EASTBIO: Development of inducible protein targeting to regulate G-protein-coupled receptor function.

Supervisors:

Dr James Hislop (University of Aberdeen)
https://www.abdn.ac.uk/people/james.hislop

Dr Gopal Sapkota (University of Dundee)
https://www.ppu.mrc.ac.uk/research/principal-investigator/gopal-sapkota


The group of signalling receptors known as G-protein-coupled receptors (GPCRs) represent the largest family of signalling proteins in the cell and play important roles in the regulation of most physiological systems. Controlling the functional complement of GPCRs is critical for maintaining fidelity of signalling which is critical for health. For example, too many receptors lead to overstimulation of the system, whereas too few receptors and the cell will not respond to the demands of the environment. Both of these outcomes are deleterious to health and underlie a wide range of disease conditions, making it of vital importance to understand how cells control receptor number.

Further, what impact changing receptor levels has on the cell function is critical in establishing how cells can respond to a range of physiological and pathophysiological conditions. Current techniques such as deletion of GPCRs by siRNA or CRISPR/Cas9 gene editing or the overexpression of specific receptors do not fully capture the dynamic nature of GPCR regulation, which are able to increase and decrease expression levels by changing the balance of synthesis and downregulation to match the unique demands of the environment. Therefore, identifying the mechanisms responsible for reducing receptor number (downregulation) is vital to our understanding of cellular signalling.

This studentship will combine the expertise in GPCR pharmacology of the Hislop lab with the protein engineering of the Sapkota lab to identify critical regulatory checkpoints in GPCR function and the development of intervention strategies for development as therapeutics. We will use muscarinic acetylcholine receptors as model examples of GPCRs whose downregulation is clinically relevant in a number of neurological disorders (1). As a starting point, we will investigate the role of the ubiquitin-lysosome system in controlling downregulation of muscarinic acetylcholine receptors. Direct ubiquitination is implicated in GPCR downregulation and is regulated by a balance of activities of ubiquitin ligases and deubiquitinating enzymes (2). Using molecular cloning, mutagenesis confocal microscopy and biochemical analysis, we will determine the role of ubiquitination in the regulation of muscarinic receptor levels. We will also utilise novel techniques such as TurboID combined with mass spectrometry to identify the specific protein-protein interactions responsible. We will go on to use novel protein engineering technologies developed by the Sapkota lab (3) to control the expression level of muscarinic receptors. This technology uses engineered protein connectivity to specifically target proteins for ubiquitination on demand to drive downregulation. This project will utilise this technology to downregulate the muscarinic receptors and allow us to determine exactly how this dynamic process influences receptor signalling both acutely and at the transcriptional level.

This studentship will use a diverse array of modern scientific techniques including state of the art protein engineering combined with biochemical analysis and fluorescent imaging techniques to fully determine how muscarinic receptors are regulated and identifying potential targets for therapeutic intervention.

Application Procedure:
http://www.eastscotbiodtp.ac.uk/how-apply-0

Please send your completed EASTBIO application form, along with academic transcripts and CV to Alison McLeod at [Email Address Removed]. Two references should be provided by the deadline using the EASTBIO reference form. Please advise your referees to return the reference form to [Email Address Removed].