Chapter 10 - Label-free impedance measurements to unravel biomolecular interactions involved in G protein-coupled receptor signaling

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Abstract

G protein-coupled receptors (GPCRs) are among the most heavily addressed drug targets in medicinal chemistry and pharmacology. The screening for new agonists or antagonists has been largely based on genetically engineered cells overexpressing the receptor to study binding of ligands directly or via intracellular signaling events downstream of receptor activation. These approaches are often invasive in nature, need to be conducted as endpoint assays, require isotope- or fluorophore-labeling and significant genetic manipulation. In contrast to that, non-invasive and label-free impedance measurements are capable of monitoring ligand-receptor interactions in target cells with endogenous receptor expression in real time. The cells expressing the receptor are grown on planar gold-film electrodes that are integrated into regular cell culture dishes. This article will highlight several impedance-based assay formats to characterize biomolecular interactions between ligands and their GPCRs in vitro, comprising agonist and antagonist characterization, dose-response relationships, receptor desensitization, and signal transduction profiling.

Introduction

G protein-coupled receptors (GPCRs) form the largest family of cell surface receptors in the human body. To date, genes for about 800 GPCRs have been identified in the human genome (Sriram & Insel, 2018). They all share the structural hallmark of having seven transmembrane domains within one polypeptide chain, an extracellular ligand binding domain and an intracellular domain. The latter is capable of interacting with a family of peripheral membrane proteins, called G proteins, upon receptor activation by endogenous or exogenous agonists. This interaction between activated GPCR and G protein transmits the signal carried by the agonists into the intracellular signaling cascades, which eventually leads to a concerted cell response (Luttrell, 2008). G proteins are αβγ-heterotrimeric proteins, holding a GTP/GDP binding site with an inherent GTPase enzyme activity in their α-subunit. The inactive protein carries GDP, which is exchanged against GTP upon binding to the activated receptor. GTP binding triggers the dissociation of the G protein into the α-subunit and the βγ-complex. The α-subunit is predominantly responsible for the onward signal transduction as it binds and activates a specific target enzyme. The human genome allows for the expression of 16 different Gα, 5 Gβ and 14 Gγ protein isoforms (Downes & Gautam, 1999; Preininger & Hamm, 2004). It depends on the specific Gα-subunit which enzyme is primarily targeted and activated for the onward signaling. While the αs subunit activates adenylate cyclase (AC), which catalyzes the conversion of ATP to cAMP, the αi subunit inhibits this enzyme and, thereby, reduces the intracellular cAMP. When the activated receptor has bound to G proteins with an αq subunit, the latter activates phospholipase C that converts the membrane lipid phosphatidylinositol to diacylglycerol (DAG) and inositol-triphosphate (IP3), followed by a release of Ca2 + ions from intracellular stores into the cytoplasm. Those receptors coupled to G proteins with α12/13 subunit target the so-called RhoA protein, a member of the Ras superfamily that is involved in regulating the cytoskeleton, transcription and cell cycle progression (Wu, Xie, Zhao, Nice, & Huang, 2012). So dependent on the specific G-protein that the receptor is coupled to, an individual extracellular signal (agonist) is transmitted into a specific intracellular signaling cascade, producing a corresponding cell response. Due to their inherent GTPase activity, the α subunits are capable of deactivating themselves by GTP hydrolysis, which brings them back to the inactive GDP-form that recombines with the βγ-complex to regenerate the inactive heterotrimeric G protein (Kobilka, 2007). Fig. 1 illustrates the signaling cascade on the example of a GPCR that is coupled to a G protein with a Gαs subunit. Receptor activation leads to an increase of the cytosolic cAMP concentration and the subsequent activation of protein kinase A (PKA), followed by distinct cell responses downstream of the signaling cascade, like cell morphology changes or altered gene expression. Please note, there are additional or alternative signaling modes that are induced by the βγ-complex or the recruitment of β-arrestin, another signal mediator protein, to the receptor, but it is beyond the scope of this article to include and discuss these pathways in detail, too.

It is not surprising from the sheer number of genes encoding GPCRs that they are involved in countless physiological processes and their dysfunction has been assigned to a myriad of severe diseases, as, for example, diabetes, some forms of night and color blindness, allergies, depression and certain forms of cancer, to mention just a few (Catapano & Manji, 2007; Reimann & Gribble, 2016; Schöneberg et al., 2004; Sriram & Insel, 2018). Accordingly, GPCRs are one of the most highly addressed drug targets. It has been estimated that up to 35% of all prescription pharmaceuticals on the market address GPCRs or associated effector proteins interacting with the receptors as agonists, antagonists or allosteric modulators (Sriram & Insel, 2018). The known ligands comprise a huge variety of biological species, ranging from hormones, pheromones, neurotransmitters to odorants and flavoring substances (Insel et al., 2019). Among the 350 human non-sensory GPCRs, there is a fraction of around 120 receptors with unknown physiological function and unknown endogenous ligand. These are termed orphan-GPCRs and they are subject to intense research activities (Alexander et al., 2019; Sriram & Insel, 2018). Very well-known and characterized GPCRs are the adrenaline receptors (AR), the dopamine receptors (DR) and the histamine receptors (HR) which will be used as examples in the experiments described below.

Since the characterization of ligands for GPCRs has gained an ever-growing interest in basic research as well as in drug discovery, a variety of different assays has been developed that monitor the biomolecular interaction between ligand and receptor either directly or indirectly. The most direct approach uses radioligand binding as readout (cf. Fig. 1, ①). Either the ligand of interest is radiolabeled itself or it is used in a competition binding assay with a known competing radioligand. The assays provide the equilibrium binding or dissociation constant with high accuracies and sensitivity. However, this assay only returns the binding characteristics, but does not reveal whether a given ligand is an agonist, activating the receptor, or an antagonist that binds without activation. Moreover, the assay typically requires genetically engineered cells that overexpress the receptor of interest and a lab infrastructure that complies with isotope work (Flanagan, 2016).

An alternative assay—also based on isotopes though—measures receptor activation via the binding of radio-labeled GTP to G proteins that are activated by the receptor. It is referred to as GTPγS*-assay (Fig. 1, ②). The GTP-analogue used in this assay holds a 35S instead of an oxygen atom in the γ-phosphate, making it resistant to hydrolysis. Thus, the G protein is no longer able to deactivate itself by its GTPase activity and the radiolabeled GTP analogue remains tightly bound to the G protein for quantification (Harrison & Traynor, 2003; Milligan, 2003).

More recently, genetically encoded sensors have been developed to monitor GPCR activation (Fig. 1, ③). These assays are based on the heterologous expression of engineered GPCRs that are modified by adding a fragment or domain of a luciferase protein at an appropriate site of the intracellular loops. At the same time, the cells are engineered to express a chimeric G protein, which holds the complementary fragment of the same luciferase. Upon agonist binding, the G proteins are recruited to the activated receptor, which leads to the reunion of the two complementary luciferase fragments restoring their enzymatic activity. Bioluminescence indicates G protein recruitment and, thus, receptor activation. These assays are called protein (fragment) complementation assays (PCA) and they have been developed in many different variants of the same assay principle (Laschet, Dupuis, & Hanson, 2019; Wan et al., 2018). It is obvious that these assays are incompatible with the study of endogenous receptor expression and require quite sophisticated genetic engineering.

Further down the signaling cascade, the concentration of the second messengers like cAMP, Ca2 + or IP3 may serve as an indicator for receptor activation (Fig. 1, ④). Sensitive readouts have been developed for these most heavily studied second messengers that are typically based on changes in fluorescence emission of molecular or protein probes (Grynkiewicz, Poenie, & Tsien, 1985; Lundstrom, 2013; Prystay, Gagné, Kasila, Yeh, & Banks, 2001). In particular for cAMP quantification, a considerable number of genetically encoded sensors has been described (Paramonov, Mamaeva, Sahlgren, & Rivero-Müller, 2015). The latter show the advantage of undisputed biocompatibility whereas small molecule synthetic probes may interfere with the signaling cascades.

Another big group of assays to report on receptor activation are reporter gene assays (Fig. 1, ⑤). Here, an easy-to-quantify protein, such as a luciferase or a fluorescent protein, is expressed under the control of a promotor that is controlled by the concentration of second messengers. Examples for such second messenger sensitive regulatory sequences are the cAMP response element (CRE) or the Ca2 +-sensitive nuclear factor of activated T-cell response element (NFAT-RE). If the receptor is activated and the second messenger concentration changes subsequently, the cells respond by expressing the reporter gene. Since these assays are based on protein biosynthesis, they will report receptor activation with a considerable time delay. Moreover, it requires significant genetic engineering of the cells under study and the assay is not applicable to wild type cells (Cheng et al., 2010; Lundstrom, 2013).

All of these assays have been developed to monitor the biomolecular interaction of a given ligand with a G protein-coupled receptor—as an agonist or an antagonist. Whereas assays ① and ② are typical endpoint assays that do not provide any time-resolved information, the assays ③, ④ and ⑤ are available in time-resolved formats but require genetic engineering for the most part and can’t be applied to wild type cells with endogenous receptor expression (Lundstrom, 2013). In contrast, monitoring the receptor expressing cells by electrochemical impedance measurements provides completely non-invasive, label-free, and real-time readouts of receptor activation, independent of any labeling or engineering of the cells. This technique is sensitive enough to be applied to wild type (Lukic & Wegener, 2015; Wegener, Zink, Rösen, & Galla, 1999) and even primary cells with endogenous receptor density. In subsequent paragraphs, the principle of the technique will be described, followed by the description of several studies illustrating that impedance measurements are perfectly well-suited to study various aspects of ligand-receptor interactions, comprising agonist and antagonist studies, dose-response relationships as well as experiments that unravel the underlying signaling cascade or highly specific receptor phenomena like desensitization.

Section snippets

Electrochemical impedance measurements to study ligand-receptor interactions

The impedance-based monitoring of receptor-mediated signaling is limited to adherent cells but does not have any further restrictions or requirements with respect to the cells under study. It is referred to as electric cell-substrate impedance sensing (ECIS) in the literature (Giaever & Keese, 1991). For the assay, the cells expressing the receptors are grown on thin gold-film electrodes that are shaped by photolithography to provide a small working electrode and larger counter electrode on the

Agonist and antagonist assays

The data presented in Fig. 3 summarizes agonist and antagonist assays for three very well-known and characterized GPCRs: (A) the β-adrenergic receptor (βAR), (B) the histamine receptor 1 (H1R) and (C) the dopamine receptor 2 (D2R). D2R is known to be expressed in two splicing variants that differ with respect to the size of an intracellular loop of the protein. All data reported here have been recorded for the longer D2R variant referred to as D2Rlong. Activation of βAR is generally considered

Dose-response studies

Impedance time course data provides a solid basis for a quantitative analysis of ligand-receptor interactions. Fig. 4 summarizes such a quantitative analysis on the example of CHO cells, overexpressing the D2R receptor, for the endogenous agonist dopamine (Fig. 4A and B) as well as for the antagonist haloperidol (Fig. 4C and D). Upon activation by dopamine, the D2R is predominantly Gαi-coupled inducing a reduction of cellular cAMP. Thus, a constant concentration of 0.4 μM forskolin is added to

In-depth analysis of the signal transduction pathway

The preceding paragraphs have demonstrated that impedance-based assays are capable of characterizing the binding of agonists and antagonists to their cell-surface receptors. In conjunction with highly specific pharmacological tools, it is moreover possible to identify the underlying signal transduction cascade(s) (Wegener et al., 1999). This strategy will be demonstrated by example of the βAR that is considered to be predominantly coupled to the Gαs-controlled pathways. Fig. 5 compares the

Receptor desensitization

One of the hallmarks of GPCR-mediated signaling is receptor desensitization. It means that activated receptors get phosphorylated and, thereby, inactivated before they are internalized into the cells for later digestion or regeneration. This mechanism ensures that there is no constant activation of the receptor and the subsequent signaling cascade upon prolonged exposure to an agonist. The system down-regulates its sensitivity to avoid exhaustion and overstimulation of both, the individual

Further experimental options and current developments

The preceding studies have highlighted some of the experimental options with respect to ligand-receptor interaction analysis that are available from impedance monitoring of cell-based assays. The strengths of this approach are its label-free nature, its time resolution, and its independence of genetic engineering. The technology is applicable to primary cultures or finite cell lines with endogenous receptor density. Cell growth on ECIS electrodes does not show any difference to their growth on

Acknowledgments

The authors would like to acknowledge financial support of this study by the Research Training Group RTG 1910 “Medicinal chemistry of selective GPCR ligands” funded by the German Research Foundation (DFG). A special thank you is dedicated to Peter Gmeiner, Harald Hübner, Stefan Loeber and Dorothée Weikert for providing us CHO cells expressing the D2R.

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