Investigating G-protein coupled receptor signalling with light-emitting biosensors

Abstract

G protein-coupled receptors (GPCRs) are the most frequent target of currently approved drugs and play a central role in both physiological and pathophysiological processes. Beyond the canonical understanding of GPCR signal transduction, the importance of receptor conformation, beta-arrestin (β-arr) biased signalling, and signalling from intracellular locations other than the plasma membrane is becoming more apparent, along with the tight spatiotemporal compartmentalisation of downstream signals. Fluorescent and bioluminescent biosensors have played a pivotal role in elucidating GPCR signalling events in live cells. To understand the mechanisms of action of the GPCR-targeted drugs currently available, and to develop new and better GPCR-targeted therapeutics, understanding these novel aspects of GPCR signalling is critical. In this review, we present some of the tools available to interrogate each of these features of GPCR signalling, we illustrate some of the key findings which have been made possible by these tools and we discuss their limitations and possible developments.

Keywords: BRET; FRET; G protein; GPCR; bioluminescence; fluorescent biosensor; signalling.

FIGURE 1

Schematic representations of sensors for GPCR activation (A) Generic intramolecular conformational RET sensor with energy donor X fused to intracellular loop 3 (ICL3) and energy acceptor Y fused to C-terminus. X and Y can be a CFP/YFP or CFP/FlAsH FRET pair or an RLuc/YFP or RLuc/FlAsH BRET pair (B) Intramolecular conformational sensor with circularly permuted GFP (cpGFP) fused to ICL3. (C) Fluorescent nanobody-based biosensor with GFP fused to camelid nanobody which binds to active-conformation ICL3.

FIGURE 2

Schematic representations of sensors for GPCR/G-Protein coupling (A) Intermolecular FRET sensor for G-protein recruitment with CFP donor fused to Gα subunit and YFP acceptor fused to Gβγ subunit. (B) Intermolecular BRET sensor for Gβγ/GRK interaction with NanoLuc donor fused to GRK and Venus acceptor formed by bimolecular fluorescence complementation (BiFC) between Gβ and Gγ subunits. (C) Intermolecular FRET sensor for G-protein recruitment with CFP donor bound to Gβγ subunit and YFP acceptor bound to receptor C-terminus. (D) Systematic protein affinity strength modulation (SPASM) sensor consisting, from N- to C-terminus, of a GPCR, mCit FRET acceptor, ER/K linker, mCer FRET donor, and peptide which binds ICL3 upon receptor activation. (E) BRET sensor with ER/K linker and YFP (BERKY) consisting, from N- to C-terminus, of membrane anchor, NanoLuc BRET donor, ER/K linker, YFP BRET acceptor and detector peptide which selectively binds the Gα subunit in its GTP bound state. (F) NanoBiT sensor for G-protein recruitment with the large NanoLuc fragment LgBiT fused to the Gα subunit and the small NanoLuc fragment SmBiT fused to the receptor C-terminus. (G) NanoBiT sensor for G-protein dissociation with LgBiT fused to the Gα subunit and SmBiT fused to the Gβγ heterodimer. (H) MiniG BRET sensor with RLuc BRET donor fused to receptor C-terminus and Venus BRET acceptor fused to MiniG. (I) MiniG bystander BRET sensor with RLuc donor fused to the MiniG protein and BRET acceptor anchored to the membrane by a membrane localisation sequence.

FIGURE 3

Schematic representations of sensors for GPCR/beta-arrestin (β-arr) and GPCR kinase (GRK) interaction (A) Generic intermolecular RET sensor for β-arr recruitment with energy donor X fused to GPCR C-terminus and energy acceptor Y bound to β-arr. X and Y can be and RLuc/YFP BRET pair, a CFP/YFP FRET pair, or a FlAsH/ReAsH FRET pair (B) Bystander BRET sensor for β-arr recruitment with RLuc donor fused to β-arr and BRET acceptor anchored to the membrane by a membrane localisation sequence. (C) BRET sensor for GRK recruitment with RLuc donor fused to GPCR C-terminus and BRET acceptor fused to GRK. (D) Generic intramolecular RET sensor for β-arr conformational change with energy donor X fused to β-arr N-terminus and energy acceptor Y fused to β-arr C-terminus. X and Y can be an RLuc/YFP BRET pair, an RLuc/FlAsH BRET pair, NanoLuc/cyOFP1 BRET pair, or a CFP/FlAsH FRET pair. (E) NanoBiT sensor for β-arr recruitment with large NanoLuc fragment LgBiT fused to β-arr and small NanoLuc fragment SmBiT fused to GPCR C-terminus. (F) Bystander split NanoLuc sensor for β-arr recruitment with one NanoLuc fragment fused to β-arr and the other complementary fragment anchored to the membrane by a membrane localisation sequence.

FIGURE 4

Schematic representations of sensors for cAMP and PKA (A) Generic PKA heterotetrameric cAMP FRET sensor with donor fluorophore X bound to PKA regulatory subunit and acceptor fluorophore Y bound to PKA catalytic subunit. X and Y can be a fluorescein/rhodamine FRET pair or YFP/CFP FRET pair (B) Generic gain of FRET cAMP sensor with donor and acceptor fluorophores X and Y bound to cyclic nucleotide binding domain (CNBD). See Table 4 for examples of CNBD and fluorophores. (C) Generic loss of FRET cAMP sensor with donor and acceptor fluorophores X and Y bound to cyclic nucleotide binding domain (CNBD). See Table 4 for examples of CNBD and fluorophores. (D) PKA activity reporter (AKAR) with donor and acceptor fluorophores X and Y bound to phosphoamino acid binding domain (dark blue) and PKA-specific phosphorylatable sequence (purple line). See Table 4 for example, fluorophores.

FIGURE 5

Schematic representations of sensors for IP3, DAG, and PKC (A) IP3 RET sensor with energy donor and acceptor X and Y fused to IP3 binding domain from IP3 receptor. (B) DAGR FRET sensor for DAG with donor and acceptor fluorophore X and Y fused to C1 DAG binding domain. (C) Daglas FRET sensor for DAG consisting from N- to C-terminal, of a membrane anchor, α-helical linker, YFP FRET acceptor, α-helical linker with Gly-Gly hinge (grey circle), cysteine rich domain or DAG binding, α-helical linker, and CFP FRET donor. (D) Unimolecular DAG BRET sensor consisting from N- to C-terminus of a membrane anchor, GFP BRET acceptor, unstructured linker, RLuc BRET donor, and C1b DAG binding domain. (E) PKC activity reporter (CKAR) with energy donor and acceptor X and Y fused to phosphothreonine binding domain (dark blue) and PKC-specific phosphorylatable sequence (red line).

FIGURE 6

Schematic representations of sensors for intracellular Ca²⁺ (A) Pericam/G-CaMP single wavelength calcium indicator with circularly permuted fluorophore (cpFP) sandwiched between calmodulin (CaM) and CaM-binding peptide M13. (B) Cameleon Ca²⁺ FRET reporter with CaM and M13 sandwiched between CFP FRET donor and YFP FRET acceptor. (C) FRET reporter for Ca²⁺ with donor and acceptor fluorophores bound to troponin C (TNC).