G Protein-coupled Receptors Function Research Articles

G Protein-Coupled Receptor–Ligand Pose and Functional Class Prediction

G protein-coupled receptor (GPCR) transmembrane protein family members play essential roles in physiology. Numerous pharmaceuticals target GPCRs, and many drug discovery programs utilize virtual screening (VS) against GPCR targets. Improvements in the accuracy of predicting new molecules that bind to and either activate or inhibit GPCR function would accelerate such drug discovery programs. This work addresses two significant research questions. First, do ligand interaction fingerprints provide a substantial advantage over automated methods of binding site selection for classical docking? Second, can the functional status of prospective screening candidates be predicted from ligand interaction fingerprints using a random forest classifier? Ligand interaction fingerprints were found to offer modest advantages in sampling accurate poses, but no substantial advantage in the final set of top-ranked poses after scoring, and, thus, were not used in the generation of the ligand–receptor complexes used to train and test the random forest classifier. A binary classifier which treated agonists, antagonists, and inverse agonists as active and all other ligands as inactive proved highly effective in ligand function prediction in an external test set of GPR31 and TAAR2 candidate ligands with a hit rate of 82.6% actual actives within the set of predicted actives.

A universal method for surface-based binding assays by preparing immobilized β2-adrenergic receptor stationary phase using solid binding peptide as a fusion tag

Protein functionalized surface has the potential to develop new assays for determining the drug-like properties of potential compounds and discovering specific partners of G protein-coupled receptors (GPCRs). However, a universal method for purifying and immobilizing functional GPCRs has remained elusive. To this end, we developed a general and rapid way to purify and immobilize β2-adrenergic receptor (β2AR) by silicon-specific peptide. We screened CotB1p as a tag from six silica-binding peptides (minTBP-1, CotB1p, SB7, Car9, and Si4–1) by examining their affinity to macroporous silica gel. We investigated the adsorption and desorption of CotB1p-tagged β2-adrenoceptor (β2AR-CotB1p) under diverse conditions to propose a protocol for receptor purification and immobilization. Under optimized conditions, β2AR immobilization were achieved by directly immersing cell lysates harboring the receptor with silica gel, and the elution of the receptor without demonstratable contaminants was realized by including l-arginine/L-lysine in the elutes. This allows purification of the receptor from Escherichia coli (E.coli) lysates with a purity of 95 %. The immobilized receptor was utilized as a stationary phase to reveal the tag impact on ligand-binding outputs by comparing the CotB1p-strategy with a typical covalent method. The KAs of salbutamol, chlorprenaline, tulobuterol, and terbutaline on β2AR-CotB1p column were 1.26 × 106, 6.59 × 106, 7.90 × 106, and 8.97 × 105 M−1 respectively, which were two orders of magnitude higher than those on the Halo-β2AR column. The whole immobilization was accomplished within 30 min without the requirement of any special treatment, resulting in enhanced accuracy for determining receptor-ligand binding parameters. Taken together, CotB1p-mediated strategy is simple, rapid, and universal for purification or immobilization of unstable biomolecules like GPCRs for analytical and biological applications.

Differential regulation of G protein-coupled receptor-associated proteins in the caudate and the putamen of cynomolgus macaques following chronic ethanol drinking.

The dorsal striatum is composed of the caudate nucleus and the putamen in human and non-human primates. These two regions receive different cortical projections and are functionally distinct. The caudate is involved in the control of goal-directed behaviors, while the putamen is implicated in habit learning and formation. Previous reports indicate that ethanol differentially influences neurotransmission in these two regions. Because neurotransmitters primarily signal through G protein-coupled receptors (GPCRs) to modulate neuronal activity, the present study aimed to determine whether ethanol had a region-dependent impact on the expression of proteins that are involved in the trafficking and function of GPCRs, including G protein subunits and their effectors, protein kinases, and elements of the cytoskeleton. Western blotting was performed to examine protein levels in the caudate and the putamen of male cynomolgus macaques that self-administered ethanol for 1 year under free access conditions, along with control animals that self-administered an isocaloric sweetened solution under identical operant conditions. Among the 18 proteins studied, we found that the levels of one protein (PKCβ) were increased, and 13 proteins (Gαi1/3, Gαi2, Gαo, Gβ1γ, PKCα, PKCε, CaMKII, GSK3β, β-actin, cofilin, α-tubulin, and tubulin polymerization promoting protein) were reduced in the caudate of alcohol-drinking macaques. However, ethanol did not alter the expression of any proteins examined in the putamen. These observations underscore the unique vulnerability of the caudate nucleus to changes in protein expression induced by chronic ethanol exposure. Whether these alterations are associated with ethanol-induced dysregulation of GPCR function and neurotransmission warrants future investigation.

Bilayer lipids modulate ligand binding to atypical chemokine receptor 3.

Chemokine receptors belong to the large class of G protein-coupled receptors (GPCRs) and are involved in a number of (patho)physiological processes. Previous studies highlighted the importance of membrane lipids for modulating GPCR structure and function. However, the underlying mechanisms of how lipids regulate GPCRs are often poorly understood. Here, we report that anionic lipid bilayers increase the binding affinity of the chemokine CXCL12 for the atypical chemokine receptor 3 (ACKR3) by modulating the CXCL12 binding kinetics. Notably, the anionic bilayer favors CXCL12 over the more positively charged chemokine CXCL11, which we explained by bilayer interactions orienting CXCL12 but not CXCL11 for productive ACKR3 binding. Furthermore, our data suggest a stabilization of active ACKR3 conformations in anionic bilayers. Taken together, the described regulation of chemokine selectivity of ACKR3 by the lipid bilayer proposes an extended version of the classical model of chemokine binding including the lipid environment of the receptor.

Deciphering the role of glycosaminoglycans in GPCR signaling

G protein-coupled receptors (GPCR) and glycosaminoglycans (GAGs) are two essential components of the cell surface that regulate physiological processes in the body. GPCRs are the most extensive family of transmembrane receptors that control cellular responses to extracellular stimuli, while GAGs are polysaccharides that contribute to the function of the extracellular matrix (ECM). Due to their proximity to the plasma membrane, GAGs participate in signal transduction by interacting with various extracellular molecules and cell surface receptors. GAGs can directly interact with certain GPCRs or their ligands (chemokines, peptide hormones and neuropeptides, structural proteins, and enzymes) from the glutamate receptor family, the rhodopsin receptor family, the adhesion receptor family, and the secretin receptor family. These interactions have recently become an emerging topic, providing a new avenue for understanding how GPCR signaling is regulated. This review discusses our current state of knowledge about the role of GAGs in GPCR signaling and function.

Cryo-EM advances in GPCR structure determination.

G-protein-coupled receptors (GPCRs) constitute a prominent superfamily in humans, and are categorized into six classes (A through F) that play indispensable roles in cellular communication and therapeutics. Nonetheless, their structural comprehension has been limited by challenges in high-resolution data acquisition. This review highlights the transformative impact of cryogenic electron microscopy (cryo-EM) in the structural determinations of GPCR-G-protein complexes. Specific technologies such as nanobodies and mini-G-proteins stabilize complexes and facilitate structural determination. We discuss the structural alterations upon receptor activation in different GPCR classes, revealing their diverse mechanisms. These cryo-EM structures provide a robust foundation for comprehending GPCR function and pave the way for future breakthroughs in drug discovery and therapeutic targeting.

2023 Julius Axelrod Symposium: Plant-Derived Molecules Acting on G Protein-Coupled Receptors.

Plant extracts have played a significant role in traditional medicine for centuries, contributing to improved health and the treatment of various human illnesses. G protein-coupled receptors (GPCRs) are crucial in numerous physiologic functions, and there is growing evidence suggesting their involvement in the therapeutic effects of many plant extracts. In recent years, scientists have identified an expanding number of isolated molecules responsible for the biologic activity of these extracts, with many believed to act on GPCRs. This article critically reviews the evidence supporting the modulation of GPCR function by these plant-derived molecules through direct binding. Structural information is now available for some of these molecules, allowing for a comparison of their binding mode with that of endogenous GPCR ligands. The final section explores future trends and challenges, focusing on the identification of new plant-derived molecules with both orthosteric and allosteric binding modes, as well as innovative strategies for designing GPCR ligands inspired by these plant-derived compounds. In conclusion, plant-derived molecules are anticipated to play an increasingly vital role as therapeutic drugs and serve as templates for drug design. SIGNIFICANCE STATEMENT: This minireview summarizes the most pertinent publications on isolated plant-derived molecules interacting with G protein-coupled receptors (GPCRs) and comments on available structural information on GPCR/plant-derived ligand pairs. Future challenges and trends for the isolation and characterization of plant-derived molecules and drug design are discussed.

Membrane mimetic-dependence of GPCR energy landscapes

We leveraged variable-temperature 19F-NMR spectroscopy to compare the conformational equilibria of the human A2A adenosine receptor (A2AAR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19F-NMR experiments to physiological temperature. A2AAR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A2AAR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A2AAR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function.

GPCRs in the round: SMA-like copolymers and SMALPs as a platform for investigating GPCRs

G-protein-coupled receptors (GPCRs) are the largest family of membrane proteins, regulate a plethora of physiological responses and are the therapeutic target for 30–40% of clinically-prescribed drugs. They are integral membrane proteins deeply embedded in the plasma membrane where they activate intracellular signalling via coupling to G-proteins and β-arrestin. GPCRs are in intimate association with the bilayer lipids and that lipid environment regulates the signalling functions of GPCRs. This complex lipid ‘landscape’ is both heterogeneous and dynamic. GPCR function is modulated by bulk membrane properties including membrane fluidity, microdomains, curvature, thickness and asymmetry but GPCRs are also regulated by specific lipid:GPCR binding, including cholesterol and anionic lipids. Understanding the molecular mechanisms whereby GPCR signalling is regulated by lipids is a very active area of research currently. A major advance in membrane protein research in recent years was the application of poly(styrene-co-maleic acid) (SMA) copolymers. These spontaneously generate SMA lipid particles (SMALPs) encapsulating membrane protein in a nano-scale disc of cell membrane, thereby removing the historical need for detergent and preserving lipid:GPCR interaction. The focus of this review is how GPCR-SMALPs are increasing our understanding of GPCR structure and function at the molecular level. Furthermore, an increasing number of ‘second generation’ SMA-like copolymers have been reported recently. These are reviewed from the context of increasing our understanding of GPCR molecular mechanisms. Moreover, their potential as a novel platform for downstream biophysical and structural analyses is assessed and looking ahead, the translational application of SMA-like copolymers to GPCR drug discovery programmes in the future is considered.

Autoantibodies Targeting G-Protein-Coupled Receptors: Pathogenetic, Clinical and Therapeutic Implications in Systemic Sclerosis.

Systemic sclerosis (SSc) is a multifaceted connective tissue disease whose aetiology remains largely unknown. Autoimmunity is thought to play a pivotal role in the development of the disease, but the direct pathogenic role of SSc-specific autoantibodies remains to be established. The recent discovery of functional antibodies targeting G-protein-coupled receptors (GPCRs), whose presence has been demonstrated in different autoimmune conditions, has shed some light on SSc pathogenesis. These antibodies bind to GPCRs expressed on immune and non-immune cells as their endogenous ligands, exerting either a stimulatory or inhibitory effect on corresponding intracellular pathways. Growing evidence suggests that, in SSc, the presence of anti-GPCRs antibodies correlates with specific clinical manifestations. Autoantibodies targeting endothelin receptor type A (ETAR) and angiotensin type 1 receptor (AT1R) are associated with severe vasculopathic SSc-related manifestations, while anti-C-X-C motif chemokine receptors (CXCR) antibodies seem to be predictive of interstitial lung involvement; anti-muscarinic-3 acetylcholine receptor (M3R) antibodies have been found in patients with severe gastrointestinal involvement and anti-protease-activated receptor 1 (PAR1) antibodies have been detected in patients experiencing scleroderma renal crisis. This review aims to clarify the potential pathogenetic significance of GPCR-targeting autoantibodies in SSc, focusing on their associations with the different clinical manifestations of scleroderma. An extensive examination of functional autoimmunity targeting GPCRs might provide valuable insights into the underlying pathogenetic mechanisms of SSc, thus enabling the development of novel therapeutic strategies tailored to target GPCR-mediated pathways.

The orphan G protein-coupled receptor 141 expressed in myeloid cells functions as an inflammation suppressor.

G protein-coupled receptors (GPCRs) regulate many cellular processes in response to various stimuli, including light, hormones, neurotransmitters, and odorants, some of which play critical roles in innate and adaptive immune responses. However, the physiological functions of many GPCRs and the involvement of them in autoimmune diseases of the central nervous system remain unclear. Here, we demonstrate that GPR141, an orphan GPCR belonging to the class A receptor family, suppresses immune responses. High GPR141 messenger RNA levels were expressed in myeloid-lineage cells, including neutrophils (CD11b + Gr1+), monocytes (CD11b + Gr1-Ly6C+ and CD11b + Gr1-Ly6C-), macrophages (F4/80+), and dendritic cells (CD11c+). Gpr141 -/- mice, which we independently generated, displayed almost no abnormalities in myeloid cell differentiation and compartmentalization in the spleen and bone marrow under steady-state conditions. However, Gpr141 deficiency exacerbated disease conditions of experimental autoimmune encephalomyelitis, an autoimmune disease model for multiple sclerosis, with increased inflammation in the spinal cord. Gpr141 -/- mice showed increased CD11b + Gr1+ neutrophils, CD11b + Gr1- monocytes, CD11c+ dendritic cells, and CD4+ T cell infiltration into the experimental autoimmune encephalomyelitis-induced spinal cord compared with littermate control mice. Lymphocytes enriched from Gpr141 -/- mice immunized with myelin oligodendrocyte glycoprotein 35-55 produced high amounts of interferon-γ, interleukin-17A, and interleukin-6 compared with those from wild-type mice. Moreover, CD11c+ dendritic cells (DCs) purified from Gpr141 -/- mice increased cytokine production of myelin oligodendrocyte glycoprotein 35-55-specific T cells. These findings suggest that GPR141 functions as a negative regulator of immune responses by controlling the functions of monocytes and dendritic cells and that targeting GPR141 may be a possible therapeutic intervention for modulating chronic inflammatory diseases.

Functional selectivity of Receptor Tyrosine Kinases regulates distinct cellular outputs.

Functional selectivity refers to the activation of differential signalling and cellular outputs downstream of the same membrane-bound receptor when activated by two or more different ligands. Functional selectivity has been described and extensively studied for G-protein Coupled Receptors (GPCRs), leading to specific therapeutic options for dysregulated GPCRs functions. However, studies regarding the functional selectivity of Receptor Tyrosine Kinases (RTKs) remain sparse. Here, we will summarize recent data about RTK functional selectivity focusing on how the nature and the amount of RTK ligands and the crosstalk of RTKs with other membrane proteins regulate the specificity of RTK signalling. In addition, we will discuss how structural changes in RTKs upon ligand binding affects selective signalling pathways. Much remains to be known about the integration of different signals affecting RTK signalling specificity to orchestrate long-term cellular outcomes. Recent advancements in omics, specifically quantitative phosphoproteomics, and in systems biology methods to study, model and integrate different types of large-scale omics data have increased our ability to compare several signals affecting RTK functional selectivity in a global, system-wide fashion. We will discuss how such methods facilitate the exploration of important signalling hubs and enable data-driven predictions aiming at improving the efficacy of therapeutics for diseases like cancer, where redundant RTK signalling pathways often compromise treatment efficacy.

The Underlying Rab Network of MRGPRX2-Stimulated Secretion Unveils the Impact of Receptor Trafficking on Secretory Granule Biogenesis and Secretion.

MRGPRX2, the human member of the MAS-related G-protein-coupled receptors (GPCRs), mediates the immunoglobulin E (IgE)-independent responses of a subset of mast cells (MCs) that are associated with itch, pain, neurogenic inflammation, and pseudoallergy to drugs. The mechanisms underlying the responses of MRGPRX2 to its multiple and diverse ligands are still not completely understood. Given the close association between GPCR location and function, and the key role played by Rab GTPases in controlling discrete steps along vesicular trafficking, we aimed to reveal the vesicular pathways that directly impact MRGPRX2-mediated exocytosis by identifying the Rabs that influence this process. For this purpose, we screened 43 Rabs for their functional and phenotypic impacts on MC degranulation in response to the synthetic MRGPRX2 ligand compound 48/80 (c48/80), which is often used as the gold standard of MRGPRX2 ligands, or to substance P (SP), an important trigger of neuroinflammatory MC responses. Results of this study highlight the important roles played by macropinocytosis and autophagy in controlling MRGPRX2-mediated exocytosis, demonstrating a close feedback control between the internalization and post-endocytic trafficking of MRGPRX2 and its triggered exocytosis.

Dissecting the genetic landscape of GPCR signaling through phenotypic profiling in C. elegans

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C. elegans. These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts.

Single-molecule visualization of human A2A adenosine receptor activation by a G protein and constitutively activating mutations

Mutations that constitutively activate G protein-coupled receptors (GPCRs), known as constitutively activating mutations (CAMs), modify cell signaling and interfere with drugs, resulting in diseases with limited treatment options. We utilize fluorescence imaging at the single-molecule level to visualize the dynamic process of CAM-mediated activation of the human A2A adenosine receptor (A2AAR) in real time. We observe an active-state population for all CAMs without agonist stimulation. Importantly, activating mutations significantly increase the population of an intermediate state crucial for receptor activation, notably distinct from the addition of a partner G protein. Activation kinetics show that while CAMs increase the frequency of transitions to the intermediate state, mutations altering sodium sensitivity increase transitions away from it. These findings indicate changes in GPCR function caused by mutations may be predicted based on whether they favor or disfavor formation of an intermediate state, providing a framework for designing receptors with altered functions or therapies that target intermediate states.

Functional GPCR Expression in Eukaryotic LEXSY System

G protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins in the human genome, and represent one of the most important classes of drug targets. Their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Therefore, in this study, we explored the efficacy of the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We selected the human A2A adenosine receptor (A2AAR), as a model protein, expressed it in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression. Overall, our results suggest that LEXSY is a promising platform for large-scale production of GPCRs for structural studies.

Unraveling the Functional Significance of Unstructured Regions in G Protein-Coupled Receptors.

Unstructured regions in functional proteins have gained attention in recent years due to advancements in informatics tools and biophysical methods. G protein-coupled receptors (GPCRs), a large family of cell surface receptors, contain unstructured regions in the form of the i3 loop and C-terminus. This review provides an overview of the functional significance of these regions in GPCRs. GPCRs transmit signals from the extracellular environment to the cell interior, regulating various physiological processes. The i3 loop, located between the fifth and sixth transmembrane helices, and the C-terminus, connected to the seventh transmembrane helix, are determinant of interactions with G proteins and with other intracellular partners such as arrestins. Recent studies demonstrate that the i3 loop and C-terminus play critical roles in allosterically regulating GPCR activation. They can act as autoregulators, adopting conformations that, by restricting G protein access, modulate receptor coupling specificity. The length and unstructured nature of the i3 loop and C-terminus provide unique advantages in GPCR interactions with intracellular protein partners. They act as "fishing lines", expanding the radius of interaction and enabling GPCRs to tether scaffolding proteins, thus facilitating receptor stability during cell membrane movements. Additionally, the i3 loop may be involved in domain swapping between GPCRs, generating novel receptor dimers with distinct binding and coupling characteristics. Overall, the i3 loop and C-terminus are now widely recognized as crucial elements in GPCR function and regulation. Understanding their functional roles enhances our comprehension of GPCR structure and signaling complexity and holds promise for advancements in receptor pharmacology and drug development.

RNAi-mediated knockdown of two orphan G protein-coupled receptors reduces fecundity in the yellow fever mosquito Aedes aegypti.

G protein-coupled receptors (GPCRs) control numerous physiological processes in insects, including reproduction. While many GPCRs have known ligands, orphan GPCRs do not have identified ligands in which they bind. Advances in genomic sequencing and phylogenetics provide the ability to compare orphan receptor protein sequences to sequences of characterized GPCRs, and thus gain a better understanding of the potential functions of orphan GPCRs. Our study sought to investigate the functions of two orphan GPCRs, AAEL003647 and AAEL019988, in the yellow fever mosquito, Aedes aegypti. From our phylogenetic investigation, we found that AAEL003647 is orthologous to the SIFamide-2/SMYamide receptor. We also found that AAEL019988 is orthologous to the Trapped in endoderm (Tre1) receptor of Drosophila melanogaster. Next, we conducted a tissue-specific expression analysis and found that both receptors had highest expression in the ovaries, suggesting they may be important for reproduction. We then used RNA interference (RNAi) to knock down both genes and found a significant reduction in the number of eggs laid per individual female mosquito, suggesting both receptors are important for Ae. aegypti reproduction.

Endosome positioning coordinates spatially selective GPCR signaling.

G-protein-coupled receptors (GPCRs) can initiate unique functional responses depending on the subcellular site of activation. Efforts to uncover the mechanistic basis of compartmentalized GPCR signaling have concentrated on the biochemical aspect of this regulation. Here we assess the biophysical positioning of receptor-containing endosomes as an alternative salient mechanism. We devise a strategy to rapidly and selectively redistribute receptor-containing endosomes 'on command' in intact cells without perturbing their biochemical composition. Next, we present two complementary optical readouts that enable robust measurements of bulk- and gene-specific GPCR/cyclic AMP (cAMP)-dependent transcriptional signaling with single-cell resolution. With these, we establish that disruption of native endosome positioning inhibits the initiation of the endosome-dependent transcriptional responses. Finally, we demonstrate a prominent mechanistic role of PDE-mediated cAMP hydrolysis and local protein kinase A activity in this process. Our study, therefore, illuminates a new mechanism regulating GPCR function by identifying endosome positioning as the principal mediator of spatially selective receptor signaling.

Accessible and Generalizable in Vitro Luminescence Assay for Detecting GPCR Activation.

G protein-coupled receptors (GPCRs) serve critical physiological roles as the most abundant family of receptors. Here, we describe the design of a generalizable and cell lysate-based method that leverages the interaction between an agonist-activated GPCR and a conformation-specific binder to reconstitute split nanoluciferase (NanoLuc) in vitro. This tool, In vitro GPCR split NanoLuc ligand Triggered Reporter (IGNiTR), has broad applications. We have demonstrated IGNiTR's use with three Gs-coupled GPCRs, two Gi-coupled GPCRs and three classes of conformation-specific binders: nanobodies, miniG proteins, and G protein peptidomimetics. As an in vitro method, IGNiTR enables the use of synthetic G protein peptidomimetics and provides easily scalable and portable reagents for characterizing GPCRs and ligands. We tested three diverse applications of IGNiTR: (1) proof-of-concept GPCR ligand screening using dopamine receptor D1 IGNiTR; (2) detection of opioids for point-of-care testing; and (3) characterizing GPCR functionality during Nanodisc-based reconstitution processes. Due to IGNiTR's unique advantages and the convenience of its cell lysate-based format, this tool will find extensive applications in GPCR ligand detection, screening, and GPCR characterization.