Impact of macrophage differentiation on adenosine receptor expression and function

Adenosine receptors are expressed in many tissues throughout the body and play an important role in tissue homeostasis. There are four adenosine receptors (A1, A2A, A2B and A3), with tissue-specific expression which is altered in a wide variety of disease states. We and others have demonstrated that different adenosine receptors interact with each other and this interaction leads to altered downstream cellular function. However, the mechanisms and exact effects of these interactions are poorly understood. Given the ubiquitous expression of these receptors and their importance in many different diseases, understanding these mechanisms is a key goal to understand their role in disease states and enable effective adenosine-receptor targeting. Important cells where adenosine receptors have been shown to be differentially expressed and play a role in cellular function are immune cells, specifically macrophages.
This project aims to:
1) Define how macrophage polarisation affects adenosine receptor expression
2) Assess adenosine receptor interactions, degradation and processing in macrophages under a variety of conditions
3) Explore how differential adenosine receptor expression in macrophages affects downstream signalling and cellular function
Project plan:
Work will be carried out in THP1 cells as a human monocyte-macrophage model system and primary human monocyte-derived macrophages.
Aim 1: We have developed a variety of novel techniques to visualise adenosine receptor expression in primary cells including ligand-directed covalent labelling, receptor-selective nanobodies and fluorescently-tagged ligands (Comeo et al., 2020; Soave et al., 2020a,b; Stoddart et al., 2020). These techniques will be used to define adenosine receptor expression in different types of monocyte-derived macrophages.
Aims 2 &3: We will also use CRISPR/Cas9 genome editing in conjunction with nucleofector based electroporation to introduce the nanoluciferase fragments HiBiT or SmBiT onto the N or C terminus of each adenosine receptor endogenously expressed in THP1 cells. Following addition of purified LgBiT, the nanoluciferase can be re-complemented to form a bioluminescent protein. This approach will be used to dynamically study ligand-receptor and receptor-receptor interactions using bioluminescence resonance energy transfer (BRET), and receptor degradation and processing following differentiation into macrophages. Real- time receptor degradation can be followed using C-terminal HiBiT or SmBiT tags in cells expressing cytosolic LgBiT. Macrophage function with differential adenosine receptor expression (both in transfected cells and primary macrophages) will be studied by monitoring cAMP accumulation, CREB phosphorylation and cytokine/chemokine production.
This project is a multi-disciplinary collaboration across several Schools within the Centre of Membrane Proteins and Receptors (COMPARE) University of Nottingham, utilising cutting-edge techniques to dissect fundamental biological processes. As well as furthering our understanding ofadenosine receptor pharmacology, the results from this project will have implications in a wide variety of diseases. COMPARE has a renowned Team Science approach for postgraduate training and the student will benefit from working within this environment.