Bright Fluorescence Monitoring System Utilizing Zoanthus sp. Green Fluorescent Protein (ZsGreen) for Human G-Protein-Coupled Receptor Signaling in Microbial Yeast Cells

G-protein-coupled receptors (GPCRs) are currently the most important pharmaceutical targets for drug discovery because they regulate a wide variety of physiological processes. Consequently, simple and convenient detection systems for ligands that regulate the function of GPCR have attracted attention as powerful tools for new drug development. We previously developed a yeast-based fluorescence reporter ligand detection system using flow cytometry. However, using this conventional detection system, fluorescence from a cell expressing GFP and responding to a ligand is weak, making detection of these cells by fluorescence microscopy difficult. We here report improvements to the conventional yeast fluorescence reporter assay system resulting in the development of a new highly-sensitive fluorescence reporter assay system with extremely bright fluorescence and high signal-to-noise (S/N) ratio. This new system allowed the easy detection of GPCR signaling in yeast using fluorescence microscopy. Somatostatin receptor and neurotensin receptor (implicated in Alzheimer’s disease and Parkinson’s disease, respectively) were chosen as human GPCR(s). The facile detection of binding to these receptors by cognate peptide ligands was demonstrated. In addition, we established a highly sensitive ligand detection system using yeast cell surface display technology that is applicable to peptide screening, and demonstrate that the display of various peptide analogs of neurotensin can activate signaling through the neurotensin receptor in yeast cells. Our system could be useful for identifying lead peptides with agonistic activity towards targeted human GPCR(s).     

Regulation of G protein‐coupled receptor signaling by plasma membrane organization and endocytosis

The trafficking of G protein coupled‐receptors (GPCRs) is one of the most exciting areas in cell biology because of recent advances demonstrating that GPCR signaling is spatially encoded. GPCRs, acting in a diverse array of physiological systems, can have differential signaling consequences depending on their subcellular localization. At the plasma membrane, GPCR organization could fine‐tune the initial stages of receptor signaling by determining the magnitude of signaling and the type of effectors to which receptors can couple. This organization is mediated by the lipid composition of the plasma membrane, receptor‐receptor interactions, and receptor interactions with intracellular scaffolding proteins. GPCR organization is subsequently changed by ligand binding and the regulated endocytosis of these receptors. Activated GPCRs can modulate the dynamics of their own endocytosis through changing clathrin‐coated pit dynamics, and through the scaffolding adaptor protein β‐arrestin. This endocytic regulation has signaling consequences, predominantly through modulation of the MAPK cascade. This review explores what is known about receptor sorting at the plasma membrane, protein partners that control receptor endocytosis, and the ways in which receptor sorting at the plasma membrane regulates downstream trafficking and signaling.      

Dynamic imaging of cannabinoid receptor 1 vesicular trafficking in cultured astrocytes

Astrocytes possess GPCRs (G-protein-coupled receptors) for neuroactive substances and can respond via these receptors to signals originating from neurons as well as astrocytes. Like many transmembrane proteins, GPCRs exist in a dynamic equilibrium between receptors expressed at the plasma membrane and those present within intracellular trafficking compartments. The characteristics of GPCR trafficking within astrocytes have not been investigated. We therefore monitored the trafficking of recombinant fluorescent protein chimeras of the CB1R (cannabinoid receptor 1) that is thought to be expressed natively in astrocytes. CB1R chimeras displayed a marked punctate intracellular localization when expressed in cultured rat visual cortex astrocytes, an expression pattern reminiscent of native CB1R expression in these cells. Based upon trafficking characteristics, we found the existence of two populations of vesicular CB1R puncta: (i) relatively immobile puncta with movement characteristic of diffusion and (ii) mobile puncta with movement characteristic of active transport along cytoskeletal elements. The predominant direction of active transport is oriented radially to/from the nuclear region, which can be abolished by disruption of the microtubule cytoskeleton. CB1R puncta are localized within intracellular acidic organelles, mainly co-localizing with endocytic compartments. Constitutive trafficking of CB1R to and from the plasma membrane is an energetically costly endeavour whose function is at present unclear in astrocytes. However, given that intracellular CB1Rs can engage cell signalling pathways, it is likely that this process plays an important regulatory role.     

Soluble mimics of a chemokine receptor: chemokine binding by receptor elements juxtaposed on a soluble scaffold

Despite the broad biological importance of G protein-coupled receptors (GPCRs), ligand recognition by GPCRs remains poorly understood. To explore the roles of GPCR extracellular elements in ligand binding and to provide a tractable system for structural analyses of GPCR/ligand interactions, we have developed a soluble protein that mimics ligand recognition by a GPCR. This receptor analog, dubbed CROSS5, consists of the N-terminal and third extracellular loop regions of CC chemokine receptor 3 (CCR3) displayed on the surface of a small soluble protein, the B1 domain of Streptococcal protein G. CROSS5 binds to the CCR3 ligand eotaxin with a dissociation equilibrium constant of 2.9 +/- 0.8 microM and competes with CCR3 for eotaxin binding. Control proteins indicate that juxtaposition of both CCR3 elements is required for optimal binding to eotaxin. Moreover, the affinities of CROSS5 for a series of eotaxin mutants are highly correlated with the apparent affinities of CCR3 for the same mutants, demonstrating that CROSS5 uses many of the same interactions as does the native receptor. The strategy used to develop CROSS5 could be applied to many other GPCRs, with a variety of potential applications.     

A cellular perspective of bias at G protein‐coupled receptors

G protein‐coupled receptors (GPCRs) modulate cell function over short‐ and long‐term timescales. GPCR signaling depends on biochemical parameters that define the what, when, and where of receptor function: what proteins mediate and regulate receptor signaling, where within the cell these interactions occur, and how long these interactions persist. These parameters can vary significantly depending on the activating ligand. Collectivity, differential agonist activity at a GPCR is called bias or functional selectivity. Here we review agonist bias at GPCRs with a focus on ligands that show dramatically different cellular responses from their unbiased counterparts.     

Monitoring ligand-mediated internalization of G protein-coupled receptor as a novel pharmacological approach

Agonist activation of a G protein-coupled receptor (GPCR) results in the redistribution of the receptor protein away from the cell surface into internal cellular compartments through a process of endocytosis known as internalization. Visualization of receptor internalization has become experimentally practicable by using fluorescent reagents such as green fluorescent protein (GFP). In this study, we examined whether the ligand-mediated internalization of a GPCR can be exploited for pharmacological evaluations. We acquired fluorescent images of cells expressing GFP-labeled GPCRs and evaluated the ligand-mediated internalization quantitatively by image processing. Using beta2-adrenoceptor and vasopressin V1a receptor as model GPCRs that couple to Gs and Gq, respectively, we first examined whether these GFP-tagged GPCRs exhibited appropriate pharmacology. The rank order of receptor internalization potency for a variety of agonists and antagonists specific to each receptor corresponded well with that previously observed in ligand binding studies. In addition to chemical ligand-induced internalization, this cell-based fluorescence imaging system successfully monitored the internalization of the proton-sensing GPCR TDAG8, and that of the free fatty acid-sensitive GPCR GPR120. The results show that monitoring receptor internalization can be a useful approach for pharmacological characterization of GPCRs and in fishing for ligands of orphan GPCRs.     

Inhibition of serotonin 5-hydroxytryptamine2c receptor function through heterodimerization: receptor dimers bind two molecules of ligand and one G-protein

Although dimerization appears to be a common property of G-protein-coupled receptors (GPCRs), it remains unclear whether a GPCR dimer binds one or two molecules of ligand and whether ligand binding results in activation of one or two G-proteins when measured using functional assays in intact living cells. Previously, we demonstrated that serotonin 5-hydroxytryptamine2C (5-HT2C) receptors form homodimers (Herrick-Davis, K., Grinde, E., and Mazurkiewicz, J. (2004) Biochemistry 43, 13963-13971). In the present study, an inactive 5-HT(2C) receptor was created and coexpressed with wild-type 5-HT2C receptors to determine whether dimerization regulates receptor function and to determine the ligand/dimer/G-protein stoichiometry in living cells. Mutagenesis of Ser138 to Arg (S138R) produced a 5-HT2C receptor incapable of binding ligand or stimulating inositol phosphate (IP) signaling. Confocal fluorescence imaging revealed plasma membrane expression of yellow fluorescent protein-tagged S138R receptors. Expression of wild-type 5-HT2C receptors in an S138R-expressing stable cell line had no effect on ligand binding to wild-type 5-HT2C receptors, but inhibited basal and 5-HT-stimulated IP signaling as well as constitutive and 5-HT-stimulated endocytosis of wild-type 5-HT2C receptors. M1 muscarinic receptor activation of IP production was normal in the S138R-expressing cells. Heterodimerization of S138R with wild-type 5-HT2C receptors was visualized in living cells using confocal fluorescence resonance energy transfer (FRET). FRET was dependent on the donor/acceptor ratio and independent of the receptor expression level. Therefore, inactive 5-HT2C receptors inhibit wild-type 5-HT2C receptor function by forming nonfunctional heterodimers expressed on the plasma membrane. These results are consistent with a model in which one GPCR dimer binds two molecules of ligand and one G-protein and indicate that dimerization is essential for 5-HT receptor function.     

Lipid Raft-Mediated Regulation of G-Protein Coupled Receptor Signaling by Ligands which Influence Receptor Dimerization: A Computational Study

G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors; they activate heterotrimeric G-proteins in response to ligand stimulation. Although many GPCRs have been shown to form homo- and/or heterodimers on the cell membrane, the purpose of this dimerization is not known. Recent research has shown that receptor dimerization may have a role in organization of receptors on the cell surface. In addition, microdomains on the cell membrane termed lipid rafts have been shown to play a role in GPCR localization. Using a combination of stochastic (Monte Carlo) and deterministic modeling, we propose a novel mechanism for lipid raft partitioning of GPCRs based on reversible dimerization of receptors and then demonstrate that such localization can affect GPCR signaling. Modeling results are consistent with a variety of experimental data indicating that lipid rafts have a role in amplification or attenuation of G-protein signaling. Thus our work suggests a new mechanism by which dimerization-inducing or inhibiting characteristics of ligands can influence GPCR signaling by controlling receptor organization on the cell membrane.     

Oligomerization of yeast α-factor receptor detected by fluorescent energy transfer between ligands

G-protein-coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes; however, in many cases, the role of oligomerization and the extent to which it occurs at low physiological levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently labeled cell-impermeant ligands bound to yeast α-mating pheromone receptors that are members of the GPCR superfamily. A novel, to our knowledge, procedure was used to analyze energy transfer as a function of receptor occupancy by donor and acceptor ligands. Measurements of loss of donor fluorescence due to energy transfer in cells expressing high levels of receptors were used to calibrate measurements of enhanced acceptor emission due to energy transfer in cells expressing low levels of receptors. The procedure allows determination of energy transfer efficiencies over a 50-fold range of expression of full-length receptors at the surface of living cells without the need to create fluorescent or bioluminescent fusion proteins. Energy transfer efficiencies for fluorescently labeled derivatives of the receptor agonist α-factor do not depend on receptor expression level and are unaffected by C-terminal truncation of receptors. Fluorescently labeled derivatives of α-factor that act as receptor antagonists exhibit higher transfer efficiencies than those for labeled agonists. Although the approach cannot determine the number of receptors per oligomer, these results demonstrate that ligand-bound, native α-factor receptors exist as stable oligomers in the cell membranes of intact yeast cells at normal physiological expression levels and that the extent of oligomer formation is not dependent on the concentration of receptors in the membrane.     

Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.     

Emerging structural biology of lipid G protein‐coupled receptors

The first crystal structure of a G protein‐coupled receptor (GPCR) was that of the bovine rhodopsin, solved in 2000, and is a light receptor within retina rode cells that enables vision by transducing a conformational signal from the light‐induced isomerization of retinal covalently bound to the receptor. More than 7 years after this initial discovery and following more than 20 years of technological developments in GPCR expression, stabilization, and crystallography, the high‐resolution structure of the adrenaline binding β₂‐adrenergic receptor, a ligand diffusible receptor, was discovered. Since then, high‐resolution structures of more than 53 unique GPCRs have been determined leading to a significant improvement in our understanding of the basic mechanisms of ligand‐binding and ligand‐mediated receptor activation that revolutionized the field of structural molecular pharmacology of GPCRs. Recently, several structures of eight unique lipid‐binding receptors, one of the most difficult GPCR families to study, have been reported. This review presents the outstanding structural and pharmacological features that have emerged from these new lipid receptor structures. The impact of these findings goes beyond mechanistic insights, providing evidence of the fundamental role of GPCRs in the physiological integration of the lipid signaling system, and highlighting the importance of sustained research into the structural biology of GPCRs for the development of new therapeutics targeting lipid receptors.     

Use of Kaede fusions to visualize recycling of G protein-coupled receptors

The heptahelical G protein-coupled receptors (GPCRs) are internalized following agonist treatment and either recycle rapidly to the plasma membrane or enter the lysosomal degradation pathway. Many conventional GPCR recycling assays suffer from the fact that receptors arriving from the secretory pathway may interfere with recycling receptors. In this study, we introduce a new methodology to study post-endocytotic GPCR trafficking using fusions with the recently cloned Kaede protein. In contrast to the widely used green fluorescent protein, the fluorescence of Kaede can be converted from green to red using ultraviolet irradiation. Our methodology allows to study recycling of GPCRs microscopically in real-time bypassing problems with secretory pathway receptors. Initially, receptors are internalized using an agonist. Fluorescence signals in endosomes are switched, and trafficking of the receptors to the plasma membrane can be easily visualized by monitoring their new fluorescence. Using this methodology, we show that the corticotropin-releasing factor receptor type 1 belongs to the family of recycling GPCRs. Moreover, we demonstrate by fluorescence correlation spectroscopy that Kaede does not oligomerize when fused to membrane proteins, representing an additional advantage of this technique. The Kaede technology may be a powerful tool to study membrane protein trafficking in general.     

Biochemical and biophysical characterization of serotonin 5-HT2C receptor homodimers on the plasma membrane of living cells

While many studies have provided evidence of homodimerization and heterodimerization of G-protein-coupled receptors (GPCRs), few studies have used fluorescence resonance energy transfer (FRET) combined with confocal microscopy to visualize receptor dimerization on the plasma membrane, and there have been no reports demonstrating the expression of serotonin receptor dimers/oligomers on the plasma membrane of living cells. In the study presented here, biochemical and biophysical techniques were used to determine if 5-HT(2C) receptors exist as homodimers on the plasma membrane of living cells. Immunoprecipitation followed by Western blotting revealed the presence of immunoreactive bands the predicted size of 5-HT(2C) receptor monomers and homodimers that were detergent and cross-linker sensitive. Bioluminescence resonance energy transfer (BRET) was assessed in HEK293 cells expressing 5-HT(2C) receptors labeled with Renilla luciferase and yellow fluorescent protein. BRET levels were not altered by pretreatment with serotonin. Confocal microscopy provided direct visualization of FRET on the plasma membrane of live cells expressing 5-HT(2C) receptors labeled with cyan (donor) and yellow (acceptor) fluorescent proteins. FRET, assessed by acceptor photobleaching, was dependent on the donor/acceptor ratio and independent of acceptor expression levels, indicating that FRET resulted from receptor clustering and not from overexpression of randomly distributed receptors, providing evidence for GPCR dimers/oligomers in a clustered distribution on the plasma membrane. The results of this study suggest that 5-HT(2C) receptors exist as constitutive homodimers on the plasma membrane of living cells. In addition, a confocal-based FRET method for monitoring receptor dimerization directly on the plasma membrane of living cells is described.     

The expanding repertoire of receptor activity modifying protein (RAMP) function

Receptor activity modifying proteins (RAMPs) associate with G-protein-coupled receptors (GPCRs) at the plasma membrane and together bind a variety of peptide ligands, serving as a communication interface between the extracellular and intracellular environments. The collection of RAMP-interacting GPCRs continues to expand and now consists of GPCRs from families A, B and C, suggesting that RAMP activity is extremely prevalent. RAMP association with GPCRs can regulate GPCR function by altering ligand binding, receptor trafficking and desensitization, and downstream signaling pathways. Here, we elaborate on these RAMP-dependent mechanisms of GPCR regulation, which provide opportunities for pharmacological intervention.     

Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding consistent with a role for receptor recycling in functional resensitization.     

Contributions of fluorescence techniques to understanding G protein-coupled receptor dimerisation

G protein-coupled receptors (GPCRs) are the largest class of eukaryotic cell-surface receptors and, over the last decade, it has become clear that they are capable of dimerisation. Whilst many biochemical and biophysical approaches have been used to study dimerisation, fluorescence techniques, including Förster resonance energy transfer and single molecule fluorescence, have been key players. Here we review recent contributions of fluorescence techniques to investigate GPCR dimers, including dimerisation in cell membranes and native tissues, the effect of ligand binding on dimerisation and the kinetics of dimer formation and dissociation. The challenges of studying multicomponent membrane protein systems have led to the development and refinement of many fluorescence assays, allowing the functional consequences of receptor dimerisation to be investigated and individual protein molecules to be imaged in the membranes of living cells. It is likely that the fluorescence techniques described here will be of use for investigating many other multicomponent membrane protein systems.     

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.     

Exploring a role for heteromerization in GPCR signalling specificity

The critical involvement of GPCRs (G-protein-coupled receptors) in nearly all physiological processes, and the presence of these receptors at the interface between the extracellular and the intracellular milieu, has positioned these receptors as pivotal therapeutic targets. Although a large number of drugs targeting GPCRs are currently available, significant efforts have been directed towards understanding receptor properties, with the goal of identifying and designing improved receptor ligands. Recent advances in GPCR pharmacology have demonstrated that different ligands binding to the same receptor can activate discrete sets of downstream effectors, a phenomenon known as 'ligand-directed signal specificity', which is currently being explored for drug development due to its potential therapeutic advantage. Emerging studies suggest that GPCR responses can also be modulated by contextual factors, such as interactions with other GPCRs. Association between different GPCR types leads to the formation of complexes, or GPCR heteromers, with distinct and unique signalling properties. Some of these heteromers activate discrete sets of signalling effectors upon activation by the same ligand, a phenomenon termed 'heteromer-directed signalling specificity'. This has been shown to be involved in the physiological role of receptors and, in some cases, in disease-specific dysregulation of a receptor effect. Hence targeting GPCR heteromers constitutes an emerging strategy to select receptor-specific responses and is likely to be useful in achieving specific beneficial therapeutic effects.     

Quantification of Ligand Binding to G-Protein Coupled Receptors on Cell Membranes by Ellipsometry

G-protein-coupled receptors (GPCRs) are prime drug targets and targeted by approximately 60% of current therapeutic drugs such as β-blockers, antipsychotics and analgesics. However, no biophysical methods are available to quantify their interactions with ligand binding in a native environment. Here, we use ellipsometry to quantify specific interactions of receptors within native cell membranes. As a model system, the GPCR-ligand CXCL12α and its receptor CXCR4 are used. Human-derived Ishikawa cells were deposited onto gold coated slides via Langmuir-Schaefer film deposition and interactions between the receptor CXCR4 on these cells and its ligand CXCL12α were detected via total internal reflection ellipsometry (TIRE). This interaction could be inhibited by application of the CXCR4-binding drug AMD3100. Advantages of this approach are that it allows measurement of interactions in a lipid environment without the need for labelling, protein purification or reconstitution of membrane proteins. This technique is potentially applicable to a wide variety of cell types and their membrane receptors, providing a novel method to determine ligand or drug interactions targeting GPCRs and other membrane proteins.     

Cell-free assay of G-protein-coupled receptors using fluorescence polarization

A recently developed nanotechnology, the Integral Molecular lipoparticle, provides an essentially soluble cell-free system in which G-protein-coupled receptors (GPCRs) in their native conformations are concentrated within virus-like particles. As a result, the lipoparticle provides a means to overcome 2 common obstacles to the development of homogeneous, nonradioactive GPCR ligand-binding assays: membrane protein solubilization and low receptor density. The work reported here describes the first application of this nanotechnology to a fluorescence polarization (FP) molecular binding assay format. The GPCR chosen for these studies was the well-studied chemokine receptor CXCR4 for which a peptide ligand (T-22) has been previously characterized. The EC50 determined for the CXCR4-T-22 peptide interaction via FP with CXCR4 lipoparticles (15 nM) is consistent with the IC50 determined for the unlabeled T-22 peptide via competitive binding (59 nM).