Prof D Gorecki, Dr G Tozzi
Applications accepted all year round
Self-Funded PhD Students Only
About the Project
Project ref: PHBM3771018
P2X7 is an ATP receptor with a number of physiological functions but also involved in acute and chronic diseases, e.g. inflammation, cancer, bone loss and psychiatric disorders. P2X7 is therefore an important therapeutic target. However, P2X7 trimeric subunit organization and its interacting partners (signalling and scaffolding proteins) can be highly diverse in various cells and pathologies. Moreover, cell-specific and sub-cellular localisation of P2X7 is still controversial. Understanding these multiple modes of action and cell -specific receptor functions is essential for developing targeted and effective treatments.
Given the importance of the P2X7 receptor in health and disease, our laboratory has created a unique P2X7 knock-in/knock-out mouse. It expresses endogenous levels of tagged P2X7 isoforms allowing immunoaffinity purification of the tagged P2RX7 followed by mass spectrometry to identify the associated proteins (the “genetic-proteomic”, Bienvenu et al, 2010). These tags also allow immunodetection of specific isoforms with anti-tag antibodies in situ.
Moreover, this mouse P2X7 gene is flanked by loxP sites allowing cell-specific and/or conditional knockouts by crossing this strain with specific Cre recombinase-expressing mice or by targeted expression of Cre recombinase.
This model takes functional analyses to a new level by defining the roles of P2X7 in any desired tissue or at any disease stage in vivo. These will be explored in this PhD project using molecular biology and proteomics methods to identify receptor assemblies and interacting proteins, confocal and advanced X-ray microscopy (http://www.port.ac.uk/school-of-engineering/zeiss-global-centre/) to unravel cellular and sub-cellular localisation of this receptor and functional analyses in specific cells and tissues (e.g. bone, brain) following cell-specific ablation by Cre recombinase.
Funding Notes
This is a self-funded project
References
1. Bienvenu F et al., Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen. Nature. 463:374-8, 2010.
2. Boumechache et al., Analysis of assembly and trafficking of native P2X4 and P2X7 receptor complexes in rodent immune cells. J Biol Chem. 284: 13446-54. 2009.
3. Khakh BS. and North RA P2X receptors as cell-surface ATP sensors in health and disease. (2006) Nature 442, 527-532
4. Masin M et al., Expression, assembly and function of novel C-terminal truncated variants of the mouse P2X7 receptor: Re-evaluation of P2X7 knockouts. Brit. J. Pharmacol. 165: 978-93, 2012.
5. Nicke et al., A functional P2X7 splice variant with an alternative transmembrane domain 1 escapes gene inactivation in P2X7 KO mice. J Biol Chem. 284:25813-22. 2009.
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