Since GPCRs are the most valuable and successful targets currently being investigated by the pharmaceutical industry, the development of a new approach for ligand screening for orphan GPCRs has great potential. In this article, we describe a novel method to detect GPCR signals in living cells using surface plasmon resonance (SPR) techniques. The SPR assay evaluates GPCR-mediated cytoskeletal rearrangement, and is suitable for screening for ligands of orphan GPCRs.
by Kexin Chen, Hideru Obinata and Takashi Izumi
The superfamily of cell surface receptors known as G protein-coupled receptors (GPCRs), plays a fundamental role both in physiological and pathological conditions by mediating a wide variety of biological processes in response to their agonists. GPCRs are one of the most successful targets for drug discovery and the GPCR signalling process has been intensively investigated over the last decades. However, there are about 100 GPCRs for which endogenous ligands still remain unidentified — the so-called ‘orphan GPCRs’. Such orphan GPCRs have huge potential in the pharmaceutical industry, so the development of suitable ligand screening systems has itself significant commercial value [1]. In this article, we introduce a novel method to evaluate the activation of GPCRs using a surface plasmon resonance (SPR) sensor. Detecting GPCR-mediated cytoskeletal rearrangements as a new cellular indicator, the new assay could be useful for ligand screening of GPCRs.
G proteins and methods for GPCR ligand screening
GPCRs regulate a variety of biological processes through binding with their ligands and activation of G proteins, which trigger the activation of intracellular effectors as molecular switches. The Alpha subunits of G proteins are classified into subfamilies based on intracellular signalling pathways: Gt, Golf, Gs, Gi, Gq and G12/13. Of these, Gt and Golf specifically couple with rhodopsin and odourant receptors, respectively. Gs activates adenylyl cyclase and increases intracellular cAMP level, whereas Gi inhibits adenylyl cyclase. Gq activates phospholipase C and increases intracellular calcium concentration. G12/13 activates Rho guanine nucleotide exchange factor (RhoGEF), and induces the formation of actin stress fibres through a small G-protein, Rho, of GTP-form [2]. Gi can also activate another small G-protein, Rac, resulting in the formation of actin stress fibres as shown in Figure 1.
Over the past 30 years, various strategies have been developed to evaluate the GPCR signaling process. The guanine nucleotide binding assay is a radioactive method, which evaluates GPCR activation by measuring [35S] GTPgS, and is mainly suited to Gi/o-coupled GPCRs. The cAMP assay is designed for Gs- and Gi-coupled receptors by measuring the cAMP level in whole cells. The inositol phosphate accumulation assay is another radioactive method for Gq-coupled GPCRs. Yet another assay, the intracellular calcium assay is designed for Gq-coupled receptors by calcium-sensitive fluorescent reagents and is extensively used for GPCR screening. As ligand binding induces receptor internalisation in some GPCRs, an internalisation assay of tagged-GPCR can also be used for ligand screening of GPCRs. A reporter gene assay targets GPCR-mediated gene expression using exogenous reporter proteins such as luciferase and galactosidase [3]. Of the various methods for the detection of the activation of GPCR, conventional assays for calcium and cAMP are the most popular. Although various methods have been developed, there still remain numerous orphan GPCRs, which has encouraged us to develop a new approach based on a different principle.
SPR assay
We have established a new method to evaluate GPCR-mediated cytoskeletal rearrangement using SPR [4]. The SPR assay was carried out using a dual-channel SPR670-MACS system (Moritex). CHO cells expressing an exogenous GPCR were directly seeded onto the SPR sensor chips without any chemical treatment of the surface of the chips, and cultured overnight in 35 mm culture dishes [Figure 2a]. After incubation in HEPES-Tyrode’s-BSA (HTB) buffer, the cells were mounted on the SPR system and further washed with HTB buffer at a flow rate of 30 µL/min through the flow channels until the base-line signal became stable. SPR measurements were performed using two flow channels; ligands for GPCRs dissolved in HTB buffer were added to one channel, the sensing channel, and the corresponding vehicle was added to the other, reference channel. As shown in Figure 2b, a rapid SPR response was evoked by 1 nM LTB4 in CHO-BLT1 cells, but not by HTB buffer.
Cells seeded on a sensor chip provide a physical possibility for monitoring the density change of cellular components in the evanescent field. In theory, the detection range of SPR is limited to the evanescent field, but in practice it is considered possible to detect density changes within a distance of approximately 300 nm of the sensor chip surface [5]. It should be noted that the plasma membrane has a thickness of several nanometres [6] and is structurally supported by cytoskeleton. The cellular cortex is an actin-rich layer just beneath the plasma membrane, responsible for maintaining cell shape and motility. It is rapidly rearranged in response to various stimuli via the polymerisation-depolymerisation cycle of actin [7], which leads to density changes in the cellular cortex. The SPR response reflects the total output of the density change in the evanescent field [Figure 3].
Advantages and disadvantages of the SPR assay
A successful screening method should be simple, sensitive and low-cost, and if possible rapid. The SPR assay is simple and fast: for the cell preparation, all that is needed is to seed the cells onto the sensor chip without any chemical modification of the chip surface. At a flow rate of 30 µL/min, it generally takes 15 minutes for one assay. The dual-channel nature of the system increases the reliability of each assay. The sensitivity of SPR detection is comparable with those of the conventional methods [Table 1]. In addition to endogenous GPCRs, SPR successfully detected endogenous receptors-mediated GPCR signals at nano molar concentration of ligands (LPA and S1P) as shown in Table 1. Usually, observation of the cytoskeleton is based on fluorescence or chemiluminescence detection using probes. In contrast, the SPR assay does not need any molecular labelling or cellular indicators and can thus save cost and time.
One of the disadvantages of the new assay is that suspended cell lines are not suitable for the SPR assay. Some treatments, such as starvation and permeabilisation, which result in destruction of the cell shape and the cytoskeleton, can result in measurement instability. SPR detection is dependent on cytoskeletal rearrangement, so it is only applicable for Gi- and/or G12/13-coupled receptors [Table 1]. It should be noted that detection of G12/13-coupled GPCR-mediated cellular response has always been extremely difficult.
Future prospects
We have developed a new method for detecting GPCR signals using a SPR sensor system. The new assay supports the possibility that cytoskeletal rearrangements could be a novel indicator of cellular responses. The assay could provide a new approach for ligand screening of orphan GPCRs. It is especially useful for G12/13-coupled receptors, for which an efficient screening method has not previously been developed. In addition, the use of the SPR detection method in ligand screening has the significant advantage of monitoring cellular response in living cells without using any label. We speculate that additional biosensors for living cells, such as SPR, will lead to the development of other new screening methodologies for basic medical science and the pharmaceutical industry.
References
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The authors
Kexin Chen, Hideru Obinata
and Takashi Izumi*
Department of Biochemistry,
Gunma University Graduate School of Medicine, 3-39-22 Showa-machi,
Maebashi, Gunma 371-8511, Japan.
*Corresponding author:
Takashi Izumi*
e-mail: takizumi@med.gunma-u.ac.jp
