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Mechanism of GPCR recruitment to clathrin-coated vesicles

Principal Supervisor: Dr. Corinne Smith, School of Life Sciences

Co-supervisor: Professor Mark Wheatley, Chair of Biochemical Pharmacology, University of Birmingham

PhD project title: Mechanism of GPCR recruitment to clathrin-coated vesicles

University of Registration: University of Warwick

Project outline:

G-protein-coupled receptors (GPCRs) play a vital role in multiple physiological processes and as a result are key pharmaceutical targets. β-arrestin, a protein which interacts both with GPCRs and clathrin, plays a vital role in coordinating the signalling activity of these important receptors. Following phosphorylation of the activated receptor, β-arrestin binds to the receptor and displaces the heterotrimeric G protein, causing signalling to be silenced. β-arrestin then acts to recruit the GPCR into clathrin-coated vesicles via interactions with clathrin and the adapter protein AP2, thus removing the GPCRs from the cell surface. In addition to this role in terminating receptor signalling, β-arrestin can also mediate GPCR signalling that is independent of G-protein activation, for example through interactions with members of the Src and MAP families of kinases. This has led to the development of ‘biased agonists’ by the pharmaceutical industry, which preferentially stimulate one signalling cascade over another. In order to understand how β-arrestin performs these functions, this project aims to discover the nature of its interactions with clathrin both alone and in combination with the main clathrin adaptor protein complex, AP2 using state-of-the-art high resolution cryo-electron microscopy and biophysical analysis.

In recent years, newly developed electron detectors have revolutionised the field of structure determination by cryo-electron microscopy allowing sub-4Å structures of challenging targets such as membrane proteins and ribosomes to be obtained without using X-ray crystallography. At Warwick we are exploiting this improvement in capability to carry out high resolution structural analysis of clathrin cage complexes using new electron microscopy and direct detection instrumentation available through the Advanced Bioimaging Research Technology Platform. While there are a number of crystal structures of β-arrestin alone and complexed with a small subdomain of clathrin there are no structures available for β-arrestin bound to an entire clathrin cage, and little high resolution structural information is available for the GPCR-β-arrestin complex. The aim of this project, therefore, is to obtain a high resolution structure of clathrin associated β-arrestin both alone and in the presence of AP2 using the new cryo-electron microscopy instrumentation available and the latest single particle imaging processing methods. This project combines the expertise of the Wheatley laboratory in GPCR signalling mechanisms and the Smith laboratory’s expertise in cryoEM and biophysical analysis of clathrin cage assemblies providing a solid basis for successful completion of this project.

BBSRC Strategic Research Priority: Molecules, Cells and Systems

Techniques that will be undertaken during the project:

  • High resolution electron microscopy
  • Image analysis of large data sets
  • 3D imaging of structural data
  • Cell biology
  • Mutagenesis, expression and purification

 Contact: Dr. Corinne Smith, School of Life Sciences