Novel pharmacological tools for investigating the pharmacology of free fatty acid receptor 2

Dedeo, Domonkos (2023) Novel pharmacological tools for investigating the pharmacology of free fatty acid receptor 2. PhD thesis, University of Glasgow.

Abstract

The G protein-coupled receptor FFA2 acts as a receptor for short chain fatty acids produced by commensal bacteria through the fermentation of dietary fibre. While FFA2 is implicated in several disease conditions, such as obesity and ulcerative colitis, the receptor has so far not been successfully exploited therapeutically. This may reflect marked species selectivity of antagonists for the human receptor, which hinder pre-clinical in vivo studies in rodent models of disease. Herein a novel transgenic mouse strain expressing human FFA2 with an integrated HA epitope tag (hFFA2-HA) was characterised as a novel animal model. To assess the expression of hFFA2-HA at the messenger RNA and protein levels, quantitative reverse-transcription PCR and immunohistochemistry approaches were utilised. hFFA2-HA was found to be expressed in adipose, colon, spleen and bone marrow-derived neutrophils, at equivalent levels as mouse FFA2 in C57BL/6N mice. In addition, hFFA2-HA expression was confirmed in subsets of enteroendocrine and haematopoietic cells in the intestine and spleen, respectively. Following immunocytochemical confirmation of hFFA2-HA protein expression in bone marrow-derived neutrophils, these were used in functional studies ex vivo. The functionality of hFFA2-HA on neutrophils was established with the use of the hFFA2-selective radioligand [3H]GLPG0974, which was found to specifically bind membranes from hFFA2-HA mouse neutrophils, and with the use of the endogenous ligand propionate in [35S]GTPγS incorporation assays, where a concentration-dependent response was observed. Importantly, hFFA2-selective antagonists were able to inhibit C3-induced [35S]GTPγS incorporation and chemotaxis in hFFA2-HA-expressing, but not in C57BL/6N neutrophils. For the investigation of hFFA2 phosphorylation, novel, potentially phosphosite-specific antisera were evaluated through Western Blot and immunocytochemistry methods. Here, residues Thr306 and Thr310 were observed to be phosphorylated in an agonist-dependent manner, while residues Ser296 and Ser297 appeared to be constitutively phosphorylated. Replacement of these residues with alanine abolished antiserum binding but did not fully ablate β-arrestin-2 interactions. The development of novel pharmacological research tools, here in the form of transgenic mouse models and phosphosite-specific antisera, can not only aid understanding of FFA2 signalling and pharmacology but can also bridge the translational gap between in vitro and clinical studies.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: R Medicine > RM Therapeutics. Pharmacology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Milligan, Professor Graeme and Tobin, Professor Andrew
• Date of Award: 2023
• Depositing User: Theses Team
• Unique ID: glathesis:2023-83550
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 21 Apr 2023 15:37
• Last Modified: 21 Apr 2023 15:57
• Thesis DOI: 10.5525/gla.thesis.83550
• URI: https://theses.gla.ac.uk/id/eprint/83550

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