The molecular basis of the impact of GPCR phosphorylation on arrestin interactions and receptor endocytosis

This thesis describes the influence of G protein-coupled receptor (GPCR) phosphorylation on its interaction with arrestin2, and subsequent role in the formation of arrestin2 signaling complexes and receptor internalization.
Chapter 1 provides a general introduction to GPCRs and their intracellular partner proteins: G proteins, G protein-coupled receptor kinases (GRKs) and arrestins. It describes the role of phosphorylation motifs in the arrestin interaction, introduces biased signaling and receptor internalization. Furthermore, recent advancements in solving GPCR-GRK and GPCR-arrestin complex structures are presented.
Chapter 2 focuses on the functional dynamics of arrestin2. It provides a protocol for the stable expression and purification of arrestin2 and an advanced NMR analysis comprising amide backbone assignments and relaxation-dependent functional dynamics. This revealed pronounced micro- to millisecond motions of apo arrestin2, which may lie on the pathway to activation.
Chapter 3 describes the interaction of arrestin2 with various CC chemokine receptor 5 (CCR5) C-terminal tail phosphopeptides. The presented structural and functional analysis revealed a key phosphorylation motif, pXpp, responsible for robust arrestin2 recruitment. This motif is found in many other receptors and appears as one of the determinants of specific arrestin2 vs. arrestin3 recognition.
Chapter 4 presents insights into clathrin-mediated endocytosis (CME), which is the dominant mechanism of GPCR internalization. The interactions of arrestin2 with the main constituents of CME, the clathrin protein and the adaptor protein 2 (AP2), were investigated, the role of receptor phosphorylation in this process analyzed, and the in vitro results further validated through cellular functional assays.
Chapter 5 describes new findings on the molecular mechanism of arrestin-biased signaling in the 1-adrenergic receptor (1AR) by carvedilol. A structural and biochemical analysis of the 1AR-arrestin2 complex revealed that arrestin-biased signaling occurs by conformational exclusion of G protein binding, rather than by selection of arrestin2 binding.
Chapter 6 is a mini-review focusing on recent advances in solving GPCR-arrestin complex structures. The review also provides future directions of possible research to obtain a deeper understanding of arrestin signaling.
Chapter 7 describes attempts to form stable complexes of full-length CCR5 with the G protein receptor kinase GRK2 as well as arrestin2, and subsequent analyses of the complexes by negative-stain EM and cryo-EM.
Chapter 8 is a protocol for the formation of a stable complex of full-length CCR5 with the super-agonist chemokine [6P4]CCL5 and the Gi protein for cryo-EM analysis. Additionally, the chapter describes the backbone NMR assignment of [6P4]CCL5.