Patent Publication Number: US-2021179692-A1

Title: G protein-coupled receptor (gpcr) ligand assay

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims benefit of priority from U.S. Provisional Application No. 62/646,534, filed Mar. 22, 2018, and U.S. Provisional Application No. 62/594,356, filed Dec. 4, 2017, which are incorporated herein by reference in their entirety. 
    
    
     STATEMENT AS TO FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under GM105646-05 and CA186752-02 awarded by the National Institutes of Health. The government has certain rights in the invention. 
    
    
     TECHNICAL FIELD 
     This document relates to materials and methods for assessing the effects of various ligands on their receptors, including, for example, materials and methods for assessing the intrinsic effects of various ligands on G protein-coupled receptors (GPCRs). 
     BACKGROUND 
     GPCRs are the target of over one-third of all pharmaceuticals in modern drug therapies. Small-molecule therapeutics that bind GPCRs can influence cellular processes by either enhancing downstream signaling (agonist) or blocking the effects of endogenous ligands (antagonist/inverse agonist) (Tyndall and Sandilya (2005)  Med. Chem.  1, 405-421). The precise effects of GPCR therapeutics on cell signaling are tissue and cell-type dependent, however, thereby limiting the ability to translate information derived from cell-based assays to the on-target effects in physiological and pathophysiological contexts. 
     To date, the stimulating effect of a ligand at the level of a single GPCR, referred to as “intrinsic efficacy,” has best been approximated by measuring the activation rate of G proteins that couple to the GPCR (Harrison and Traynor (2003)  Life Sci.  74, 489-508; Denis et al. (2012)  Curr. Pharm. Des.  18, 128-144; and Gregorio et al. (2017)  Nature  10.1038/nature22354). G protein activation assays that utilize either live cells or crude membrane preparations containing the GPCR are complicated by the heterogeneity inherent in different cell or membrane batches. An alternative approach (Gregorio et al., supra) uses extensive single molecule measurements with GPCRs and G proteins purified to homogeneity to define a “molecular efficacy” that currently is the closest approximation in the field to the intrinsic efficacy, but this approach requires procedure-optimization for each GPCR and is resource-intensive for examining large numbers of compounds. Other studies have used a Forster resonance energy transfer- (FRET-) based biosensor based on Systematic Protein Affinity Strength Modulation (SPASM) technology to detect ligand-induced interaction between a GPCR and the C-terminus of the Ga subunit of a G protein in live cells (Malik et al. (2013)  J Biol. Chem.  288, 17167-17178), and to examine the selectivity of G protein activation by GPCRs (Semack et al. (2016)  J. Biol. Chem.  291, 17929-17940). 
     SUMMARY 
     This document is based, at least in part, on the development of a system and an assay that can effectively compare the intrinsic effects of ligands on GPCRs. The systems and methods described herein include the use of Giant Plasma Membrane Vesicles (GPMVs) derived from cells expressing SPASM sensors (e.g., SPASM GPCR sensors). Purified GPMVs incorporate the SPASM sensors within their lipid bilayer, while maintaining protein function. As discussed herein, the GPMV-incorporated SPASM sensors (“GPMV sensors”) are highly stable, making them a scalable reagent suitable for high-throughput drug screening and analysis. Since they are incorporated into the GPMV membrane, the SPASM GPCR sensors are in their native environment and can maintain their native conformation. FRET measurements using SPASM GPCR sensors in live cells can be sensitive to expression levels, cell handling, and ligand stimulation times. Unlike live cells, GPMV sensors provide consistency and reliability in FRET measurements, regardless of expression levels, handling, and ligand stimulation time. Using this novel reagent, the relationship between ligand-induced changes in FRET intensity and intrinsic efficacy has been investigated for GPCRs, demonstrating that FRET measurements correlate directly with reported intrinsic efficacy (R 2 =0.99) and activation rate of G proteins that couple to a GPCR (R 2 =0.97) (Gregorio et al., supra). The data described in the Examples herein support the combined application of GPMVs and SPASM GPCR sensors as a novel FRET-based assay for precisely measuring the intrinsic efficacy of GPCR ligands. 
     In a first aspect, this document provides a giant plasma membrane vesicle (GPMV) sensor containing a membrane-associated polypeptide, where the membrane-associated polypeptide contains a target G protein-coupled receptor (GPCR), a linker including an amino acid sequence flanked by a FRET donor and a FRET acceptor, and a receptor partner for the target GPCR, where the linker separates the target GPCR from the receptor partner. The linker can be about 10 nm to about 30 nm in length. The linker can include an ER/K sequence having a substantially repeating sequence of glutamic acid, arginine, and lysine residues, and can be about 50 to about 250 amino acids in length. In some cases, the linker can include the amino acid sequence set forth in SEQ ID NO:2, or a sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:2. The receptor partner can be a peptide, polypeptide, protein, or protein domain. The membrane-associated polypeptide can further contain a (Gly-Ser-Gly) 2 -4 amino acid sequence on either side of the FRET donor and the FRET acceptor. The FRET donor can be monomeric Cerulean or monomeric enhanced green fluorescent protein (eGFP), and the FRET acceptor can be monomeric Citrine or monomeric Cherry. In some cases, the membrane-associated polypeptide can include, in order from amino terminus to carboxy terminus, the target GPCR, a first (Gly-Ser-Gly) 2-4  amino acid sequence, a FRET donor or acceptor, a second (Gly-Ser-Gly) 2-4  amino acid sequence, the linker, a third (Gly-Ser-Gly) 2-4  amino acid sequence, a partner for the FRET donor or acceptor, a fourth (Gly-Ser-Gly) 2-4  amino acid sequence, and the receptor partner for the target GPCR. 
     In another aspect, this document features a method for quantifying the intrinsic efficacy of a compound for a target GPCR. The method can include (a) providing a GPMV sensor, where the sensor contains a GPMV that includes a membrane-associated polypeptide, the membrane-associated polypeptide containing a target GPCR, a linker including an amino acid sequence flanked by a FRET donor and a FRET acceptor, and a receptor partner for the target GPCR, where the linker separates the target GPCR from the receptor partner; (b) measuring an initial FRET ratio for the sensor; (c) contacting the sensor with a compound that interacts with the target GPCR; (d) measuring a test FRET ratio for the sensor; and (e) calculating the intrinsic efficacy of the compound based on the difference between the test FRET ratio and the initial FRET ratio. The linker can be about 10 nm to about 30 nm in length. The linker can contain an ER/K sequence having a substantially repeating sequence of glutamic acid, arginine, and lysine residues, and can be about 50 to about 250 amino acids in length. In some cases, the linker can contain the amino acid sequence set forth in SEQ ID NO:2, or a sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:2. The receptor partner can be a peptide, polypeptide, protein, or protein domain. The membrane-associated polypeptide can further contain a (Gly-Ser-Gly) 2-4  amino acid sequence on either side of the FRET donor and the FRET acceptor. The FRET donor can be monomeric Cerulean or monomeric eGFP, and the FRET acceptor can be monomeric Citrine or monomeric Cherry. In some cases, the membrane-associated polypeptide can contain, in order from amino terminus to carboxy terminus, the target GPCR, a first (Gly-Ser-Gly) 2-4  amino acid sequence, a FRET donor or acceptor, a second (Gly-Ser-Gly) 2-4  amino acid sequence, the linker, a third (Gly-Ser-Gly) 2-4  amino acid sequence, a partner for the FRET donor or acceptor, a fourth (Gly-Ser-Gly) 2-4  amino acid sequence, and the receptor partner for the target GPCR. 
     In another aspect, this document features a method for identifying a compound that interacts with a target GPCR. The method can include (a) providing a GPMV sensor, where the sensor contains a GPMV that includes a membrane-associated polypeptide, the membrane-associated polypeptide containing a target GPCR, a linker including an amino acid sequence flanked by a FRET donor and a FRET acceptor, and a receptor partner for the target GPCR, where the linker separates the target GPCR from the receptor partner; (b) measuring an initial FRET ratio for the sensor; (c) contacting the sensor with a candidate compound; (d) measuring a test FRET ratio for the sensor; and (e) identifying the candidate compound as interacting with the target GPCR when the test FRET ratio is altered by at least 0.5% as compared to the initial FRET ratio. The linker can be about 10 nm to about 30 nm in length. The linker can include an ER/K sequence having a substantially repeating sequence of glutamic acid, arginine, and lysine residues, and can be about 50 to about 250 amino acids in length. In some cases, the linker can include the amino acid sequence set forth in SEQ ID NO:2, or a sequence that is at least 95% identical to the sequence set forth in SEQ ID NO:2. The receptor partner can be a peptide, polypeptide, protein, or protein domain. The membrane-associated polypeptide can further contain a (Gly-Ser-Gly) 2-4  amino acid sequence on either side of the FRET donor and the FRET acceptor. The FRET donor can be monomeric Cerulean or monomeric eGFP, and the FRET acceptor can be monomeric Citrine or monomeric Cherry. In some cases, the membrane-associated polypeptide can contain, in order from amino terminus to carboxy terminus, the target GPCR, a first (Gly-Ser-Gly) 2-4  amino acid sequence, a FRET donor or acceptor, a second (Gly-Ser-Gly) 2-4  amino acid sequence, the linker, a third (Gly-Ser-Gly) 24  amino acid sequence, a partner for the FRET donor or acceptor, a fourth (Gly-Ser-Gly) 2-4  amino acid sequence, and the receptor partner for the target GPCR. The method can further include providing two or more GPMV sensors, each containing a different target GPCR, measuring initial and test FRET ratios for each GPMV sensor, and identifying the candidate compound as a GPCR-specific ligand when at least some of the test FRET ratios are not altered by at least 0.5% as compared to the initial FRET ratios. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. 
     The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. 
    
    
     
       DESCRIPTION OF DRAWINGS 
         FIG. 1A  is a schematic of the GPCR-SPASM sensors used in the studies described herein (top), and a diagram of the sensors in dissociated (low FRET) and associated (high FRET) states (bottom). 
         FIG. 1B  is a schematic diagram depicting a process for synthesizing and purifying GPMVs. 
         FIG. 1C  is a series of fluorescence images of GPMVs incorporating a GPCR-SPASM sensor. GPMVs were synthesized from HEK293 cells expressing a beta-2 adrenergic receptor- (β2AR-) Spep sensor. Purified GPMVs were imaged with mCerulean. Scale bar=2 um. 
         FIG. 2A  is a graph plotting competition of radioligand binding to GPMV-β2AR-Spep sensors via isoproterenol. GPMVs incorporating 20 fmol of indicated β2AR-Spep sensors were stimulated with increasing concentrations of isoproterenol, in the presence of I 125 -(±)-Cyanopindolol (500 nM). 
         FIG. 2B  is a graph plotting cAMP accumulation in GPMV-β2AR-Spep sensors stimulated with saturating concentrations of isoproterenol (Iso; 10 μM), fenoterol (Feno; 10 μM), salbutamol (Salb; 10 μM), clenbuterol (Clen; 10 μM), and isoproterenol with ICI 118,551 (Iso+ICI; 10 μM and 1 μM, respectively). The increase in cAMP is expressed as a percentage of the forskolin-stimulated cAMP response (Fsk). 
         FIG. 3A  is a graph plotting the change in FRET ratio following isoproterenol stimulation. FRET measurements were taken with GPMV-β2AR-SPASM sensors tethered to the C terminal α5 helix of the GαS subunit (Spep) or a no-peptide control (−). The S-pep sensors exhibited a gain in FRET following isoproterenol (100 μM) stimulation, whereas the No-pep sensors did not show an increase. 
         FIG. 3B  is a graph plotting dose-dependent changes in GPMV-β2AR-Spep FRET measurements. GPMV-β2AR-Spep sensors were stimulated with increasing concentrations of isoproterenol (black) or increasing concentrations of ICI 118,551 (gray). GPMV-β2AR-Spep sensors were stimulated with a saturating concentration of isoproterenol (100 μM) and 
         FIG. 4A  is a graph plotting GPMV sensitivity to GPCR-SPASM sensor expression. GPMVs were synthesized from HEK293 cells expressing (24 hour expression time) or extensively overexpressing (48 hour expression time) β2AR-Spep sensors, and stimulated with isoproterenol. Extensively overexpressing β2AR-Spep sensors drastically decreased the level of ΔFRET in live cells. GPMV-β2AR-Spep sensors synthesized from extensively overexpressing HEK293 cells did not exhibit a significant decrease in ΔFRET levels. GPMV FRET measurements were not sensitive to duration of either sensor expression in cells or ligand stimulation. 
         FIG. 4B  is a graph plotting FRET measurements for GPMV-β2AR-Spep sensors that were maintained on ice or stored frozen with 10% sucrose, 10% glycerol, or 50% glycerol for twelve days. Stored GPMV-β2AR-Spep sensors were thawed and stimulated with isoproterenol (100 uM). ΔFRET levels of GPMV-β2AR-Spep sensors in all storage conditions were comparable with that of a fresh batch. Thus, the GPMVs were stable over a long period of time and post freeze-thaw. 
         FIGS. 5A and 5B  are graphs showing that GPMV-β2AR-Spep ΔFRET correlates linearly with molecular efficacy. GPMV-β2AR-Spep sensors were stimulated with various ligands, and the ΔFRET values were plotted against previously documented measurements of ligand intrinsic efficacies (Gregorio et al., supra). Ligand induced ΔFRET correlated strongly with both the molecular efficacy ( FIG. 5A , R 2 =0.96˜0.99) and the ligands&#39; ability to induce GTP turnover via purified β2AR-GaS protein complex in vitro ( FIG. 5B , R 2 =0.79˜0.97). Upper lines (black): optimized buffer (20 mM HEPES pH 7.4, 25 mM KCl, 5 mM MgCl 2 ). Lower lines (gray): HBS buffer (20 mM HEPES pH 7.4, 5 mM KCl, 145 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 ). Error bars denote s.e.m. Data are derived from at least three independent experiments, with at least three replicates per condition. 
         FIG. 6  is a graph demonstrating that GPMV-β2AR-Spep ΔFRET does not correlate with classical efficacy measurements. GPMV-β2AR-Spep sensors were stimulated with various ligands, and the ΔFRET were plotted against previously documented ligand efficacy ratios (Baker (2010)  Br. J. Pharmacol.  160(5):1048-61). Error bars denote s.e.m. Data are derived from at least three independent experiments, with at least three replicates per condition. R 2 =0.29 for efficacy ratio vs ΔFRET. 
         FIGS. 7A-7C  are a series of graphs showing that GPMV-β2AR-Spep ΔFRET does not correlate with factors that dictate classical efficacy. GPMV-β2AR-Spep sensors were stimulated with various ligands and the ΔFRET were plotted against previously documented measurements of experimental factors that dictate ligand efficacy ratio (Baker, supra). Error bars denote s.e.m. Data are derived from at least three independent experiments, with at least three replicates per condition. R 2 =0.61 for cAMP vs ΔFRET ( FIG. 7A ), 0.32 for −log[EC 50 ] vs ΔFRET ( FIG. 7B ), and 0.09 for −log K d  vs ΔFRET ( FIG. 7C ). In addition, R 2 =0.25 for EC 50  vs ΔFRET and 0.04 for K d  vs ΔFRET (not shown). 
         FIG. 8  is a graph showing that GPMV-GPCR sensors can be utilized in a high-throughput manner. Fluorescence measurements of ligand-treated GPMV-β2AR-Spep sensors were obtained using a high-throughput 96-well plate reader. ΔFRET efficiencies as described elsewhere (Toropova et al. (2014)  Elife  3:e03372 DOI:10.7554/eLife.03372) were calculated and plotted against the ΔFRET ratios shown in  FIGS. 5A and 5B . Calculated ΔFRET efficacies correlates linearly with ΔFRET ratios (R 2 =0.94). 
         FIG. 9  is a graph demonstrating that GPMV-GPCR sensors are generally applicable to a variety of GPCR and G proteins. GPMVs, incorporating the β2AR-Spep, vasopressin) receptor- (V1R-) Qpep, and alpha-1A adrenergic receptor- (α1AR-) Ipep sensors, were synthesized by the bicine-mediated method. The sensors were treated with their respective full agonists (100 μM isoproterenol, 100 nM AVP, and 100 μM epinephrine, respectively), and changes in FRET were measured. 
     
    
    
     DETAILED DESCRIPTION 
     The materials and methods provided herein can effectively be used to identify ligands for protein targets, and to compare the intrinsic effects of ligands on GPCRs. In particular, as described herein, GPMVs derived from cells expressing SPASM sensors incorporate the SPASM sensors within their lipid bilayer, and the SPASM molecules remain functional since the receptor portion of the SPASM molecule can be present in its native environment within the membrane. The GPMV sensors are a highly stable, consistent, reliable, and scalable reagent suitable for high-throughput drug screening and analysis. 
     Thus, the materials and methods described herein provide means for identifying and assessing interactions between molecules, such as ligands and receptors, or other proteins having a function that can be modulated by interaction with an activator or inhibitor, for example. Thus, this document provides, inter alia, sensors that include a GPMV and a chimeric polypeptide that can be used as a tool to determine whether a candidate compound (e.g., a peptide, nucleic acid, small molecule, or biologic) interacts with a particular target, and to assess the intrinsic effects of the candidate compound on the target. 
     Treating live cells with the organic compound N-ethylmaleimide (NEM) induces vesiculation of the cell membrane, yielding large (“giant”) vesicles that are composed of cytoplasm and plasma membrane. These GPMVs incorporate membrane-associated proteins from the originating cell, but lack any intracellular organelles. GPMVs have been used as a tool to, for example, investigate phase separation in eukaryotic membranes (Sezgin et al. (2012)  Nat. Protoc.  7, 1042-1051), and for the study of membrane proteins such as the immunoglobulin E receptor (IgE-R) and fibroblast growth factor receptor (FGFR3) (Sarabipour and Hristova (2016)  Biochim. Biophys. Acta—Biomembr.  1858, 1436-1442; and Holowka and Baird (1983)  Biochemistry  22, 3466-3474). The lack of intracellular membranous structures in GPMVs provides an adaptable platform for segregating membrane integrated receptors such as GPCRs from the regulation machinery of the cytoplasm. 
     SPASM is a protein engineering technique for modulating and analyzing the interaction between two proteins and/or peptides (Malik et al., supra; and Sivaramakrishnan and Spudich (2011)  Proc. Natl. Acad. Sci. USA  108, 20467-20472. A SPASM sensor is a single polypeptide composed of two proteins of interest fused together at either end of an ER/K α-helix linker (e.g., as depicted in  FIG. 1A ). The presence of a FRET donor and a FRET acceptor that can be present on either side of the linker (between the linker and the proteins of interest), or at the N- and C-termini of the sensor, allows for quantification of protein-protein interaction in live cells. The length of the ER/K linker dictates the relative concentration of the N and C terminal proteins by regulating the on-rate of the proteins, without affecting the off-rate (Sivaramakrishnan and Spudich, supra). Thus, SPASM provides pairwise control over stoichiometry (1:1), co-localization, and relative concentration. SPASM sensors are modular and can be adapted to control and/or monitor interactions between a receptor (e.g., a GPCR) and a peptide or a protein domain derived from a different binding partner, such as a G protein subunit for a SPASM GPCR sensor. 
     As described herein, this document provides GPMVs containing SPASM polypeptides, nucleic acids encoding the polypeptides, host cells containing the nucleic acids, and methods for making and using the GPMVs, cells, polypeptides, and nucleic acids. 
     The term “polypeptide” as used herein refers to a compound of two or more subunit amino acids, regardless of post-translational modification (e.g., phosphorylation or glycosylation). The subunits may be linked by peptide bonds or other bonds such as, for example, ester or ether bonds. The term “amino acid” refers to either natural and/or unnatural or synthetic amino acids, including D/L optical isomers. 
     By “isolated” or “purified” with respect to a polypeptide it is meant that the polypeptide is separated to some extent from cellular components with which it normally is found in nature (e.g., other polypeptides, lipids, carbohydrates, and nucleic acids). A purified polypeptide can yield a single major band on a non-reducing polyacrylamide gel. A purified polypeptide can be at least about 75% pure (e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% pure). Purified polypeptides can be obtained by, for example, extraction from a natural source, by chemical synthesis, or by recombinant production in a host cell or transgenic plant, and can be purified using, for example, affinity chromatography, immunoprecipitation, size exclusion chromatography, and ion exchange chromatography. The extent of purification can be measured using any appropriate method, including, without limitation, column chromatography, polyacrylamide gel electrophoresis, or high-performance liquid chromatography. 
     The terms “nucleic acid” and “polynucleotide” can be used interchangeably, and refer to both RNA and DNA, including cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, and DNA (or RNA) containing nucleic acid analogs. Polynucleotides can have any three-dimensional structure. A nucleic acid can be double-stranded or single-stranded (i.e., a sense strand or an antisense single strand). Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers, as well as nucleic acid analogs. 
     The percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This stand-alone version of BLASTZ can be obtained online at fr.com/blast or at ncbi.nlm.nih.gov. Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C:\seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to −1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq c:\seq1.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q −1 -r 2. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C: \seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt); and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences. 
     Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence (e.g., SEQ ID NO:1), or by an articulated length (e.g., 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100. For example, an amino acid sequence that has 400 matches when aligned with the sequence set forth in SEQ ID NO:1 is 96.7 percent identical to the sequence set forth in SEQ ID NO:1 (i.e., 400±413×100=96.7). It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It also is noted that the length value will always be an integer. 
     The nucleic acids may be incorporated into or contained within recombinant nucleic acid constructs such as vectors. A “vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term “vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes one or more “expression control sequences” that control or regulate the transcription and/or translation of another DNA sequence. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.). 
     Host cells containing a nucleic acid or vector also are provided herein. Suitable host cells can include, without limitation, bacterial cells, yeast cells, and human or non-human mammalian cells (e.g., HEK 293 cells, 3T3 cells, or HeLa cells). Methods for making GPMVs containing GPCR sensor polypeptides from such host cells also are provided herein. Suitable methods for preparing GPMVs include, without limitation, those described in the Examples below. 
     In some embodiments, this document provides GPMVs containing chimeric sensor polypeptides that contain (a) a target GPCR; (b) a linker containing an amino acid sequence flanked by a FRET donor and a FRET acceptor; and (c) a receptor partner for the target GPCR. As used herein, the “receptor partner” can be a peptide, polypeptide, protein, or protein domain having affinity for the target GPCR. Interactions between the target GPCR and the receptor partner can be detected based on fluorescence that results when the FRET donor and FRET acceptor interact. Interaction between a test compound and the target GPCR can be detected based on a change in the FRET ratio for the sensor before and after addition of the test compound to the GPMV sensor. 
     The target GPCR may be any member of the GPCR superfamily. Amino acid sequences for GPCRs include those known in the art and available in, for example, GENBANK®. In some cases, a chimeric polypeptide can include an amino acid sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to a known amino acid sequence for a GPCR. Non-limiting examples of GPCRs include, without limitation, β2AR, vasopressin1 receptor (V1R), and α1AR. A non-limiting example of a representative amino acid sequence for a GPCR is set forth in SEQ ID NO:1, which is the sequence of the human beta 2 adrenergic receptor: 
     
       
         
           
               
            
               
                 (SEQ ID NO: 1) 
               
               
                 MGQPGNGSAFLLAPNRSHAPDHDVTQQRDEVWVVGMGIVMSLIVLAIVF 
               
               
                   
               
               
                 GNVLVITAIAKFERLQTVTNYFITSLACADLVMGLAVVPFGAAHILMKM 
               
               
                   
               
               
                 WTFGNFWCEFWTSIDVLCVTASIETLCVIAVDRYFAITSPFKYQSLLTK 
               
               
                   
               
               
                 NKARVIILMVWIVSGLTSFLPIQMHWYRATHQEAINCYANETCCDFFTN 
               
               
                   
               
               
                 QAYAIASSIVSFYVPLVIMVFVYSRVFQEAKRQLQKIDKSEGRFHVQNL 
               
               
                   
               
               
                 SQVEQDGRTGHGLRRSSKFCLKEHKALKTLGIIMGTFTLCWLPFFIVNI 
               
               
                   
               
               
                 VHVIQDNLIRKEVYILLNWIGYVNSGFNPLIYCRSPDFRIAFQELLCLR 
               
               
                   
               
               
                 RSSLKAYGNGYSSNGNTGEQSGYHVEQEKENKLLCEDLPGTEDFVGHQG 
               
               
                   
               
               
                 TVPSDNIDSQGRNCSTNDSLL. 
               
            
           
         
       
     
     The linker can be positioned between the target GPCR and the receptor partner. Such positioning can permit the target GPCR and the receptor partner to move relative to one another. The linker can have a length of about 8 nm to about 35 nm (e.g., about 8 to 10 nm, about 10 to 15 nm, about 15 to 20 nm, about 20 to 25 nm, about 25 to 30 nm, about 30 to 35 nm, or about 10 to 30 nm). In some embodiments, the polypeptide linker can include an “ER/K” sequence that includes a substantially repeating sequence of glutamic acid, arginine, and lysine residues. For example, the linker may include the sequence set forth in SEQ ID NO:2 (EEEEKKKQQEEEAE RLRRIQEEMEKERKRREEDEERRRKEEEERRMKLEMEAKRKQEEEERKKRED DEKRKKK), or a sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:2, or the linker may include the sequence set forth in SEQ ID NO:3 (EEEEKKKEEEEKKQKEEQERLAKEEAERKQKEEQERLAKE EAERKQKEEEERKQKEEEERKQKEEEERKLKEEQERKAAEEKKAKEEAERK AKEEQERKAEEERKKKEEEERLERERKEREEQEKKAKEEAERIAKLEAEKKA EEERKAKEEEERKAKEEEERKKKEEQERLAKEKEEAERKAAEEKKAKEEQER KEKEEAERKQR), or a sequence that is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO:3. Typically, the linker will be about 50 to about 250 (e.g., about 50 to about 75, about 75 to about 100, about 50 to about 100, about 100 to about 150, about 150 to about 200, about 100 to about 200, or about 200 to about 250) amino acids in length. 
     It is to be noted that the GPCR sensors in the GPMVs provided herein can include any suitable resonance energy transfer donor and acceptor pairs. As described in the Examples herein, for example, mCitrine and mCerulean can be used, but other useful fluorophores include cyan fluorescent proteins (e.g., AmCyan1), green fluorescent proteins (e.g., eGFP, AcGFP1, and ZsGreen1), yellow fluorescent proteins (e.g., ZsYellow1 and mBanana), orange fluorescent proteins (e.g., mOrange and mOrange2), red fluorescent proteins (e.g., DsRed-Express2, DsRed-Express, tdTomato, DsRed-Monomer, DsRed2, AsRed2, and mStrawberry), far-red fluorescent proteins (e.g., HcRedl, mRaspberry, E2-Crimson, and mPlum), and switchable fluorescent proteins (e.g., Dendra2, Timer, and PAmCherry). All of the aforementioned are available from, for example, Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.; Mountain View, Calif.). Other types of resonance energy transfer donors and acceptors can be used, including bioluminescence resonance energy transfer (BRET) donors and/or receptors. 
     In addition, one or more (e.g., two, three, four, or more than four) Gly-Ser-Gly sequences can be present on either side of the FRET acceptor and the FRET donor, and/or between the FRET donor and acceptor and the ER/K sequence. The inclusion of one or more Gly-Ser-Gly sequences can provide rotational freedom between the target GPCR and the receptor partner, which may facilitate interaction between those regions of the chimeric polypeptide. 
     Thus, in some cases, the chimeric polypeptides provided herein can include a target GPCR, a linker amino acid sequence that contains a sequence flanked by a FRET donor and a FRET acceptor, and a receptor partner, in addition to one or more optional components (e.g., one or more Gly-Ser-Gly sequences). For example, in some embodiments a chimeric polypeptide contained in a GPMV sensor may contain, in order from amino terminus to carboxy terminus, a target GPCR having affinity for a receptor partner, a (Gly-Ser-Gly) 2-4  amino acid sequence, a resonance energy transfer donor or acceptor, a (Gly-Ser-Gly) 2-4  amino acid sequence, a linker containing an amino acid sequence at least 90% identical to the sequence set forth in SEQ ID NO:2 or SEQ ID NO:3, a (Gly-Ser-Gly) 24  amino acid sequence, a partner for the resonance energy transfer donor or acceptor, a (Gly-Ser-Gly) 2-4  amino acid sequence, and the receptor partner, which has affinity for the target GPCR. 
     This document also provides nucleic acids that encode the chimeric polypeptides described herein, as well as host cells containing the nucleic acids, and methods for generating GPMV sensors by expressing a chimeric polypeptide in a host cell and then generating GPMVs. Useful methods for transfecting or transforming cells with an expression vector include those known in the art, as well as the methods described in the Examples herein. The methods described in the Examples can be particularly useful, as they include optimized steps. 
     Also provided herein are methods that can be used to identify compounds (e.g., small molecule compounds or biologics, including peptides and nucleic acids) that interact with target GPCRs. In some embodiments, for example, this document provides methods for identifying a compound that can interact with a receptor. The methods can include providing a GPMV sensor as disclosed herein, for example, where the GPMV sensor includes a chimeric polypeptide that contains (1) a target GPCR that contains at least the ligand binding domain from a selected receptor; (2) a linker that includes an amino acid sequence flanked by a FRET donor and a FRET acceptor; and (3) a receptor partner that binds to the target GPCR, such that the linker separates the target GPCR from the receptor partner. The methods also can include measuring an initial FRET ratio for the sensor, contacting the sensor with a candidate compound (e.g., a small molecule or a biologic), measuring a second FRET ratio for the sensor, and identifying the candidate compound as having affinity for the target if the second FRET ratio is increased by at least 0.5% (e.g., at least 0.5%, 1%, 2%, 3%, 4%, 5%, or more than 5%) as compared to the initial FRET ratio. 
     In some cases, this method can be used to identify a ligand specific for a particular target GPCR (i.e., a ligand that can detectably bind to a selected GPCR, but that does not bind detectably to other GPCRs). For example, the above-described method can be conducted using a plurality (e.g., two, three, four, five, or more than five) GPMV sensors, where each sensor contains a different target GPCR. In such methods, an initial FRET ratio and a second FRET ratio can be determined for each sensor in the presence of a candidate compound. If a particular sensor exhibits a FRET ratio that is increased by at least 0.5% after addition of the candidate compound (as determined by comparing the second FRET ratio to the initial FRET ratio), but other sensors do not exhibit FRET ratios that are decreased by at least 0.5%, the candidate compound can be identified as a being specific for the target GPCR of the sensor that demonstrated the decreased FRET ratio. 
     In addition, the methods provided herein can be used to evaluate the intrinsic efficacy of a compound that interacts with a target GPCR. In some cases, such methods can include the following steps: 
     (a) providing a GPMV sensor containing (i) a target GPCR that contains at least the ligand binding domain from a selected receptor, (ii) a linker that includes an amino acid sequence flanked by a FRET donor and a FRET acceptor, and (iii) a receptor partner that binds to the target GPCR, such that the linker separates the target GPCR from the receptor partner; 
     (b) measuring an initial FRET ratio for the sensor; 
     (c) contacting the GPMV sensor with a compound that interacts with the target GPCR; 
     (d) measuring a second FRET ratio for the GPMV sensor; and 
     (e) determining the intrinsic efficacy of the compound for the target based on the difference between the test FRET ratio and the initial FRET ratio. In addition to or instead of FRET, the readout can include BRET between a luciferase and a fluorophore flanking the linker, and/or changes in fluorescence, bioluminescence, or enzymatic activity triggered by interactions between polypeptides flanking the linker. 
     It is to be noted that in addition to providing a technology for identifying ligands and evaluating ligand intrinsic efficacy as described herein, the GPMV technology can be more broadly used to maintain GPCR molecules in their native membrane state. This can, in turn, be used to identify GPCR-targeting therapeutics. For example, an anti-GPCR-based drug discovery method may include immunizing a mammal with a GPMV that does not include a GPCR, and then immunizing the mammal with a GPMV that includes a GPCR, in order to raise antibodies against the GPCR. The antibodies can then be used for drug discovery. Such methods would obviate the need to purify individual GPCR proteins in order to use them for raising antibodies, since the GPMV-associated GPCR molecules would be present in their native environment (the lipid bilayer) and, most likely, their native conformation. 
     The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. 
     EXAMPLES 
     Example 1—Materials and Methods 
     Sensor construct: The plasmid used in the studies described herein included the following sequences in the pcDNA®5 vector (ThermoFisher Scientific; Waltham, Mass.). 
     
       
         
           
               
               
            
               
                 N-terminal HA tag and  GSG linker : 
                   
               
               
                 (SEQ ID NO: 4) 
                   
               
               
                 ATGTATCCTTATGATGTTCCTGATTACGCT GGAAGTGGA ; 
                   
               
               
                   
               
               
                 β2AR (can be substituted into any other GPCR) and  GSG linkers : 
               
               
                 (SEQ ID NO: 5) 
                   
               
               
                 ATGGGGCAACCCGGGAACGGCAGCGCCTTCTTGCTGGCACCCAATAGAAGCCAT 
                   
               
               
                   
               
               
                 GCGCCGGACCACGACGTCACGCAGCAAAGGGACGAGGTGTGGGTGGTGGGC 
               
               
                   
               
               
                 ATGGGCATCGTCATGTCTCTCATCGTCCTGGCCATCGTGTTTGGCAATGTG 
               
               
                   
               
               
                 CTGGTCATCACAGCCATTGCCAAGTTCGAGCGTCTGCAGACGGTCACCAA 
               
               
                   
               
               
                 CTACTTCATCACTTCACTGGCCTGTGCTGATCTGGTCATGGGCCTGGCAGT 
               
               
                   
               
               
                 GGTGCCCTTTGGGGCCGCCCATATTCTTATGAAAATGTGGACTTTTGGCAA 
               
               
                   
               
               
                 CTTCTGGTGCGAGTTTTGGACTTCCATTGATGTGCTGTGCGTCACGGCCAG 
               
               
                   
               
               
                 CATTGAGACCCTGTGCGTGATCGCAGTGGATCGCTACTTTGCCATTACTTC 
               
               
                   
               
               
                 ACCTTTCAAGTACCAGAGCCTGCTGACCAAGAATAAGGCCCGGGTGATCA 
               
               
                   
               
               
                 TTCTGATGGTGTGGATTGTGTCAGGCCTTACCTCCTTCTTGCCCATTCAGA 
               
               
                   
               
               
                 TGCACTGGTACCGGGCCACCCACCAGGAAGCCATCAACTGCTATGCCAAT 
               
               
                   
               
               
                 GAGACCTGCTGTGACTTCTTCACGAACCAAGCCTATGCCATTGCCTCTTCC 
               
               
                   
               
               
                 ATCGTGTCCTTCTACGTTCCCCTGGTGATCATGGTCTTCGTCTACTCCAGG 
               
               
                   
               
               
                 GTCTTTCAGGAGGCCAAAAGGCAGCTCCAGAAGATTGACAAATCTGAGGG 
               
               
                   
               
               
                 CCGCTTCCATGTCCAGAACCTTAGCCAGGTGGAGCAGGATGGGCGGACGG 
               
               
                   
               
               
                 GGCATGGACTCCGCAGATCTTCCAAGTTCTGCTTGAAGGAGCACAAAGCC 
               
               
                   
               
               
                 CTCAAGACGTTAGGCATCATCATGGGCACTTTCACCCTCTGCTGGCTGCCC 
               
               
                   
               
               
                 TTCTTCATCGTTAACATTGTGCATGTGATCCAGGATAACCTCATCCGTAAG 
               
               
                   
               
               
                 GAAGTTTACATCCTCCTAAATTGGATAGGCTATGTCAATTCTGGTTTCAAT 
               
               
                   
               
               
                 CCCCTTATCTACTGCCGGAGCCCAGATTTCAGGATTGCCTTCCAGGAGCTT 
               
               
                   
               
               
                 CTGTGCCTGCGCAGGTCTTCTTTGAAGGCCTATGGGAATGGCTACTCCAGC 
               
               
                   
               
               
                 AACGGCAACACAGGGGAGCAGAGTGGATATCACGTGGAACAGGAGAAAG 
               
               
                   
               
               
                 AAAATAAACTGCTGTGTGAAGACCTCCCAGGCACGGAAGACTTTGTGGGC 
               
               
                   
               
               
                 CATCAAGGTACTGTGCCTAGCGATAACATTGATTCACAAGGGAGGAATTG 
               
               
                   
               
               
                 TAGTACAAATGACTCACTGCTGTCTAGA GGAAGCGGAGGAAGCGGAGGA   
               
               
                   
               
               
                   AGCGGAGGAAGCGGA ; 
               
               
                   
               
               
                 mCitrine,  TEV cleavage site , and  GSG linkers : 
               
               
                 (SEQ ID NO: 6) 
                   
               
               
                 GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGAC 
                   
               
               
                   
               
               
                 GGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCT 
               
               
                   
               
               
                 ACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTG 
               
               
                   
               
               
                 CCCTGGCCCACCCTCGTGACCACCTTCGGCTACGGCCTGATGTGCTTCGCC 
               
               
                   
               
               
                 CGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCC 
               
               
                   
               
               
                 CGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACT 
               
               
                   
               
               
                 ACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCG 
               
               
                   
               
               
                 CATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGG 
               
               
                   
               
               
                 CACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGA 
               
               
                   
               
               
                 CAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATC 
               
               
                   
               
               
                 GAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCAT 
               
               
                   
               
               
                 CGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCTACCAGT 
               
               
                   
               
               
                 CCAAACTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTG 
               
               
                   
               
               
                 GAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAA 
               
               
                   
               
               
                 GGAATTC GGAAGCGGAGGAAGCGGA   GAAAACCTGTATTTTCAGGGC   GGAAG   
               
               
                   
               
               
                   CGGA ; 
               
               
                   
               
               
                 10 nm ER/K linker and  GSG linkers : 
               
               
                 (SEQ ID NO: 7) 
                   
               
               
                 GAAGAGGAAGAGAAAAAAAAA 
                   
               
               
                   
               
               
                 CAGCAGGAAGAGGAAGCAGAAAGGCTGAGGCGTATTCAAGAAGAAATGG 
               
               
                   
               
               
                 AAAAGGAAAGAAAAAGACGTGAAGATGACGAACAACGTCGAAGAAAGG 
               
               
                   
               
               
                 AAGAGGAGGAAAGGCGGATGAAACTTGAGATGGAAGCAAAGAGAAAAC 
               
               
                   
               
               
                 AAGAAGAAGAAGAGAGAAAGAAAAGGGAAGATGATGAAAAACGCAAGA 
               
               
                   
               
               
                 AGAAG GGCTCTGGCGGCTCTGGC ; 
               
               
                   
               
               
                 mCerulean and  GSG linkers : 
               
               
                 (SEQ ID NO: 8) 
                   
               
               
                 GTGAGCAAGGGCGAGGAGCTGTTCACCG 
                   
               
               
                   
               
               
                 GGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAG 
               
               
                   
               
               
                 TTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGAC 
               
               
                   
               
               
                 CCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCT 
               
               
                   
               
               
                 CGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCGCCCGCTACCCCGACC 
               
               
                   
               
               
                 ACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTC 
               
               
                   
               
               
                 CAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGC 
               
               
                   
               
               
                 CGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAG 
               
               
                   
               
               
                 GGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGT 
               
               
                   
               
               
                 ACAACGCCATCAGCGACAACGTCTATATCACCGCCGACAAGCAGAAGAA 
               
               
                   
               
               
                 CGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC 
               
               
                   
               
               
                 GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC 
               
               
                   
               
               
                 CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCAAGCTGAGCA 
               
               
                   
               
               
                 AAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACC 
               
               
                   
               
               
                 GCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTTAATTAAC GG   
               
               
                   
               
               
                   AAGCGGCGGGTCAGGTGGAAGTGGA ; 
               
               
                 and 
               
               
                   
               
               
                 GαS C-terminal α-5 peptide (can be substituted for other binding partners): 
               
               
                 (SEQ ID NO: 9) 
                   
               
               
                 GACACTGAGAACATCCGCCGTGTGTTCAACGACTGCCGTGACATCATTCA 
                   
               
               
                 GCGCATGCACCTTCGTCAGTACGAGCTGCTC. 
               
            
           
         
       
     
     Cell culture preparation: HEK-293T-Flp-n cells were cultured in DMEM containing 4.5 g/L D-glucose, with 10% FBS (heat inactivated) (v/v), and 1% L-glutamine, 20 mM HEPES, pH 7.5. Cells were incubated at 37° C. in a humidified atmosphere at 5% CO 2 . Cells were allowed to grow to 95% confluency. The time to achieve confluency typically took about 24 hours when plated with 2×10 6  cells/ml in 10 ml media, and a confluent 10 cm tissue culture treated dish typically had a density of 4-6×10 6  cells/ml. 
     Transfection: The following reagents were combined in the listed order: 1 ml OPTI-MEM™, 20 μg DNA, 60 μg polyethylenimine (PEI). The mixture was incubated at room temperature for 15 to 30 minutes. It is noted that other transfection agents (e.g., X-TREMEGENE™, Sigma) may be used in place of PEI, but the described protocol has been optimized for PEI as a transfection agent. Cells to be transfected were washed with 10 ml PBS, and then detached from culture dishes with 1 ml 0.25% trypsin. Detached cells were resuspended in DMEM (pre-warmed to 37° C.) to a concentration of 3×10 6  cells/ml. The DNA+PEI mixture was added into 10 ml of resuspended cells and inverted gently 5 times. Transfection in suspension saved time and assured that the ratio of cells to the amount of transfection reagent remained consistent. The amount of DNA and the density of the cells was optimal for maximum cell transfection efficiency. In fact, compared to transfecting cells on a surface, transfecting cells in suspension resulted in a 51% increase in GPCR-SPASM sensor incorporation into GPMVs when using PEI. In some cases, a leucine-rich, cleavable “Lucy” tag (MRPQILLLLALLTLGLA; SEQ ID NO:10) (Chan et al. (2011) BMC Biochem 12:27; and Shepard et al. (2013)  PLoS One  8:e68758, https://doi.org/10.1371/journal.pone.0068758) was added at the N-terminus of the GPCR to enhance surface GPCR expression and therefore increase GPCR expression in GPVMs. 
     Transfected cells were then plated onto a 15 cm tissue culture treated dish and incubated at 37° C. in a humidified atmosphere at 5% CO 2  for 4 hours. The 10 ml of media containing PEI was then exchanged with 20 ml of fresh DMEM (pre-warmed to 37° C. prior to exchange). The 4 hour incubation prior to media exchange was optimal for maximum transfection efficiency and minimal cell toxicity due to the PEI. In addition, the 10 ml media volume was optimal for facilitating adhesion to the surface within the 4 hour timeframe. After 24 hours of expression, fluorescence intensity and localization within the cells was assessed using a tissue culture fluorescence microscope. The fluorescence was checked every 2 hours until optimal fluorescence intensity vs. internalization was achieved. 
     GPMV synthesis and purification: Cells were scraped off the 15 cm tissue culture dish and transferred to a 50 ml conical tube. The standard protocol (see, Sezgin et al., 2012) synthesizes GPMVs from adherent cells, but the optimized protocol described herein synthesized GPMVs in suspension. GPMVs synthesized in suspension were purified straight from solution, thus increasing yield and eliminating the need for additional washes to release GPMVs lingering on the surface of tissue culture treated dishes when they are synthesized from adherent cells. 
     For 2 mM N-ethylmaleimide- (NEM-) mediated GPMV synthesis, cells were centrifuged at 300×g and resuspended in 20 ml GPMV buffer (10 mM HEPES pH 7.5, 150 mM NaCl, 2 mM CaCl 2 ), and then spun and resuspended once more. Cells were transferred to a glass Erlenmeyer flask and spiked with 2 mM NEM before being incubated in a shaking incubator at 28° C. for 2 hours. For bicine-mediated GPMV synthesis, cells were spun at 300×g and resuspended in 20 ml commercial PBS pH 7.4 (ThermoFisher Scientific). Cells were then resuspended in bicine-GPMV buffer (200 mM NaCl, 5 mM KCl, 0.5 mM MgCl 2 , 0.75 mM CaCl 2 , 100 mM bicine pH 8.5), transferred to a glass Erlenmeyer flask, and incubated in a shaking incubator at 28° C. for 2 hours. 
     For either method, the GPMV+cell solution was centrifuged at 100×g 4° C. for 10 minutes to pellet dead cells and debris, and the supernatant containing GPMVs was transferred to a 50 ml conical tube. GPMVs were maintained on ice for subsequent procedures. The GPMV solution was centrifuged at 3000×g 4° C. for 30 minutes to pellet GPMVs, which were resuspended in 10 ml HBS buffer and transferred to a 15 ml conical tube. It is noted that the standard protocol (Sezgin et al., 2012) pellets GPMVs at 20,000×g for 1 hour, whereas the present protocol has been optimized to 3000×g for 30 minutes, which is sufficient for GPMV pelleting without clumping and deformation of GPMVs. This protocol thus saves time (30 minutes vs. 1 hour) and does not require high-cost ultracentrifuge instruments. The GPMV solution was then centrifuged at 100×g 4° C. for 10 minutes to pellet any leftover dead cells and debris, since the presence of dead cells in FRET experiments can negate the change in FRET induced by ligands, due to internalized non-functional sensors within the dead cells. Thus, the protocol utilized as described herein included an additional low speed spin to exclude as many dead cells and as much debris from the solution as possible. The resulting GPMV solution was centrifuged at 3000×g 4° C. for 30 minutes to pellet GPMVs, which were resuspended in 1 ml HBS buffer or optimized buffer (20 mM HEPES pH 7.5, 25 mM KCl, 5 mM MgCl 2 ) and maintained on ice. 
     Experiment and data collection: mCerulean fluorescence counts (peak emission at 475 nm) of GPMVs were measured using a fluorometer (excitation 430 nm, bandpass 8 nm, emission 450-600 nm, bandpass 4 nm). GPMVs were diluted in HBS or optimized buffer to achieve 600,000 mCerulean fluorescence counts. One (1) mM ascorbic acid was added to the GPMV solution. The drug buffer was prepared by adding 1 mM ascorbic acid to 1 ml HBS or optimized buffer; the buffer was maintained on ice for subsequent procedures. GPCR ligands were diluted in the drug buffer to 10×[desired concentration]. 400 μl of GPMV solution was mixed with 44 μl of 10×ligand, and incubated at room temperature for 5 minutes. FRET spectra of GPMVs were acquired using a fluorometer (excitation 430 nm, bandpass 8 nm, emission 450-600 nm, bandpass 4 nm). The scatter background counts of the 475 nm and 525 nm emission counts were calculated by referencing the 450 nm scatter count and subtracting background emission counts from the total 475 nm and 525 nm emission counts. The FRET ratio (475 nm emission/525 nm emission) also was calculated. 
     Experiment and data collection in 96 well plate reader: mCerulean fluorescence counts (peak emission at 475 nm) for GPMVs in optimized buffer were acquired using a fluorometer (excitation 430 nm, bandpass 8 nm, emission 450-600 nm, bandpass 4 nm). Specifically, GPMVs were diluted in optimized buffer to achieve 2,000,000 mCerulean fluorescence counts. Ascorbic acid (1 mM) was added to the GPMV solution. Drug buffer was prepared by adding 1 mM ascorbic acid to 1 ml optimized buffer. The mixture was maintained on ice for subsequent procedures. GPCR ligands were diluted in the drug buffer to 10×[desired concentration]. In a clear-bottom 96 well plate, 10 μl of GPCR ligand or buffer was added to each well, and then 90 μl GPMVs was added to each ligand-containing well. Plates were incubated at room temperature for 5 minutes. The plates were then placed in a FLEXSTATION® 3 Multi-Mode Microplate Reader (Molecular Devices; San Jose, Calif.). The donor fluorophore (mCerulean, excitation 435 nm) was excited and a spectrum read was taken (bottom read, emission 500-550 nm, emission step 5 nm, cutoff 515 nm, sensitivity 40 reads per well). The acceptor fluorophore (mCitrine, excitation 485 nm) was excited and a spectrum read was taken (bottom read, emission 500-550 nm, emission step 5 nm, cutoff 515 nm, sensitivity 40 reads per well). The optical density (600 nm) was read for each well. The 525 nm emission counts from both the 435 nm and 485 nm excited spectrum were isolated. The scatter background 525 nm emission counts were calculated by referencing the optical density and subtracting background emission counts from the total 525 nm emission counts. The FRET efficiency was calculated using the following equation: 
     
       
         
           
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                       f 
                       
                         
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                         .485 
                       
                     
                   
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     F A.435 =525 nm emission fluorescence counts when excited at 435 nm. F A.485 =525 nm emission fluorescence counts when excited at 485 nm. ε A.435 =molar extinction coefficient of acceptor fluorescent protein at 435 nm. ε A.485 =molar extinction coefficient of acceptor fluorescent protein at 485 nm. ε D.435 =molar extinction coefficient of donor fluorescent protein at 435 nm. ε A.435 /ε A.485 =0.08096. ε A.485 /ε D.435 =0.713. 
     Example 2—Purified GPMVs Incorporate SPASM GPCR Sensors 
     As described elsewhere, SPASM GPCR sensors can be used to probe G protein-selective GPCR conformation in live cells (Malik et al., supra; and Semack et al., supra). GPMVs provide a convenient way of extracting a cell&#39;s plasma membrane from the cytoplasm regulation machinery, and possess the potential to incorporate GPCRs. To test the utility of GPMVs with SPASM sensors as a cell-derived agent, GPMVs were synthesized and purified from HEK293 cells expressing a β2AR-SPASM sensor (Malik et al., supra). The sensor (β2AR-Spep) contains the following from N- to C-terminus: full-length β2AR, mCitrine (FRET acceptor), a 10 nm ER/K α-helix linker, mCerulean (FRET donor), and a 27-amino acid peptide derived from the C-terminal α5 helix of the GaS subunit, all of which are separated via (Gly-Ser-Gly) 4  linkers ( FIG. 1A ). A schematic of the GPMV purification process is illustrated in  FIG. 1B . Briefly, HEK293 cells expressing the β2AR-Spep sensors were washed and resuspended in GPMV buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2 mM CaCl 2 ). Cells were then incubated with 2 mM NEM for 2 hours at 30° C. Dead cells and debris were pelleted at 100×g for 10 minutes, and the supernatant containing GPMVs was extracted. GPMVs were further washed with HBS via three rounds of pelleting (3000×g for 30 minutes) and resuspension. Fluorescent imaging was then used to demonstrate that β2AR-Spep sensors were incorporated into GPMVs. The sensors were clearly visible and exhibited a nearly homogenous distribution for each GPMV ( FIG. 1C ). The GPMVs were spherical in shape, in agreement with the literature, and exhibited varying sizes in the range of 2-6 μm ( FIG. 1C ). Together, these data report the successful incorporation of SPASM GPCR sensors into GPMVs. 
     Example 3—GPMV-Incorporated Membrane Proteins are Functional 
     The functionality of the β2AR-Spep sensors in GPMVs was then investigated. In particular, the function of the β2AR-Spep sensor was tested by a combination of radioligand binding and concentration-response analyses ( FIG. 2A ). β2AR-Spep sensors incorporated in GPMVs (GPMV-β2AR-Spep) bound robustly to both the orthosteric antagonist [ 125 I]cyanopindolol and the agonist isoproterenol. The affinities (pKd and pKi) for [ 125 I]cyanopindolol and isoproterenol were 6.59 and 8.59, respectively. Notably, under the same concentration of β2AR-Spep sensors (monitored via mCerulean fluorescence counts), GPMVs had a twenty-fold higher ligand binding capacity than purified membrane preparations (Gupte et al. (2017)  Proc. Natl. Acad. Sci. USA  114, 3756-3761), suggesting significantly higher levels of functional GPCR. This may stem from the fact that the GPMVs originated exclusively from the plasma membrane, whereas purified membrane extracts also contain intracellular membrane components such as the ER and/or golgi. β2AR-Spep sensors in the ER or golgi would be non-functional and unable to bind to GPCR ligands. Thus, a portion of the β2AR-Spep sensors included in the membrane extracts could be non-mature and non-functional, while most of the GPCRs incorporated into the GPMVs are mature/functional receptors that have been localized to the plasma membrane. Hence, the data provided herein demonstrate that the fraction of functional β2AR-Spep sensors is significantly higher in GPMVs than in membrane extracts. Additionally, stimulating GPMV-β2AR-Spep sensors with isoproterenol induced cAMP synthesis, indicating that both the endogenous Ga subunits and the endogenous adenylate cyclase present in the GPMVs were functional ( FIG. 2B ). Together, these data show that the membrane proteins incorporated in GPMVs, including SPASM GPCR sensors, retain their function. 
     Example 4—Characterization of the FRET Response in Purified GPMV-β2AR-Spep 
     Studies were then undertaken to assess the utility of GPMVs incorporating SPASM GPCR sensors (GPMV sensors) as a cell free FRET-based assay for monitoring GPCR-α5 helix interaction. Toward this goal, the FRET measurements of SPASM GPCR sensors in GPMVs were characterized. SPASM GPCR sensors report the interaction between GPCRs and the C terminal α5 helix through FRET measurements (Malik et al., supra; and Semack et al., supra). As described elsewhere, β2AR-Spep sensors exhibit an agonist-dependent gain in FRET in live cells (Semack et al. (2016)  J. Vis. Exp.  11554696, 1-11). The studies described herein showed that similarly, purified GPMV-β2AR-Spep sensors display a gain in FRET when stimulated with isoproterenol, comparable to the gain described for live cells ( FIG. 3A ). The observed gain in FRET was specific for the C terminal α5 helix of the GaS subunit, demonstrating that the sensor probes G protein-selective GPCR conformations. Specificity of the gain in FRET was further demonstrated by the concentration dependence of the isoproterenol response ( FIG. 3B ). Additionally, the orthosteric antagonist ICI 118,551 competitively suppressed the gain in FRET observed at saturating isoproterenol concentrations (100 μM). These observations exemplify that the FRET measurements observed with GPMV-β2AR-Spep sensors can probe the interaction between PAR and the C terminal α5 of the GaS subunit. Together, these data demonstrate the utility of GPMV sensors as a cell free FRET-based assay for monitoring GPCR-α5 helix interaction and G protein-selective GPCR conformations. 
     Example 5—Advantages of GPMV Over Live Cell Experiments 
     Due to the dynamic nature of the cell regulation machinery, SPASM-FRET measurements in live cells are sensitive to time-dependent factors such as expression levels and stimulation length. In contrast, GPMV-β2AR-Spep sensors showed robust response regardless of these elements. In live cells, the expression level of β2AR-Spep influenced FRET measurements, where excessive overexpression tended to saturate the system and decrease the dynamic range of ΔFRET ( FIG. 4A ). Conversely, GPMVs synthesized from HEK293 cells excessively overexpressing (48 hour expression) β2AR-Spep exhibited the same level of ΔFRET as GPMVs synthesized from much lower expressing (24 hour expression) cells. Thus, the minimal sensitivity of GPMV sensors to time-dependent factors provides a higher level of consistency in FRET measurements than live cells. Further, purified GPMV-β2AR-Spep sensors sustained a constant level of agonist-specific ΔFRET for 12 days when preserved on ice, representing a high level of stability. Additionally, these sensors could be frozen under various conditions and did not exhibit any significant loss in function post-thawing ( FIG. 4B ). The ability to stably freeze-thaw GPMV-β2AR-Spep allows for large-scale production and storage of the reagent, providing multiple experiments from a common batch. Collectively, these observations exemplified the consistency, stability, and scalability of GPMV sensors. In addition, the use of a modified low salt buffer (20 mM HEPES pH 7.4, 25 mM KCl, 5 mM MgCl 2 ) provides a higher ΔFRET (˜0.07), thereby substantially increasing the dynamic range of the GPMV-based assay. GPMVs can also be permeabilized with detergents (e.g., saponin) to modulate the intra-GPMV environment. For example, permeabilization can be used to study the influence of soluble biologics and small-molecules, such as nucleotides, on GPCR signaling in a native environment. 
     Example 6—Measurement of Intrinsic Efficacy Utilizing SPASM GPCR Sensors Integrated into GPMVs 
     A ligand&#39;s ability to elicit a specific downstream response is termed “ligand efficacy,” and is dictated by the cellular context and the ligand&#39;s “intrinsic efficacy” (Kenakin (2014)  A Pharmacology Primer: Techniques for More Effective and Strategic Drug Discovery: Fourth Edition , Elsevier, Inc.). The “intrinsic efficacy” is a ligand-specific term defining the level of stimulus that a bound ligand exerts on a single receptor (Kenakin, supra). However, measurement of intrinsic efficacy has eluded researchers due to the multitude of factors that regulate GPCR signaling (Salazar et al. (2007)  Biochim. Biophys. Acta—Biomembr.  1768, 1006-1018). The closest generalizable method of measuring intrinsic efficacy to date is monitoring G protein activation (Harrison and Traynor, supra; Denis et al., supra; and Gregorio et al., supra). GPCR-mediated activation of G proteins is measured utilizing live cells, crude membrane extracts, or purified receptors and G proteins. Both systems present technical limitations that restrict their use as reliable and scalable high-throughput assays. Purifying GPCRs and G proteins is a multiple-step process, which requires laborious optimizations for each procedure. Further, the inherent heterogeneity of live cells and crude membrane extracts introduces inconsistency between observations. As such, current methods of measuring intrinsic efficacy either requires high cost or suffers low reliability. Here 
     The utility of the GPMV sensors described herein for measuring intrinsic efficacy was therefore assessed. Toward that end, FRET measurements in GPMV-β2AR-Spep sensors were monitored following stimulation via ligands with efficacy profiles ranging from inverse agonists to full agonists ( FIGS. 5A and 5B ). FRET measurements in agonist-stimulated GPMV-β2AR-Spep sensors correlated strongly (R 2 =0.99) with intrinsic efficacies and G protein activation documented elsewhere (Gregorio et al., supra). These data demonstrated the utility of GPMV sensors as a modular assay that reports the efficacies of GPCR ligands through FRET measurements. The scalable production of GPMV sensors, alongside the high level of consistency and stability in FRET measurements, provides a novel and facile cell-derived assay for measuring the intrinsic efficacies of GPCR ligands. 
     Example 7—Buffer Optimization 
     Salts such as sodium and magnesium can affect the function of GPCR by acting as allosteric modulators (see, e.g., Ye et al. (2018)  Nat Commun  9:1372; |DOI: 10.1038/s41467-018-03314-9). The possibility of increasing the dynamic range of the GPMV-GPCR-FRET sensors by altering the experimental salt concentrations was therefore assessed. Substituting HEPES buffered saline (20 mM HEPES pH 7.4, 5 mM KCl, 145 mM NaCl, 2 mM CaCl 2 , 1 mM MgCl 2 ) with an optimized buffer (20 mM HEPES pH 7.4, 25 mM KCl, 5 mM MgCl 2 ) significantly increased the dynamic range of the FRET measurements. It is noted that the optimized buffer excluded sodium, a known negative allosteric modulator (Ye et al., supra). FRET experiments with multiple ligands were carried out in low and high salt conditions, and the results were compared with previously documented measurements of ligand intrinsic efficacies (Gregorio et al., supra). Though FRET measurement for a common ligand differed drastically between low and high salt concentrations, FRET measurements for all ligands in both low and high salt conditions correlated strongly with both the ligands&#39; ability to induce molecular efficacy ( FIG. 5A , R 2 =0.96˜0.99) and GTP turnover via purified β2AR-GaS protein complex in vitro ( FIG. 5B , R 2 =0.79˜0.97). These results indicated that salt concentration can significantly influence the intrinsic efficacy maxima of GPCR ligands, but do not alter the relative intrinsic efficacies between GPCR ligands. 
     Example 8—Comparison with Parameter that Dictate Classical Efficacy 
     FRET measurements also were compared with results of a study that measured the efficacies of various ligands using classic pharmacological methods (Baker, supra). The study utilized cell-based assays generally utilized in classical pharmacology to derive the parameter “efficacy ratio.” Though the experimental measurements and parameters described in the study are generally used in classical pharmacology as a measurement of ligand efficacy, the readout of these cell-based assays are several levels removed from the actual receptor-G protein interaction. Thus, it was observed that the FRET measurements, which represent ligand intrinsic efficacies as established above ( FIGS. 5A and 5B ), did not correlate with the documented efficacy ratios ( FIG. 6 ) or with experimental measurements dictating the efficacy ratios ( FIGS. 7A-7C ). These observations present an example of the importance of utilizing tools that investigate phenomena occurring at the level of the receptor-G protein complex when studying ligand intrinsic efficacy, advocating for the utility of the GPMV-GPCR-FRET sensors described herein, as the readout of these sensors is dictated directly by the ligand&#39;s effect on the function of the receptor. 
     Example 9—GPMV-GPCR Sensors can be Utilized in a High-Throughput Manner 
     GPMV-β2AR-Spep sensors were stimulated with various ligands, and fluorescence measurements were taken utilizing a high-throughput 96-well plate reader. Measurements taken via the plate reader were used to calculate the ΔFRET efficiencies as described in elsewhere (Toropova et al., supra), and plotted against the ΔFRET ratios shown in  FIGS. 5A and 5B . Calculated ΔFRET ratios correlated linearly (R 2 =0.97) with ΔFRET efficiencies ( FIG. 8 ), demonstrating that GPMV-GPCR sensors can be utilized in a high-throughput manner. 
     Example 10—GPMV Synthesis Utilizing Bicine 
     An alternative method of synthesizing GPMVs utilizes a buffer containing of various salts and the organic compound, bicine, as a buffering agent (see, Del Piccolo et al. (2012)  Anal Chem  84(20):8650-8655). An advantage of this method is that it does not use any chemical agents that might cross-link to free cysteines. Utilizing the bicine-mediated GPMV synthesis method, GPMVs were synthesized to incorporate various GPMV-GPCR sensors. ΔFRET measurements ( FIG. 9 ) were comparable to those observed for GPMVs synthesized with the NEM-mediated method. 
     Example 11—GPMV-GPCR Sensors are Generally Applicable to a Variety of GPCRs and G Proteins 
     GPMVs incorporating various GPCR sensors (β2AR, V1R, and α1AR) were synthesized using the bicine-mediated method. Each sensor was tethered to the C-terminal α5-helix peptide from its cognate G-protein (Gs, Gq, and Gi, respectively). The GPMV-GPCR sensors were then treated with their respective full agonists (100 μM isoproterenol, 100 nM [Are]vasopressin (AVP), and 100 μM epinephrine, respectively), and changes in FRET was measured. The GPMV-GPCR sensors exhibited ΔFRET levels ( FIG. 9 ) comparable to those obtained in the other experiments described herein, indicating that GPMV-GPCR sensors are generally applicable to a variety of GPCR, regardless of the type of cognate G protein. 
     OTHER EMBODIMENTS 
     It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.