Characterising constitutive activity and inverse agonism across the free fatty acid G protein-coupled receptors using BRET biosensors

Kenawy, Marwa (2025) Characterising constitutive activity and inverse agonism across the free fatty acid G protein-coupled receptors using BRET biosensors. PhD thesis, University of Glasgow.

Abstract

Recent studies have identified five G protein-coupled receptors (GPCRs), known as free fatty acid receptors (FFARs), as promising targets for treating metabolic and inflammatory conditions. However, the constitutive activity of these receptors has not yet been thoroughly characterised. In this study, bioluminescence resonance energy transfer (BRET) biosensors were employed to define the constitutive signalling properties of all five FFARs. The bystander-BRET technique was first used to assess β-arrestin2 recruitment to each FFAR, enabling the determination of which receptors naturally interact with β-arrestin2 in the
absence of a ligand. Among the five FFARs tested, FFA4 exhibited the highest levels of constitutive and basal β-arrestin2 recruitment. Furthermore, the extent of constitutive β-arrestin2 recruitment was directly proportional to the amount of FFA4 expressed, indicating a strong relationship between receptor abundance and constitutive signalling activity.

Although several receptors did not display measurable constitutive signalling, the bystander BRET assay proved effective for detecting agonist-stimulated β-arrestin2 recruitment across all FFARs. The magnitude of agonist-induced arrestin recruitment varied by receptor, following the order: FFA4 > GPR84 > FFA1 > FFA2/FFA3. This suggests that FFA4 is not only the most active in the absence of a ligand but also the most responsive to agonist stimulation in terms of arrestin pathway engagement. Additionally, the effects of AH-7614, a well-characterised negative allosteric modulator (NAM) of FFARs, were evaluated. The results revealed that AH 7614 acts as an inverse agonist of β-arrestin2 recruitment, thereby reducing its constitutive activity. Moreover, AH-7614 partially inhibited agonist-mediated β-arrestin2 recruitment, primarily by decreasing the recruitment rate rather than completely blocking the response. These findings highlight the value of BRET-based assays for dissecting constitutive signalling at FFARs and the complexity of their regulation by both receptor expression and
pharmacological modulators. This study provides important insights into FFAR signalling mechanisms, which may inform the development of new therapeutic strategies targeting metabolic and inflammatory diseases.

Additionally, a bystander BRET-based internalisation assay was employed to measure both ligand-dependent and constitutive internalisation of free fatty acid receptors. These studies demonstrated that FFA4 receptors displayed constitutive internalisation, whereas FFA1, FFA4, and GPR84 displayed ligand-induced internalisation. FFA4 NAM AH-7614 functions as an inverse agonist of FFA4 internalisation. The kinetics of the AH-7614 internalisation inverse agonism response were significantly slower than those of FFA4 agonists stimulating
internalisation. Furthermore, AH-7614 inhibited agonist-stimulated internalisation. However, this effect varied depending on the ligands used.

Constitutive G protein signalling of free fatty acid receptors was systematically assessed using TRUPATH BRET biosensors, enabling real-time and quantitative measurements of both ligand dependent and constitutive G protein activation.These experiments revealed that FFA2, FFA4, and GPR84 all displayed significant constitutive activity, coupled to both Gαq and Gαi2 in the absence of agonist stimulation. Importantly, the extent of constitutive G protein signalling correlated with receptor expression levels, indicating that higher receptor abundance enhances basal G protein activation. Pharmacological characterisation further demonstrated that antagonists of FFA2 and FFA4-specifically CATPB and AH-7614, respectively, function as inverse agonists by reducing basal Gαi2 activation, thereby confirming their ability to inhibit constitutive receptor signalling. The FFA4 allosteric modulator AH-7614 exhibited robust inverse agonistic properties and rapidly suppressed basal G protein activity. These findings establish that constitutive signalling is a prominent feature of FFA2, FFA4, and GPR84 andthat
inverse agonists targeting these receptors can effectively dampen their constitutive activity. This study provides new insights into the pharmacological regulation of free fatty acid receptors and underscores the importance of constitutive activity and inverse agonism in GPCR biology.

In summary, this thesis provides a comprehensive analysis of the constitutive and ligand regulated signalling properties of free fatty acid receptors, with a particular focus on their G protein coupling profiles and trafficking dynamics. By employing advanced BRET- based biosensors, including the TRUPATH platform, we systematically characterised both ligand dependent and constitutive G protein activation across the FFA receptor family. These studies revealed that FFA2, FFA4, and GPR84 display significant constitutive G protein signalling, and that the extent of this basal activity is closely linked to receptor expression levels. Furthermore, pharmacological profiling demonstrated that antagonists of FFA2 and FFA4 function as inverse agonists, effectively suppressing constitutive G protein activity, with the FFA4 allosteric modulator AH-7614 exhibiting rapid and robust inverse agonism. Overall, bystander BRET and Trupath have provided new insights into the complex trafficking, internalisation, and G protein behaviour of these receptors, highlighting the importance of characterising these receptors for validation in interpreting receptor dynamics. Collectively, these findings advance our understanding of the fundamental pharmacology and cell biology of fatty acid receptors and establish a set of sensitive quantitative tools and conceptual frameworks that will be invaluable for future drug discovery efforts targeting these receptors in metabolic and inflammatory diseases.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Hudson, Dr. Brian and Milligan, Professor Graeme
• Date of Award: 2025
• Depositing User: Theses Team
• Unique ID: glathesis:2025-85242
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Jun 2025 12:22
• Last Modified: 23 Jun 2025 12:27
• Thesis DOI: 10.5525/gla.thesis.85242
• URI: https://theses.gla.ac.uk/id/eprint/85242

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