Investigating the role of ICL3 in β1-adrenergic receptor activation

Abstract

G protein-coupled receptors (GPCRs) are 7-transmembrane (7-TM) alpha helical membrane proteins involved in a myriad of physiological functions and have implications in many diseases. GPCRs are important targets for ~35% of commercial drugs. Activation of GPCRs by external signals and through coupling to different G proteins or arrestins, results in cyclic adenosine monophosphate (cAMP) response, calcium mobilization, or phosphorylation of extracellular regulated protein kinases. Structural studies have provided detailed insight into the receptor architecture in active and inactive states of the receptors. However, GPCRs are dynamic and have a complex energy landscape as they regulate complex downstream signaling processes. The thermostable turkey β1-adrenergic receptor (tβ1AR) is a prototypical class A GPCR widely used in structural studies and is activated by catecholamine ligands, adrenaline, and noradrenaline. Although many crystal structures of tβ1AR are published, there is little known about the intracellular loop 3 (ICL3) – connecting transmembrane helices 5 and 6. It is usually truncated in crystallographic studies as it interferes with the lattice formation and is replaced with T4 lysozyme to enhance crystal contacts. In some active state crystal structures (3SN6, 3POG) of GPCRs, the ICL3 is present but cannot be resolved using X-ray diffraction because of its highly dynamic nature. Thus, a more suitable technique such as nuclear magnetic resonance (NMR) spectroscopy is needed to study the dynamic nature of this low complexity region in these receptors. In this thesis, I have investigated β1AR conformational dynamics by creating a chimera of tβ1AR transmembrane domain with the flexible ICL3 from human β1AR (hICL3). The position of ICL3 is close to the G protein binding site and hypothesized to have a role in binding specificity of the receptor with intracellular binding partners. This thesis provides a comprehensive analysis for characterization of the conformational dynamics of β1ARhICL3 chimera in response to a range of ligands and intracellular binding partners such as G protein mimetic nanobodies, miniG protein, and endophilin SH3 domains using NMR and biophysical techniques. In Chapter 3, successful cloning, expression in insect cells and purification of β1ARhICL3 chimera in detergent micelles together with expression and purification of intracellular binding partners such as endophilin SH3 domains is discussed. Optimisation of buffer conditions and preliminary NMR experiments were carried out to determine protein quality and stability. In Chapter 4, extensive NMR studies carried out using 19F NMR and 13C NMR are described. The characterization of ligand bound and ternary states with different binding partners suggests that the hICL3 imparts a more active character to β1ARhICL3 chimera as compared to tβ1AR. Intermediate conformation states can be observed indicating changes in exchange processes depending on the type of ligand bound to the receptor which indicates that the hICL3 modulates the receptor activity and interacts with the ICL2. Furthermore, distinct conformations of inactive β1ARhICL3 bound to antagonist and inactivating nanobody, active state bound to agonist and active-state nanobody, a second active state bound to miniG, and a SH3 domain bound state are observed. Chapter 5 discusses the biophysical characterisation of the β1ARhICL3 where the dissociation constant, Kd for Nb6B9, miniG and SH3 domain using microscale thermophoresis was obtained with all binding partners showing nanomolar affinities. The cAMP response in presence of a range of ligands was recorded using cellular assay comparing the chimera β1ARhICL3 with tβ1AR. In conclusion, the β1ARhICL3 construct due to the presence of hICL3 shows a more dynamic behaviour in comparison to the tβ1AR construct. This suggests that the β1ARhICL3 construct dynamics represents the wildtype β1AR more closely as the flexible loop imparts more plasticity to the receptor at the intracellular side. The β1ARhICL3 shows a smoother conformational landscape, allowing the receptor to adopt various intermediate conformations tending towards the active state.