Establishing a Biophysical Assay for the Interactions of Calcitonin Family G Protein-Coupled Receptors with Receptor Activity Modifying Proteins And The Elucidation of the Structure-Based Signalling of G Protein-Coupled Receptors via the Novel Application of Geometric Morphometrics With Principal Component Analysis

Abstract

GPCRs are implicated in a wide variety of diseases, making them particularly attractive drug targets; critical developments in structural and functional techniques have led to breakthrough discoveries, shifting paradigms of understanding with novel concepts. Select ligands are now known to induce biased signalling in GPCRs, differentially activating intracellular signalling pathways; conformational landscapes of GPCRs are heterogeneous, indicating diversity in the activation transition and intermediate states which likely correlate with biased signalling. Moreover, the majority of GPCRs signal via more than one G protein sub-type, preferentially coupling with ranked selectivity, and are allosterically modulated by a range of factors. In order to develop safe, effective and selective therapeutics against GPCRs, a holistic understanding of these concepts necessitates comprehensive, multidisciplinary approaches, combining enhanced biophysical and biochemical assays, determination and analyses of structure, and complementary computational techniques. This thesis presents the successful establishment of a FRET-based interaction assay for the calcitonin family of GPCRs and the RAMPs, with an average FRET efficiency of 87.78%, average interaction distance of 3.42 nm, and cAMP pEC50s in agreement with the literature. This promising assay will offer novel insights into GPCR-RAMP dynamics, forming the basis of a high-throughput biophysical drug discovery platform. Secondly, the structure-based signalling of GPCRs was explored with the novel application of geometric morphometrics and principal component analysis to resolved structures, consistently and reliably classifying GPCRs by their global shape morphology, supported by PERMANOVA and ANOSIM multivariate statistics. Case study examples of the β2-adrenergic, adenosine 2A, secretin-like and calcium-sensing receptors first proved this concept effective, before exploration of thermostabilisation, fusion proteins, the structural determinants of G protein coupling, and AlphaFold structures. Overall, this thesis provides novel contributions to fully elucidating the structure/function relationship of GPCRs, building a multidisciplinary model of understanding, which will enable the unprecedented discovery and development of safe, effective therapeutics.