Quantitative approaches for studying G protein-coupled receptor signalling and pharmacology

Abstract

G protein-coupled receptor (GPCR) signalling pathways underlie numerous physiological processes, are implicated in many diseases and are major targets for therapeutics. There are more than 800 GPCRs, which together transduce a vast array of extracellular stimuli into a variety of intracellular signals via heterotrimeric G protein activation and multiple downstream effectors. A key challenge in cell biology research and the pharmaceutical industry is developing tools that enable the quantitative investigation of GPCR signalling pathways to gain mechanistic insights into the varied cellular functions and pharmacology of GPCRs. Recent progress in this area has been rapid and extensive. In this Review, we provide a critical overview of these new, state-of-the-art approaches to investigate GPCR signalling pathways. These include novel sensors, Förster or bioluminescence resonance energy transfer assays, libraries of tagged G proteins and transcriptional reporters. These approaches enable improved quantitative studies of different stages of GPCR signalling, including GPCR activation, G protein activation, second messenger (cAMP and Ca2+) signalling, β-arrestin recruitment and the internalisation and intracellular trafficking of GPCRs.

Keywords: BRET; Ca2+ signalling; Cell signalling; FRET; GPCRs; IP3; cAMP.

Fig. 1.

Overview of GPCR signalling. Following agonist binding, the GPCR undergoes a conformational change, enabling it to bind and activate a heterotrimeric G protein. G protein signalling is determined primarily by the type of Gα subunit, which falls into one of four subfamilies: Gα_s, which is coupled to activation of adenylyl cyclase and production of cAMP; Gα_i/o, which is coupled to inhibition of adenylyl cyclase and activation of phosphodiesterase to decrease cAMP; Gα_q/11, which is coupled to activation of phospholipase C and subsequent mobilisation of intracellular Ca²⁺ through IP₃ receptors; and Gα_12/13, which is coupled to activation of Rho GTPases. The Gβγ complex can also mediate signalling by, for example, activating G protein-coupled inwardly rectifying potassium (GIRK) channels. Activated GPCRs are phosphorylated by GRKs, enabling recruitment of β-arrestins. β-arrestins can interact with the AP2 complex, which then interacts with clathrin. Recruitment of clathrin elicits the formation of a clathrin-coated pit, followed by endocytosis and receptor internalisation. Following internalisation, some GPCRs can continue signalling via G proteins from subcellular compartments. DAG, diacylglycerol; IP₃R, IP₃ receptor; Rho kinase, Rho-associated protein kinases. Created in BioRender by Redfern-Nichols, T., 2025. https://BioRender.com/w94r168. This figure was sublicensed under CC-BY 4.0 terms.

Fig. 2.

Fluorescence and luminescence in assays for GPCR signalling. Both luminescence and fluorescence measurements are versatile and common ways to assess GPCR signalling. Proximity sensors allow detection of appropriately oriented proteins in proximity (less than 10 nm, illustrated in A) (Kobayashi et al., 2019). These sensors often utilise (B) FRET or (C) BRET, which rely on the excitation of a fluorescent acceptor by a fluorescent or luminescent donor, respectively. (D) Another type of proximity assay uses luciferases or fluorescent proteins that are ‘split’ into two protomers, which reconstitute an intact protein when they approach close enough to bind (Bae et al., 2024). (E) Single-wavelength sensors that produce increased fluorescence or bioluminescence following a conformational change can also detect GPCR signalling events. Proximity assays can quantify upstream GPCR dynamics such as (F) ligand binding or (G) GPCR conformational changes through intramolecular proximity sensors. Created in BioRender by Redfern-Nichols, T., 2025. https://BioRender.com/y05d181. This figure was sublicensed under CC-BY 4.0 terms.

Fig. 3.

Measuring β-arrestin and GPCR kinase recruitment. (A) Biosensors to measure GRK activity during the initial steps in GPCR desensitisation are relatively limited and rely on RET between a tagged GPCR and GRK. (B–D) Biosensors to measure β-arrestin recruitment are diverse and can be broadly classified into three types: proximity based, conformational change based and transcriptional reporter based. Proximity-based sensors are commonly used to measure proximity of the recruited β-arrestin to (B) a GPCR or (C) the plasma membrane. Other sensors measure recruitment of β-arrestin by detecting a conformational change via either (D) increased signal from a fluorescent conformational sensor fused to β-arrestin, or (E) an intramolecular proximity sensor. (F) Proprietary assays use cell lines expressing a GPCR fused to a transcription factor and a β-arrestin tagged with a protease, which cleaves the transcription factor from the intracellular C terminus of the receptor when recruited, increasing the synthesis of a reporter gene. Created in BioRender by Redfern-Nichols, T., 2025. https://BioRender.com/z75j601. This figure was sublicensed under CC-BY 4.0 terms.

Fig. 4.

Proximity sensors for studying GPCR internalisation and subcellular trafficking. Internalisation can be measured by assessing the loss of a GPCR from the plasma membrane, either from (A) a reduction in the interaction between a tag on the extracellular N terminus of the GPCR and a membrane-impermeable component in the extracellular medium, or (B) a bystander interaction with a plasma membrane-associated GTPase (such as RIT or KRas). (C) Similar bystander interactions can be applied to measure the interaction of internalised GPCRs with monomeric GTPases specific to different subcellular compartments. These GTPases include Rab5a at clathrin-coated pits or early endosomes, Rab4 at fast recycling endosomes, Rab11 at slow recycling endosomes and Rab7 at late endosomes. A lysosomal marker, lysosome-associated membrane glycoprotein (LAMP, also known as LAMP1), is also used to indicate receptor degradation. Created in BioRender by Redfern-Nichols, T., 2025. https://BioRender.com/e65u934. This figure was sublicensed under CC-BY 4.0 terms.