Basis and molecular consequences of interactions between adrenomedullin and ACKR3/CXCR4

Cocchiara, Pietro (2024) Basis and molecular consequences of interactions between adrenomedullin and ACKR3/CXCR4. PhD thesis, University of Glasgow.

Abstract

C-X-C-motif chemokine Receptor 4 (CXCR4) and Atypical Chemokine Receptor 3 (ACKR3) are two 7-transmembrane domains receptors widely expressed in the body and predominantly found in immune cells. These two receptors are often studied together due to their common ligand C-X-C-motif chemokine Ligand 12 (CXCL12) and its implication in several patho-physiological processes, such as embryo differentiation, cancer development and cancer cell proliferation, and the regulation of immune system responses to inflammatory stimuli. In the past, several non-chemokine ligands have been identified to be able to bind and exert various functions once bound to these receptors. In this thesis, the role of adrenomedullin (ADM), a non-chemokine peptide with pro-angiogenic effects, was explored. Since this small peptide is considered a putative ligand for ACKR3, the effects of the interactions between this ligand and ACKR3 were explored in Chapter 3. Indeed, a possible interaction was observed in xCELLigence Real-Time Cell Analysis (RTCA) with adrenomedullin influencing the activation of the receptor. However, this activation is still functionally poorly understood, since in β-arrestin recruitment the concentrations required for this interaction to happen were over physiological levels. Chapter 4 characterized the effects of the possible interaction between CXCR4 and adrenomedullin. Since in the past, the heterodimerization between CXCR4 and ACKR3 was shown, it was investigated whether adrenomedullin would have elicited any response upon stimulation of CXCR4 with this ligand to better clarify the role of this ligand in regulating the CXCR4-CXCL12-ACKR3 axis. Several approaches were used to study G protein signalling activation, β-arrestin recruitment and phosphorylation of CXCR4, for which adrenomedullin acted as a negative allosteric modulator affecting the efficacy of CXCL12 in activating G protein signalling, and as a biased antagonist towards CXCR4, showing a preference for blocking the G protein signalling pathway. Finally, in Chapter 5 a preliminary study of the GRK-mediated phosphorylation of both receptors was carried out, by using a small antagonist to block GRKs phosphorylation and NanoBiT technology to study the recruitment of these kinases to these receptors upon ligand-mediated activation. These approaches showed that GRK2/3/5/6 have an important role in phosphorylating the receptor, although the outcomes were cell-type and assay-dependent. However, this study opens the possibility of clarifying how the phosphorylation state affects the pharmacology of these receptors and to validate an effective “barcoding” of the receptors. In this thesis, it was demonstrated that adrenomedullin may represent an interesting starting point for developing new therapeutics targeting CXCR4 and ACKR3, although important structural studies are still required to highlight the outcomes of the interactions here described.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Subjects: Q Science > QR Microbiology > QR180 Immunology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Funder's Name: European Commission (EC)
• Supervisor's Name: Milligan, Professor Graeme and Graham, Professor Gerard
• Date of Award: 2024
• Depositing User: Theses Team
• Unique ID: glathesis:2024-84377
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 20 Jun 2024 13:54
• Last Modified: 20 Jun 2024 14:19
• Thesis DOI: 10.5525/gla.thesis.84377
• URI: https://theses.gla.ac.uk/id/eprint/84377

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