G protein-coupled receptors (GPCRs) are the most abundant family of cell surface receptors and represent the largest class of therapeutic targets. Responding to a wide range of stimuli, GPCRs elicit diverse signaling events ranging from phosphorylation cascades and transcription regulation to ion channel activity and secondary messenger production. Either directly or indirectly these signaling events usually lead to changes in at least one of the following: cell size/shape, cell-substrate attachment quality, or the kinetics of cell proliferation. Because xCELLigence® real-time cell analysis (RTCA) instruments are exquisitely sensitive to these physical phenomena, they excel at quantitatively monitoring GPCR-mediated signaling. The efficacy of RTCA for studying diverse GPCRs is illustrated in Figure 1. Here Chinese hamster ovary cells expressing human receptors for histamine, dopamine, or serotonin are exposed to their respective ligands. Histamine receptor, dopamine receptor, and serotonin receptor (which act through the Gaq, Gas, and Gai classes of G proteins, respectively) all show distinct, robust, and titratable impedance responses to their respective ligands. GPCR antagonists can block these ligand-induced responses (data not shown), indicating that the phenomena being observed reflect bona fide GPCR activation. By plotting the amplitude of the impedance trace (at a given time point) as a function of ligand concentration it is possible to calculate EC50 values. Importantly, EC50 values calculated from RTCA traces match extremely well the values determined by alternative methodologies.
It has long been known that a single GPCR can impinge upon multiple signaling pathways. A more recent finding is that drugs targeting a single GPCR can differentially modulate distinct subsets of that receptor’s signaling repertoire2. This phenomenon, known as “ligand-biased signaling” or “functional selectivity”, presents the potential for achieving extremely specific biochemical modulation with therapeutic drugs.
The phenomenon of GPCR functional selectivity presents a technical challenge in drug discovery and development because it makes it difficult to comprehensively evaluate the biochemical effects of a given GPCR-ligand interaction. A unique assay may be required for evaluating how a compound influences each of a GPCR’s downstream pathways. Moreover, the complete signaling repertoire of most GPCRs is unknown. Accordingly, an assay of broader scope – capable of evaluating all of a GPCR’s output channels simultaneously – is needed. Enter xCELLigence. As one example of how RTCA meets this need, consider stimulation of the b2-adrenergic receptor (b2AR). In HEK293S cells b2AR was known to elicit at least two distinct signaling events: activation of the MEK pathway and accumulation of cAMP via adenylyl cyclase stimulation. Stimulation of b2AR with the ligand isoproterenol gives rise to the distinct impedance trace shown in Figure 2. Inhibition of adenylyl cyclase (AC) or MEK similarly reduce the impedance in the 5-100 minute time regime, but have no impact on the first (negatively sloped) phase of the response profile (0-5 minutes). This suggests that the first phase is caused by a signaling pathway other than those mediated by AC or MEK. Stallaert and coworkers used this new, holistic view of b2AR activation to hypothesize and then prove the existence of a novel b2AR–promoted Ca2+ mobilization event.
Key Benefits of xCELLigence®
- Flexibility: Capable of probing activation of GPCRs of wide structural and functional diversity.
- Broad Scope: Simultaneously screen GPCR function across all coupling classes: Gs, Gq, as well as Gi and G12/13 (which are traditionally difficult).
- Global Observation: Detection of functional selectivity.
- Cell Variety: Assay endogenous GPCRs in primary cells, stem cells, or disease relevant cell lines.