fluorescent ligands for the study of receptors. Trends Pharmacol Sci. 1996, 17: 393-399.PubMedGoogle Scholar
- Daly CJ, McGrath JC: Fluorescent ligands, antibodies, and proteins for the study of receptors. Pharmacol Ther. 2003, 100: 101-118.PubMedGoogle Scholar
- Bohn B: Flow cytometry: a novel approach for the quantitative analysis of receptor – ligand interactions on surfaces of living cells. Mol Cell Endocrinol. 1980, 20: 1-15.PubMedGoogle Scholar
- Schneider E, Mayer M, Ziemek R, Li L, Hutzler C, Bernhardt G, Buschauer A: A simple and powerful flow cytometric method for the simultaneous determination of multiple parameters at G protein-coupled receptor subtypes. Chembiochem. 2006, 7: 1400-1409.PubMedGoogle Scholar
- Medina MA, Schwille P: Fluorescence correlation spectroscopy for the detection and study of single molecules in biology. Bioessays. 2002, 24: 758-764.PubMedGoogle Scholar
- Briddon SJ, Hill SJ: Pharmacology under the microscope: the use of fluorescence correlation spectroscopy to determine the properties of ligand-receptor complexes. Trends Pharmacol Sci. 2007, 28: 637-645.PubMed CentralPubMedGoogle Scholar
- Owicki JC: Fluorescence polarization and anisotropy in high throughput screening: perspectives and primer. J Biomol Screen. 2000, 5: 297-306.PubMedGoogle Scholar
- Bettio A, Dinger MC, Beck-Sickinger AG: The neuropeptide Y monomer in solution is not folded in the pancreatic-polypeptide fold. Protein Sci. 2002, 11: 1834-1844.PubMed CentralPubMedGoogle Scholar
- Gupta A, Decaillot FM, Gomes I, Tkalych O, Heimann AS, Ferro ES, Devi LA: Conformation state-sensitive antibodies to G-protein-coupled receptors. J Biol Chem. 2007, 282: 5116-5124.PubMedGoogle Scholar
- Lohse MJ, Nikolaev VO, Hein P, Hoffmann C, Vilardaga JP, Bunemann M: Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol Sci. 2008, 29: 159-165.PubMedGoogle Scholar
- Palanche T, Ilien B, Zoffmann S, Reck MP, Bucher B, Edelstein SJ, Galzi JL: The neurokinin A receptor activates calcium and cAMP responses through distinct conformational states. J Biol Chem. 2001, 276: 34853-34861.PubMedGoogle Scholar
- Castro M, Nikolaev VO, Palm D, Lohse MJ, Vilardaga JP: Turn-on switch in parathyroid hormone receptor by a two-step parathyroid hormone binding mechanism. Proc Natl Acad Sci USA. 2005, 102: 16084-16089.PubMed CentralPubMedGoogle Scholar
- Vilardaga JP, Bunemann M, Krasel C, Castro M, Lohse MJ: Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat Biotechnol. 2003, 21: 807-812.PubMedGoogle Scholar
- Rochais F, Vilardaga JP, Nikolaev VO, Bunemann M, Lohse MJ, Engelhardt S: Real-time optical recording of beta1-adrenergic receptor activation reveals supersensitivity of the Arg389 variant to carvedilol. J Clin Invest. 2007, 117: 229-235.PubMed CentralPubMedGoogle Scholar
- Hein P, Rochais F, Hoffmann C, Dorsch S, Nikolaev VO, Engelhardt S, Berlot CH, Lohse MJ, Bunemann M: Gs activation is time-limiting in initiating receptor-mediated signaling. J Biol Chem. 2006, 281: 33345-33351.PubMedGoogle Scholar
- Gales C, Van Durm JJ, Schaak S, Pontier S, Percherancier Y, Audet M, Paris H, Bouvier M: Probing the activation-promoted structural rearrangements in preassembled receptor-G protein complexes. Nat Struct Mol Biol. 2006, 13: 778-786.PubMedGoogle Scholar
- Janetopoulos C, Jin T, Devreotes P: Receptor-mediated activation of heterotrimeric G-proteins in living cells. Science. 2001, 291: 2408-2411.PubMedGoogle Scholar
- Bunemann M, Frank M, Lohse MJ: Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc Natl Acad Sci USA. 2003, 100: 16077-16082.PubMed CentralPubMedGoogle Scholar
- Tanimura A, Nezu A, Morita T, Turner RJ, Tojyo Y: Fluorescent biosensor for quantitative real-time measurements of inositol 1,4,5-trisphosphate in single living cells. J Biol Chem. 2004, 279: 38095-38098.PubMedGoogle Scholar
- Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, Taylor SS, Tsien RY, Pozzan T: A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol. 2000, 2: 25-29.PubMedGoogle Scholar
- Ponsioen B, Zhao J, Riedl J, Zwartkruis F, Krogt van der G, Zaccolo M, Moolenaar WH, Bos JL, Jalink K: Detecting cAMP-induced Epac activation by fluorescence resonance energy transfer: Epac as a novel cAMP indicator. EMBO Rep. 2004, 5: 1176-1180.PubMed CentralPubMedGoogle Scholar
- Honda A, Adams SR, Sawyer CL, Lev-Ram V, Tsien RY, Dostmann WR: Spatiotemporal dynamics of guanosine 3',5'-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci USA. 2001, 98: 2437-2442.PubMed CentralPubMedGoogle Scholar
- Nikolaev VO, Gambaryan S, Lohse MJ: Fluorescent sensors for rapid monitoring of intracellular cGMP. Nat Methods. 2006, 3: 23-25.PubMedGoogle Scholar
- Souslova EA, Belousov VV, Lock JG, Stromblad S, Kasparov S, Bolshakov AP, Pinelis VG, Labas YA, Lukyanov S, Mayr LM, Chudakov DM: Single fluorescent protein-based Ca2+ sensors with increased dynamic range. BMC Biotechnol. 2007, 7: 37-PubMed CentralPubMedGoogle Scholar
- Piehler J: New methodologies for measuring protein interactions in vivo and in vitro. Curr Opin Struct Biol. 2005, 15: 4-14.PubMedGoogle Scholar
- Chan FK, Siegel RM, Zacharias D, Swofford R, Holmes KL, Tsien RY, Lenardo MJ: Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein. Cytometry. 2001, 44: 361-368.PubMedGoogle Scholar
- Edwards BS, Kuckuck FW, Prossnitz ER, Okun A, Ransom JT, Sklar LA: Plug flow cytometry extends analytical capabilities in cell adhesion and receptor pharmacology. Cytometry. 2001, 43: 211-216.PubMedGoogle Scholar
- Kenworthy AK: Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods. 2001, 24: 289-296.PubMedGoogle Scholar
- Pfleger KD, Eidne KA: New technologies: bioluminescence resonance energy transfer (BRET) for the detection of real time interactions involving G-protein coupled receptors. Pituitary. 2003, 6: 141-151.PubMedGoogle Scholar
- Ayoub MA, Couturier C, Lucas-Meunier E, Angers S, Fossier P, Bouvier M, Jockers R: Monitoring of ligand-independent dimerization and ligand-induced conformational changes of melatonin receptors in living cells by bioluminescence resonance energy transfer. J Biol Chem. 2002, 277: 21522-21528.PubMedGoogle Scholar
- Paulmurugan R, Massoud TF, Huang J, Gambhir SS: Molecular imaging of drug-modulated protein-protein interactions in living subjects. Cancer Res. 2004, 64: 2113-2119.PubMed CentralPubMedGoogle Scholar
- Bhaumik S, Gambhir SS: Optical imaging of Renilla luciferase reporter gene expression in living mice. Proc Natl Acad Sci USA. 2002, 99: 377-382.PubMed CentralPubMedGoogle Scholar
- George SR, O'Dowd BF, Lee SP: G-protein-coupled receptor oligomerization and its potential for drug discovery. Nat Rev Drug Discov. 2002, 1: 808-820.PubMedGoogle Scholar
- Hansen JL, Sheikh SP: Functional consequences of 7TM receptor dimerization. Eur J Pharm Sci. 2004, 23: 301-317.PubMedGoogle Scholar
- Terrillon S, Bouvier M: Roles of G-protein-coupled receptor dimerization. EMBO Rep. 2004, 5: 30-34.PubMed CentralPubMedGoogle Scholar
- Hebert TE, Gales C, Rebois RV: Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and in situ by using fluorescence-based techniques. Cell Biochem Biophys. 2006, 45: 85-109.PubMedGoogle Scholar
- Rios CD, Jordan BA, Gomes I, Devi LA: G-protein-coupled receptor dimerization: modulation of receptor function. Pharmacol Ther. 2001, 92: 71-87.PubMedGoogle Scholar
- Maurel D, Comps-Agrar L, Brock C, Rives ML, Bourrier E, Ayoub MA, Bazin H, Tinel N, Durroux T, Prezeau L, Trinquet E, Pin JP: Cell-surface protein-protein interaction analysis with time-resolved FRET and snap-tag technologies: application to GPCR oligomerization. Nat Methods. 2008, 5: 561-567.PubMed CentralPubMedGoogle Scholar
- Meyer BH, Segura JM, Martinez KL, Hovius R, George N, Johnsson K, Vogel H: FRET imaging reveals that functional neurokinin-1 receptors are monomeric and reside in membrane microdomains of live cells. Proc Natl Acad Sci USA. 2006, 103: 2138-2143.PubMed CentralPubMedGoogle Scholar
- Patel RC, Lange DC, Patel YC: Photobleaching fluorescence resonance energy transfer reveals ligand-induced oligomer formation of human somatostatin receptor subtypes. Methods. 2002, 27: 340-348.PubMedGoogle Scholar
- Patel RC, Kumar U, Lamb DC, Eid JS, Rocheville M, Grant M, Rani A, Hazlett T, Patel SC, Gratton E, Patel YC: Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells. Proc Natl Acad Sci USA. 2002, 99: 3294-3299.PubMed CentralPubMedGoogle Scholar
- Gales C, Rebois RV, Hogue M, Trieu P, Breit A, Hebert TE, Bouvier M: Real-time monitoring of receptor and G-protein interactions in living cells. Nat Methods. 2005, 2: 177-184.PubMedGoogle Scholar
- Heithier H, Frohlich M, Dees C, Baumann M, Haring M, Gierschik P, Schiltz E, Vaz WL, Hekman M, Helmreich EJ: Subunit interactions of GTP-binding proteins. Eur J Biochem. 1992, 204: 1169-1181.PubMedGoogle Scholar
- Hughes TE, Zhang H, Logothetis DE, Berlot CH: Visualization of a functional Galpha q-green fluorescent protein fusion in living cells. Association with the plasma membrane is disrupted by mutational activation and by elimination of palmitoylation sites, but not be activation mediated by receptors or AlF4. J Biol Chem. 2001, 276: 4227-4235.PubMedGoogle Scholar
- Hynes TR, Mervine SM, Yost EA, Sabo JL, Berlot CH: Live cell imaging of Gs and the beta2-adrenergic receptor demonstrates that both alphas and beta1gamma7 internalize upon stimulation and exhibit similar trafficking patterns that differ from that of the beta2-adrenergic receptor. J Biol Chem. 2004, 279: 44101-44112.PubMedGoogle Scholar
- Gaborik Z, Hunyady L: Intracellular trafficking of hormone receptors. Trends Endocrinol Metab. 2004, 15: 286-293.PubMedGoogle Scholar
- Angers S, Salahpour A, Joly E, Hilairet S, Chelsky D, Dennis M, Bouvier M: Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc Natl Acad Sci USA. 2000, 97: 3684-3689.PubMed CentralPubMedGoogle Scholar
- Berglund MM, Schober DA, Statnick MA, McDonald PH, Gehlert DR: The use of bioluminescence resonance energy transfer 2 to study neuropeptide Y receptor agonist-induced beta-arrestin 2 interaction. J Pharmacol Exp Ther. 2003, 306: 147-156.PubMedGoogle Scholar
- Terrillon S, Durroux T, Mouillac B, Breit A, Ayoub MA, Taulan M, Jockers R, Barberis C, Bouvier M: Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis. Mol Endocrinol. 2003, 17: 677-691.PubMedGoogle Scholar
- Kraft K, Olbrich H, Majoul I, Mack M, Proudfoot A, Oppermann M: Characterization of sequence determinants within the carboxyl-terminal domain of chemokine receptor CCR5 that regulate signaling and receptor internalization. J Biol Chem. 2001, 276: 34408-34418.PubMedGoogle Scholar
- Hasbi A, Devost D, Laporte SA, Zingg HH: Real-time detection of interactions between the human oxytocin receptor and G protein-coupled receptor kinase-2. Mol Endocrinol. 2004, 18: 1277-1286.PubMedGoogle Scholar
- Bohme I, Stichel J, Walther C, Morl K, Beck-Sickinger AG: Agonist induced receptor internalization of neuropeptide Y receptor subtypes depends on third intracellular loop and C-terminus. Cell Signal. 2008, 20: 1740-1749.PubMedGoogle Scholar
- Ye S, Kohrer C, Huber T, Kazmi M, Sachdev P, Yan EC, Bhagat A, RajBhandary UL, Sakmar TP: Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis. J Biol Chem. 2008, 283: 1525-1533.PubMedGoogle Scholar
- Howarth M, Liu W, Puthenveetil S, Zheng Y, Marshall LF, Schmidt MM, Wittrup KD, Bawendi MG, Ting AY: Monovalent, reduced-size quantum dots for imaging receptors on living cells. Nat Methods. 2008, 5: 397-399.PubMed CentralPubMedGoogle Scholar
- Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA: Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nat Biotechnol. 1997, 15: 772-777.PubMedGoogle Scholar
- Nordberg E, Friedman M, Gostring L, Adams GP, Brismar H, Nilsson FY, Stahl S, Glimelius B, Carlsson J: Cellular studies of binding, internalization and retention of a radiolabeled EGFR-binding affibody molecule. Nucl Med Biol. 2007, 34: 609-618.PubMedGoogle Scholar
- Cheung TC, Hearn JP: Development of a baculovirus-based fluorescence resonance energy transfer assay for measuring protein-protein interaction. Eur J Biochem. 2003, 270: 4973-4981.PubMedGoogle Scholar
- Kroeger KM, Pfleger KD, Eidne KA: G-protein coupled receptor oligomerization in neuroendocrine pathways. Front Neuroendocrinol. 2003, 24: 254-278.PubMedGoogle Scholar
- Stefan CJ, Blumer KJ: A syntaxin homolog encoded by VAM3 mediates down-regulation of a yeast G protein-coupled receptor. J Biol Chem. 1999, 274: 1835-1841.PubMedGoogle Scholar
- Sengupta P, Chou JH, Bargmann CI: odr-10 encodes a seven transmembrane domain olfactory receptor required for responses to the odorant diacetyl. Cell. 1996, 84: 899-909.PubMedGoogle Scholar
- Suter KJ, Wuarin JP, Smith BN, Dudek FE, Moenter SM: Whole-cell recordings from preoptic/hypothalamic slices reveal burst firing in gonadotropin-releasing hormone neurons identified with green fluorescent protein in transgenic mice. Endocrinology. 2000, 141: 3731-3736.PubMedGoogle Scholar
- Hadjantonakis AK, Nagy A: The color of mice: in the light of GFP-variant reporters. Histochem Cell Biol. 2001, 115: 49-58.PubMedGoogle Scholar
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