Native G Protein-coupled Receptors Research Articles

Specific pharmacological and Gi/o protein responses of some native GPCRs in neurons

G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins and are important drug targets. The discovery of drugs targeting these receptors and their G protein signaling properties are based on assays mainly performed with modified receptors expressed in heterologous cells. However, GPCR responses may differ in their native environment. Here, by using highly sensitive Gi/o sensors, we reveal specific properties of Gi/o protein-mediated responses triggered by GABAB, α2 adrenergic and cannabinoid CB1 receptors in primary neurons, different from those in heterologous cells. These include different profiles in the Gi/o protein subtypes-mediated responses, and differences in the potencies of some ligands even at similar receptor expression levels. Altogether, our results show the importance of using biosensors compatible with primary cells for evaluating the activities of endogenous GPCRs in their native environment.

Imaging flow cytometry of tumoroids: A new method for studying GPCR expression.

Fluorescence confocal microscopy is commonly used to analyze the regulation membrane proteins expression such as G protein-coupled receptors (GPCRs). With this approach, the internal movement of GPCRs within the cell can be observed with a high degree of resolution. However, these microscopy techniques led to complex and time-consuming analysis and did not allow a large population of events to be sampled. A recent approach termed imaging flow cytometry (IFC), which combines flow cytometry and fluorescence microscopy, had two main advantages to study the regulation of GPCRs expression such as orexins receptors (OXRs): the ability (1) to analyze large numbers of cells and; (2) to visualize cell integrity and fluorescent markers localization. Here, we compare these two technologies using the orexin A (OxA) ligand coupled to rhodamine (OxA-rho) to investigate anti-tumoral OX1R expression in human digestive cancers. IFC has been adapted for cancer epithelial adherent cells and also to 3D cell culture tumoroids which partially mimic tumoral structures. In the absence of specific antibody, expression of OX1R is examined in the presence of OxA-rho. 2D-culture of colon cancer cells HT-29 exhibits a maximum level of OX1R internalization induced by OxA with 19% ± 3% colocalizing to early endosomes. In 3D-culture of HT-29 cells, internalization of OX1R/OxA-rho reached its maximum at 60 min, with 30.7% ± 6.4% of OX1R colocalizing with early endosomes. This is the first application of IFC to the analysis of the expression of a native GPCR, OX1R, in both 2D and 3D cultures of adherent cancer cells.

Class-Switching of B Lymphocytes by DNA and Cell Immunization for Stereospecific Monoclonal Antibodies against Native GPCR

To develop efficient applications of monoclonal antibodies for therapeutic purposes, stereospecific recognition of the target antigens is needed. DNA immunization is one of the best methods for sensitizing B lymphocytes that can produce conformation-specific antibodies. Here we verified the class-switching of monoclonal antibodies by DNA immunization followed by cell immunization for the generation of stereospecific monoclonal antibodies against native G protein-coupled receptor (GPCR) using the optimized stereospecific targeting (SST) technique. This technology facilitates the efficient selection of sensitized B lymphocytes through specific interaction with the intact antigen via B-cell receptors (BCRs). We demonstrate that multiple DNA immunizations followed by a single cell immunization in combination with a longer sensitization period (three to four months) are an appropriate sensitizing strategy for the generation of IgG-type stereospecific monoclonal antibodies by class-switching, and the characteristics of antibody production could be transferred to hybridoma cells provided by the optimized SST technique.

A human antibody against human endothelin receptor type A that exhibits antitumor potency

Endothelin receptor A (ETA), a class A G-protein-coupled receptor (GPCR), is involved in the progression and metastasis of colorectal, breast, lung, ovarian, and prostate cancer. We overexpressed and purified human endothelin receptor type A in Escherichia coli and reconstituted it with lipid and membrane scaffold proteins to prepare an ETA nanodisc as a functional antigen with a structure similar to that of native GPCR. By screening a human naive immune single-chain variable fragment phage library constructed in-house, we successfully isolated a human anti-ETA antibody (AG8) exhibiting high specificity for ETA in the β-arrestin Tango assay and effective inhibitory activity against the ET-1-induced signaling cascade via ETA using either a CHO-K1 cell line stably expressing human ETA or HT-29 colorectal cancer cells, in which AG8 exhibited IC50 values of 56 and 51 nM, respectively. In addition, AG8 treatment repressed the transcription of inhibin βA and reduced the ETA-induced phosphorylation of protein kinase B and extracellular regulated kinase. Furthermore, tumor growth was effectively inhibited by AG8 in a colorectal cancer mouse xenograft model. The human anti-ETA antibody isolated in this study could be used as a potential therapeutic for cancers, including colorectal cancer.

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A2A receptor (A2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A2AR C-terminus from 412 to 316 amino acids (A2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A2AR levels. As cAMP production is downstream of the first activation event—coupling of G protein to its receptor—investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gαs proteins, we characterize the association of A2AR and A2AΔ316R to Gαs with and without GDP or GTPγs using surface plasmon resonance (SPR). Gαs affinity for A2AR was greatest for apo-Gαs, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A2AR C-terminus (A2AΔ316R) decreased the affinity of the unliganded receptor for Gαs by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.

Ligand-directed covalent labelling of a GPCR with a fluorescent tag in live cells

To study the localisation of G protein-coupled receptors (GPCR) in their native cellular environment requires their visualisation through fluorescent labelling. To overcome the requirement for genetic modification of the receptor or the limitations of dissociable fluorescent ligands, here we describe rational design of a compound that covalently and selectively labels a GPCR in living cells with a fluorescent moiety. We designed a fluorescent antagonist, in which the linker incorporated between pharmacophore (ZM241385) and fluorophore (sulfo-cyanine5) is able to facilitate covalent linking of the fluorophore to the adenosine A2A receptor. We pharmacologically and biochemically demonstrate irreversible fluorescent labelling without impeding access to the orthosteric binding site and demonstrate its use in endogenously expressing systems. This offers a non-invasive and selective approach to study function and localisation of native GPCRs.

Furan Cross-Linking Technology for Investigating GPCR-Ligand Interactions.

Interactions between G protein-coupled receptors and their ligands hold extensive potential for drug discovery. Studying these interactions poses technical problems due to their transient nature and the inherent difficulties when working with G protein-coupled receptors (GPCR) that are only functional in a membrane setting. Here, we describe the use of a furan-based chemical cross-linking methodology to achieve selective covalent coupling between a furan-modified peptide ligand and its native GPCR present on the surface of living cells under normal cell culture conditions. This methodology relies on the oxidation of the furan moiety, which can be achieved by either addition of an external oxidation signal or by the reactive oxygen species produced by the cell. The cross-linked ligand-GPCR complex is subsequently detected by Western blotting based on the biotin label that is incorporated in the peptide ligand.

Co-IP assays for measuring GPCR-arrestin interactions.

βarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The βarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR-arrestin interaction in cells.

Enabling STD-NMR fragment screening using stabilized native GPCR: A case study of adenosine receptor

Structural studies of integral membrane proteins have been limited by the intrinsic conformational flexibility and the need to stabilize the proteins in solution. Stabilization by mutagenesis was very successful for structural biology of G protein-coupled receptors (GPCRs). However, it requires heavy protein engineering and may introduce structural deviations. Here we describe the use of specific calixarenes-based detergents for native GPCR stabilization. Wild type, full length human adenosine A2A receptor was used to exemplify the approach. We could stabilize native, glycosylated, non-aggregated and homogenous A2AR that maintained its ligand binding capacity. The benefit of the preparation for fragment screening, using the Saturation-Transfer Difference nuclear magnetic resonance (STD-NMR) experiment is reported. The binding of the agonist adenosine and the antagonist caffeine were observed and competition experiments with CGS-21680 and ZM241385 were performed, demonstrating the feasibility of the STD-based fragment screening on the native A2A receptor. Interestingly, adenosine was shown to bind a second binding site in the presence of the agonist CGS-21680 which corroborates published results obtained with molecular dynamics simulation. Fragment-like compounds identified using STD-NMR showed antagonistic effects on A2AR in the cAMP cellular assay. Taken together, our study shows that stabilization of native GPCRs represents an attractive approach for STD-based fragment screening and drug design.

Structural Life Science towards the Regulation of Selective GPCR Signaling

G protein-coupled receptors (GPCRs) are the largest family of receptors in the human genome. They are involved in many diseases and also the target of approximately 30% of all modern medicinal drugs. GPCRs respond to a broad spectrum of chemical entities, ranging from photons, protons, and calcium ions to small organic molecules (including odorants and neurotransmitters), peptides, and glycoproteins. Many GPCRs are members of closely related subfamilies that respond to the same hormone or neurotransmitter. However, they have different physiologic functions based on the cells in which they are expressed and the different signaling pathways that they exploit (e.g., coupling through heterotrimeric G-proteins such as Gs, Gi, and Gq, as well as β-arrestins). Antibody fragments including Fab and Fv can effectively stabilize and crystallize membrane proteins. However, using the mouse hybridoma technology it has been difficult to develop monoclonal antibodies that can recognize conformational epitopes of native GPCRs. We have recently succeeded in developing antibodies against native GPCRs using this technology in combination with our improved immunization and screening methods. In this symposium review, I present a successful example of prostaglandin E2 receptor (one of the GPCRs) crystallization using antibody fragments. To avoid several adverse effects of current therapeutics, it is essential to understand the molecular mechanism of GPCR signaling in a monomeric, dimeric, or oligomeric state. Also, we are interested in selectively regulating GPCR signaling via functional antibodies developed using our methods and/or the designed small organic molecules depending on the GPCR structure.

G Protein-Coupled Receptor Multimers: A Question Still Open Despite the Use of Novel Approaches.

Heteromerization of G protein-coupled receptors (GPCRs) can significantly change the functional properties of involved receptors. Various biochemical and biophysical methodologies have been developed in the last two decades to identify and functionally evaluate GPCR heteromers in heterologous cells, with recent approaches focusing on GPCR complex stoichiometry and stability. Yet validation of these observations in native tissues is still lagging behind for the majority of GPCR heteromers. Remarkably, recent studies, particularly some involving advanced fluorescence microscopy techniques, are contributing to our current knowledge of aspects that were not well known until now, such as GPCR complex stoichiometry and stability. In parallel, a growing effort is being applied to move the field forward into native systems. This short review will highlight recent developments to study the stoichiometry and stability of GPCR complexes and methodologies to detect native GPCR dimers.

Native serotonin 5-HT2C receptors are expressed as homodimers on the apical surface of choroid plexus epithelial cells.

G protein-coupled receptors (GPCRs) are a prominent class of plasma membrane proteins that regulate physiologic responses to a wide variety of stimuli and therapeutic agents. Although GPCR oligomerization has been studied extensively in recombinant cells, it remains uncertain whether native receptors expressed in their natural cellular environment are monomers, dimers, or oligomers. The goal of this study was to determine the monomer/oligomer status of a native GPCR endogenously expressed in its natural cellular environment. Native 5-HT2C receptors in choroid plexus epithelial cells were evaluated using fluorescence correlation spectroscopy (FCS) with photon counting histogram (PCH). An anti-5-HT2C fragment antigen binding protein was used to label native 5-HT2C receptors. A known monomeric receptor (CD-86) served as a control for decoding the oligomer status of native 5-HT2C receptors by molecular brightness analysis. FCS with PCH revealed molecular brightness values for native 5-HT2C receptors equivalent to the molecular brightness of a homodimer. 5-HT2C receptors displayed a diffusion coefficient of 5 × 10(-9) cm(2)/s and were expressed at 32 receptors/μm(2) on the apical surface of choroid plexus epithelial cells. The functional significance and signaling capabilities of the homodimer were investigated in human embryonic kidney 293 cells using agonists that bind in a wash-resistant manner to one or both protomers of the homodimer. Whereas agonist binding to one protomer resulted in G protein activation, maximal stimulation required occupancy of both protomers. This study is the first to demonstrate the homodimeric structure of 5-HT2C receptors endogenously expressed in their native cellular environment, and identifies the homodimer as a functional signaling unit.

Optical control of metabotropic glutamate receptors

G-protein coupled receptors (GPCRs), the largest family of membrane signaling proteins, respond to neurotransmitters, hormones and small environmental molecules. The neuronal function of many GPCRs has been difficult to resolve because of an inability to gate them with subtype-specificity, spatial precision, speed and reversibility. To address this, we developed an approach for opto-chemical engineering native GPCRs. We applied this to the metabotropic glutamate receptors (mGluRs) to generate light-agonized and light-antagonized “LimGluRs”. The light-agonized “LimGluR2”, on which we focused, is fast, bistable, and supports multiple rounds of on/off switching. Light gates two of the primary neuronal functions of mGluR2: suppression of excitability and inhibition of neurotransmitter release. The light-antagonized “LimGluR2block” can be used to manipulate negative feedback of synaptically released glutamate on transmitter release. We generalize the optical control to two additional family members: mGluR3 and 6. The system works in rodent brain slice and in zebrafish in vivo, where we find that mGluR2 modulates the threshold for escape behavior. These light-gated mGluRs pave the way for determining the roles of mGluRs in synaptic plasticity, memory and disease.

In VivoVisualization of Delta Opioid Receptors upon Physiological Activation Uncovers a Distinct Internalization Profile

G-protein-coupled receptors (GPCRs) mediate numerous physiological functions and represent prime therapeutic targets. Receptor trafficking upon agonist stimulation is critical for GPCR function, but examining this process in vivo remains a true challenge. Using knock-in mice expressing functional fluorescent delta opioid receptors under the control of the endogenous promoter, we visualized in vivo internalization of this native GPCR upon physiological stimulation. We developed a paradigm in which animals were made dependent on morphine in a drug-paired context. When re-exposed to this context in a drug-free state, mice showed context-dependent withdrawal signs and activation of the hippocampus. Receptor internalization was transiently detected in a subset of CA1 neurons, uncovering regionally restricted opioid peptide release. Importantly, a pool of surface receptors always remained, which contrasts with the in vivo profile previously established for exogenous drug-induced internalization. Therefore, a distinct response is observed at the receptor level upon a physiological or pharmacological stimulation. Altogether, direct in vivo GPCR visualization enables mapping receptor stimulation promoted by a behavioral challenge and represents a powerful approach to study endogenous GPCR physiology.

Screening for GPCR Ligands Using Surface Plasmon Resonance

G-protein coupled receptors (GPCRs) are a class of drug targets of primary importance. However, receptor assays are based on measurement of either ligand displacement or downstream functional responses, rather than direct observation of ligand binding. Issues of allosteric modulation, probe dependence, and functional selectivity create challenges in selecting suitable assays formats. Therefore, a method that directly measures GPCR–ligand interactions, independent of binding site, probe, and signaling pathway would be a useful primary and orthogonal screening method. We have developed a GPCR biosensor assay protocol that offers the opportunity for high-throughput label-free screening that directly measures GPCR–ligand interactions. The biosensor-based direct screening method identifies the interaction of both orthosteric and allosteric ligands with solubilized, native GPCRs, in a label-free and cell-free environment, thus overcoming the limitations of indirect and displacement assay methods. We exemplify the method by the discovery of novel ligands for the chemokine receptor, CCR5, that are ligand efficient fragments.

Heterologous Expression and Purification of the Serotonin Type 4 Receptor from Transgenic Mouse Retina

Recent breakthroughs in the solution of X-ray structures for G protein-coupled receptors (GPCRs) with diffusible ligands have employed extensively mutated or recombined receptor fusion proteins heterologously expressed in conventional in vitro cell-based systems. While these advances now show that crystallization of non-rhodopsin members of this superfamily can be accomplished, the use of radically modified proteins may limit the relevance of the derived structures for precision-guided drug design. To better enable the study of native GPCR structures, we report here efforts to engineer an in vivo expression system that harnesses the photoreceptor system of the retina to express heterologous GPCRs with native human sequences in a biochemically homogeneous and pharmacologically functional conformation. As an example, we show that the human 5HT4 receptor, when placed under the influence of the mouse opsin promoter and an opsin rod outer segment (ROS) targeting sequence, localized to ROS of transgenic mouse retina. The resulting receptor protein was uniformly glycosylated and pharmacologically intact as demonstrated by immunoblotting and radioligand binding assays. Upon solubilization, the retinal 5HT4 receptor retained the binding properties of its initial state in retinal membranes. With the engineered T7 monoclonal epitope sequence, the solubilized receptor was easily purified by one-step immunoaffinity chromatography and the purified receptor in detergent solution preserved its ligand binding properties. This expression method may prove generally useful for generating functional, high-quality GPCR protein.

Changes in G Protein-coupled Receptor Sorting Protein Affinity Regulate Postendocytic Targeting of G Protein-coupled Receptors

After activation, most G protein-coupled receptors (GPCRs) are regulated by a cascade of events involving desensitization and endocytosis. Internalized receptors can then be recycled to the plasma membrane, retained in an endosomal compartment, or targeted for degradation. The GPCR-associated sorting protein, GASP, has been shown to preferentially sort a number of native GPCRs to the lysosome for degradation after endocytosis. Here we show that a mutant beta(2) adrenergic receptor and a mutant mu opioid receptor that have previously been described as lacking "recycling signals" due to mutations in their C termini in fact bind to GASP and are targeted for degradation. We also show that a mutant dopamine D1 receptor, which has likewise been described as lacking a recycling signal, does not bind to GASP and is therefore not targeted for degradation. Together, these results indicate that alteration of receptors in their C termini can expose determinants with affinity for GASP binding and consequently target receptors for degradation.

Measuring the Modulatory Effects of RGS Proteins on GIRK Channels

Discovery of "regulators of G-protein signaling" (RGS) as GTPase-activating proteins for heterotrimeric G proteins has provided a highly sought "missing link," reconciling past discrepancies between the in vitro GTPase activity of purified G proteins and the kinetics of physiological responses mediated by G-protein signaling in vivo. With the number of RGS genes in the mammalian genome at more than 30, associating specific RGS proteins to specific G-protein-coupled receptor (GPCR) signaling events has become a focus of RGS investigators. The ubiquitous expression of multiple RGS proteins has complicated this effort, yet the outlook has been encouraged with the identification of RGS9 as the determinant mediating rapid recovery of the transducin-dependent photoresponse. G-protein-gated inwardly rectifying potassium (GIRK) channels that mediate inhibitory synaptic transmission via GPCR activation of pertussis toxin-sensitive G proteins are similarly accelerated by RGS proteins when reconstituted in heterologous cell expression systems and fully reproduce the gating properties of native GIRK channels in neurons and cardiomyocytes. The endogenous neuronal and cardiac RGS protein(s) that accelerate GPCR-->GIRK channel-gating kinetics are currently not known. This article describes methods used to measure the receptor-dependent GIRK channel-gating parameters reconstituted in Chinese hamster ovary (CHO-K1) cells and Xenopus oocytes, as well as rat atrial myocytes and rat cerebellar granule neurons as model cells with native GPCR-->GIRK channel signaling. Applications of these methods for structure-function-based studies of RGS proteins, G proteins, and GPCRs are discussed. We also describe single cell reverse transcriptase polymerase chain reaction methods developed to profile atrial myocyte and neuronal RGS expression to identify specific RGS proteins for targeted knockdown or knockout.

G-protein alpha subunit isoforms couple differentially to receptors that mediate presynaptic inhibition at rat hippocampal synapses.

Presynaptic receptors that are coupled to heterotrimeric G-proteins are found throughout the brain and are responsible for modulating synaptic transmission. At least 10 G-protein-coupled receptors (GPCRs) reduce transmission in hippocampal neurons. Additionally, hippocampal neurons express up to 17 different Galpha, Gbeta, and Ggamma subunits, making for a striking array of possible heterotrimer compositions and GPCR-heterotrimer interactions. The identity of the Galpha subunit is likely a critical determinant in coupling specificity between GPCRs and their molecular effectors mediating presynaptic inhibition. We studied the role of four Galpha(i/o) subunits (Galpha(o1), Galpha(i1,) Galpha(i2), and Galpha(i3)) in mediating presynaptic inhibition in hippocampal neurons by expressing pertussis toxin-insensitive (PTx-ins) Galpha(i/o) mutants. PTx treatment of these cells disrupts coupling of endogenous subunits, leaving only the mutant Galpha subunits to couple with native GPCRs and betagamma subunits. Successful rescue of presynaptic inhibition indicates that the expressed mutant Galpha subunit can couple to the GPCR of interest. All four PTx-ins Galpha subunits rescued presynaptic inhibition by adenosine A1 receptors. A PTx-ins Galpha subunit also rescued adenosine A1-mediated inhibition of spontaneous vesicle fusion frequency. Of the remaining GPCRs tested, cannabinoid CB1, somatostatin, and GABA(B) receptors displayed an alpha subunit-dependent selectivity in binding to G-protein heterotrimers, whereas group III metabotropic glutamate receptor-mediated inhibition was not rescued by expression of any of the four PTx-ins Galpha subunits. Differential coupling of G-protein alpha subunits may be a means of achieving specificity between different GPCRs and their molecular targets for mediating presynaptic inhibition.

G-Protein α Subunit Isoforms Couple Differentially to Receptors that Mediate Presynaptic Inhibition at Rat Hippocampal Synapses

Presynaptic receptors that are coupled to heterotrimeric G-proteins are found throughout the brain and are responsible for modulating synaptic transmission. At least 10 G-protein-coupled receptors (GPCRs) reduce transmission in hippocampal neurons. Additionally, hippocampal neurons express up to 17 different Gα, Gβ, and Gγ subunits, making for a striking array of possible heterotrimer compositions and GPCR–heterotrimer interactions. The identity of the Gα subunit is likely a critical determinant in coupling specificity between GPCRs and their molecular effectors mediating presynaptic inhibition. We studied the role of four Gαi/osubunits (Gαo1, Gαi1,Gαi2, and Gαi3) in mediating presynaptic inhibition in hippocampal neurons by expressing pertussis toxin-insensitive (PTx-ins) Gαi/omutants. PTx treatment of these cells disrupts coupling of endogenous subunits, leaving only the mutant Gα subunits to couple with native GPCRs and βγ subunits. Successful rescue of presynaptic inhibition indicates that the expressed mutant Gα subunit can couple to the GPCR of interest. All four PTx-ins Gα subunits rescued presynaptic inhibition by adenosine A1 receptors. A PTx-ins Gα subunit also rescued adenosine A1-mediated inhibition of spontaneous vesicle fusion frequency. Of the remaining GPCRs tested, cannabinoid CB1, somatostatin, and GABABreceptors displayed an α subunit-dependent selectivity in binding to G-protein heterotrimers, whereas group III metabotropic glutamate receptor-mediated inhibition was not rescued by expression of any of the four PTx-ins Gα subunits. Differential coupling of G-protein α subunits may be a means of achieving specificity between different GPCRs and their molecular targets for mediating presynaptic inhibition.