Molecular mechanism of Arrestin-3 Recruitment and Activation by G Protein-Coupled Receptors
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Author
Date
2020Type
- Doctoral Thesis
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Abstract
Arrestins are 45-47 kDa cytosolic proteins that constitute a family of 4 members in mammals and are important regulators of G protein-coupled receptor (GPCR) signaling. Arrestins play key roles in desensitizing GPCRs to G protein coupling, and some arrestins also regulate GPCR internalization and cellular trafficking. Arrestin-1 and arrestin-4 are primarily expressed in the retina and regulate the activity of the opsins, the light-sensitive GPCRs involved in photo-transduction in the rod and cone cells. Arrestin-2 and arrestin-3, the so-called “β-arrestins”, are ubiquitously expressed and regulate hundreds of different GPCRs. Over the past three decades, the mechanisms of arrestin recruitment and activation by GPCRs have mainly been investigated through structural and mutagenesis studies of arrestin-1. Due to their high structural and sequence similarity, the other arrestins were assumed to operate in a similar manner as arrestin-1, although more recent studies suggest significant mechanistic and functional differences between the different arrestins. Currently the molecular processes behind arrestin-2/3/4 recruitment and activation by GPCRs is not as well understood as for arrestin-1. This thesis aims at filling this gap in knowledge, specifically regarding the molecular mechanisms underlying the recruitment and activation of the human arrestin-3 to several GPCRs.
The first part of the thesis reports on the recruitment of arrestin-3 mutants, derived from an alanine-scan library, to the human β2-adrenergic receptor (β2AR) in living cells. A novel split-nanoluciferase assay was employed, which is based on protein-fragment complementation and has several advantages over other currently available reporter enzyme strategies. The objectives were to i) validate this new split nanoluc approach and ii) to assess possible new binding modes of arrestin-3 to the β2AR. However, technical limitations of the split nanoluc assay made the assay inconclusive.
The second part of this thesis describes double and triple alanine mutants of arrestin-3, which were anticipated to simultaneously disrupt the polar core and the three-element interaction, two structural elements important in stabilizing the basal arrestin conformation. The mutants I386A+T299A and I386A+T299A+R166A in particular formed stable complexes with the β1-adrenergic receptor. The results from this study are part of the patent EP19153159 application “β-arrestin mutants” filed in January 2019.
The third part of this thesis describes a targeted mutagenesis approach, whereby key structural features of arrestin-3 involved in GPCR recruitment and arrestin activation were constrained by intramolecular cysteine crosslinking or inactivated by glycine mutations. After extensive characterization by mass spectrometry, circular dichroism and limited trypsin digest, the recruitment of different arrestins and constrained mutants of arrestin-3 to various activated states of rhodopsin were assessed by centrifugal pull-down analysis. In comparison to arrestin-1, the phosphorylated receptor C-terminus and phospholipid membrane play a more significant role in the recruitment of arrestin-2 and arrestin-3 to rhodopsin. Spontaneous intramolecular crosslinking in arrestin-3 was found to be particularly efficient for the restriction of loop movements. Restraining inter-domain rotation, a hallmark of arrestin activation, had the most dramatic effect on the recruitment of arrestin-3 to phosphorylated and light-activated rhodopsin. Show more
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https://doi.org/10.3929/ethz-b-000402014Publication status
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Contributors
Examiner: Schertler, Gebhard
Examiner: Waldhoer, Maria
Examiner: Berger, Philipp
Examiner: Werner, Sabine
Publisher
ETH ZurichSubject
Arrestin; Cell signaling; Rhodopsin; GPCR; Biased signaling; Beta-adrenergic receptors; Engineered arrestin-3Organisational unit
03866 - Schertler, Gebhard (emeritus) / Schertler, Gebhard (emeritus)
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