G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects.
G protein-coupled receptors (GPCRs) are seven transmembrane (7TM) cell surface receptors and major drug targets. GPCRs represent the largest protein family in humans, comprising 826 out of 20,283 (4%) genes. In humans, the majority of GPCRs are olfactory receptors. The remaining ~350 non-olfactory members are considered druggable, of which 165 have been identified as drug targets. Importantly, 35% of all marketed and FDA-approved drugs target GPCRs, making them the focus of intensive investment by pharmaceutical companies.
With the recent advances in multitarget drugs that regulate multiple GPCRs simultaneously and the increasing need to screen for off-target activities, it is necessary to have efficient, robust, and reproducible assays to measure the activation of GPCRs in living cells. Furthermore, it is crucial to identify the signaling pathways that a specific GPCR/drug combination regulates to understand cellular physiology. In addition, it is increasingly important for drug discovery campaigns to assess both the desired on-target and on-pathway effects, as well as the potentially detrimental off-target and off-pathway effects. This review examines the currently available cell-based assays that evaluate GPCR activities and their downstream signaling pathways.
GPCRs and GPCR-mediated signaling have been the focus of research since the pioneering work on adrenergic receptors by Robert J. Lefkowitz and Brian Kobilka, who won the 2012 Nobel Prize in Chemistry for their research on GPCRs. A given GPCR can regulate multiple signaling pathways in different cell types, depending on the cellular transducer and effector repertoire. Therefore, GPCRs play a crucial role in signal transduction in diverse physiological responses, which include sensory perception, olfaction, neurotransmission and hormone signaling, immune response, cardiovascular regulation, and essentially every other physiological function. Likewise, GPCRs are implicated in the pathogenesis or pharmacological treatment of frequently occurring disorders, such as cancer, hypertension, diabetes, and mental disorders like schizophrenia and depression, supporting their key role in health and disease.
Heterotrimeric G proteins formed of Gα, Gβ, and Gγ subunits, together with GPCR kinases (GRKs) and β-arrestins, are the three main types of protein families that directly interact with GPCRs upon activation and are called transducers. Additionally, pathway selectivity in assays can be affected by the tested ligand, a concept called biased signaling or functional selectivity. Agonist binding to a GPCR leads to conformational changes that transfer to the intracellularly bound heterotrimeric G protein and promotes a GDP/GTP exchange in Gα, leading to the dissociation of active Gα and Gβ/γ. These then initiate or modulate one of the classical second messenger cascades (cAMP, IP 3/DAG, or cGMP) and can also directly interact with effector proteins such as ion channels, e.g., GIRK channels. GPCR activation also leads to receptor phosphorylation by GPCR kinases (GRKs), β-arrestin-mediated desensitization, and endosomal internalization. Recent evidence suggests that this can lead to sustained intracellular G protein-mediated signaling from endosomes, resulting in MAP kinase (MAPK) pathway activation and nuclear gene regulation. However, β-arrestins can also activate MAPK signaling directly at the plasma membrane.
GPCRs are often called ’7TM receptors’ because they consist of seven hydrophobic alpha-helical transmembrane domains (TM1–TM7) in a common three-dimensional arrangement. Each GPCR consists of a single polypeptide with an extracellular N-terminus, an intracellular C-terminus, three extracellular loops (ECL1–ECL3), and three intracellular loops (ICL1–ICL3). The orthosteric binding site for the endogenous ligand is located either in between the transmembrane helices, in the extracellular domain, or both. Even an unusual intracellular binding site has recently been described for TAS2R14. When viewed from the extracellular side, the 7TM regions are arranged in an anti-clockwise orientation. Agonist binding and GPCR activation result in a prominent outward movement of TM6 by 7–19 Å away from TM3, thereby breaking specific interactions (‘locks’). This conformational change is transferred via the directly connected ICL3 to the bound Gα protein and leads to the release of the GDP and the concomitant dissociation of the active G protein subunits followed by downstream signaling.
Biased signaling or functional selectivity describes the observation that different ligands can preferentially activate different pathways through the same receptor with regards to potency as well as efficacy. Ligands can affect the coupling preferences of GPCRs for different G protein subtypes that modulate cAMP, calcium, or RhoA. Ligands can also affect GPCR phosphorylation via GRKs, arrestin binding, internalization, and, according to recent evidence, G protein-dependent endosomal signaling in a biased fashion. Indeed, agonists and antagonists that selectively promote either G protein-dependent or β-arrestin-dependent responses were identified in drug discovery campaigns. This concept was further extended to different combinations of G protein subtypes. However, the multiple alternative transducers and their various pathway components downstream of an activated GPCR contribute to signaling bias. Biased signaling can become apparent at any hierarchical level of signaling, i.e., at the level of transducers, effectors, second messengers, and transcription factors. Additionally, more recent reports suggest that proteins other than G proteins, GRKs, and β-arrestins can directly bind to active GPCRs and function as transducers, as discussed in more detail below—another potential source of bias that might be exploited in the future.
Ligands can induce receptor conformations with biased preferences for GRKs, arrestins, and internalization. Ligands can then also have different endosomal retainment properties. When using cell-based assays to monitor GPCR activation and downstream signaling, it is important to consider the potential complications arising from different binding and signaling kinetics under potentially non-equilibrium assay conditions.
