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The role of cytoplasmic dynein in the endosomal trafficking of activated EGF receptor

Reference	BB/C512929/1
Principal Investigator / Supervisor	Professor Philip Woodman
Co-Investigators / Co-Supervisors	Professor Viki Allan
Institution	The University of Manchester
Department	Life Sciences
Funding type	Research
Value (£)	215,919
Status	Completed
Type	Research Grant
Start date	01/08/2005
End date	31/07/2008
Duration	36 months

Abstract

The down-regulation of growth factor receptors such as epidermal growth factor receptor (EGFR) is vital for proper control of cell proliferation. This down-regulation occurs following endocytosis, when the active tyrosine kinase cytosolic domain of EGFR is sequestered within the multivesicular body (MVB). From there the receptors are trafficked to the lysosome, where they are degraded. Using fluorescence light microscopy of living cells, we have demonstrated that EGFRs move inwards along microtubules during a specific phase of EGFR transport, concomitant with the maximal rate of EGFR sorting away from transferrin receptor (which is not degraded, but recycled to the plasma membrane). This inward movement is dependent on cytoplasmic dynein 1 (dynein), and the overall aim of the project is to dissect the role of dynein in the movement of EGRF-rich MVBs, and its possible role in the separation of vacuolar from tubular regions of the endosome. The first question to be addressed will be what is dynein¿s role in the movement of EGFR-rich MVBs, and does it participate in receptor sorting? This will be investigated by following EGFR movement by light microscopy at high temporal resolution. We will develop this assay to allow us to monitor the separation of EGFR away from transferring receptor at rapid frame rates. To test the role of dynein, we will use methods that cause rapid inhibition of dynein activity (e.g. microinjection of function-blocking antibodies, peptides and proteins), rather than look at the effects of long-term inhibition of motor function that occurs following transient transfection with function-blocking constructs. These assays for endosome movement and sorting will provide a functional read-out for the presence of active dynein on endosomes, and will be used throughout the proposal. The second question to be addressed is the molecular mechanism by which dynein is recruited to the endosomal compartment. Although the paradigm in the field is that the dynein accessory complex, dynactin, provides the anchoring point for dynein on most of its cargoes, there have recently been a number of studies that suggest that dynein subunits can bind directly to cargoes, including transmembrane receptor proteins. We will use our extensive library of antibodies and constructs to dynein and dynactin subunits to determine whether the dynein light chains (LCs) and light intermediate chains (LICs) are involved in cargo binding. We will test the hypothesis that the Tctex-1 LC interacts directly with the EGFR cytoplasmic tail using biochemical and molecular approaches. We will also establish whether an interaction between LIC and the endosomal protein Hook 1 is important. The third aspect for investigation is whether the binding of EGF to its receptor, which causes receptor activation, up-regulates motility of MVBs, and if so, whether the dynein and dynactin complexes are targets for phosphorylation following EGFR activation. Finally, we will test how dynein-dependent movement might be mechanistically linked to other aspects of receptor sorting.

Summary

unavailable

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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