Single particle tracking of G protein-coupled receptor – beta-arrestin interactions

Grimes, Jak ORCID: 0000-0001-8992-8224 (2022). Single particle tracking of G protein-coupled receptor – beta-arrestin interactions. University of Birmingham. Ph.D.

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Abstract

Following activation by an agonist, G protein-coupled receptors (GPCRs) recruit b-arrestins, which mediate rapid signal desensitisation and receptor internalisation. b-arrestins can also activate non-classical signalling pathways, distinct from those triggered by G proteins. The mechanisms that govern receptor–b-arrestin interactions in the complexity of a living cell, however, remain poorly understood.
Classically, b-arrestins are thought to exist in the cytoplasm and form stable complexes with activated GPCRs at the plasma membrane, diffusing together with GPCRs to clathrin-coated pits (CCPs). Recent biophysical data has brought this model into disrepute, suggesting that transient interactions govern signalling, to meet the everchanging needs of the cell.
Here, a dynamic characterisation of GPCR–b-arrestin interactions is presented, at the plasma membrane of living cells with single-molecule resolution. b-adrenergic receptors were used as a prototypical GPCR family, given their crucial roles in cardiac physiology and pathologies dominated by aberrant b-arrestin signalling. bArr2 was shown to spontaneously bind to the plasma membrane, where it transiently interacts with b2ARs via lateral diffusion. Following receptor interaction, the plasma membrane stabilises bArr2 in a membrane-bound, active-like conformation, allowing it to reach clathrin-coated pits (CCPs) without the activating receptor.
A major lipid-anchoring site in the C-edge of bArr2 and a novel role of the finger loop region were identified – independent of a bound receptor. These results shed new light on the complex sequence of events involved in b-arrestin interaction with GPCRs and its activation, revealing a critical role for b-arrestin interactions with the lipid bilayer.
Given the importance of b-adrenergic receptors in the development and treatment of chronic heart failure, single particle tracking methodologies were then used to generate assays which would enable the imaging of endogenous receptors and their signalling proteins in physiologically relevant cells, where they exert their effects. Fluorescent ligands were employed to image adult mouse cardiac myocytes, so that the diffusion and spatial localisation of GPCRs could be observed in a complex cell model. This pushes the capabilities of single-molecule microscopy techniques to their current limits, providing insights into endogenous plasma membrane receptors in live, beating heart cells.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):