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Analysis of the function of Lamellipodin in receptor-mediated endocytosis

Reference	BB/G00319X/1
Principal Investigator / Supervisor	Dr Matthias Krause
Co-Investigators / Co-Supervisors
Institution	King's College London
Department	Randall Div of Cell and Molecular Biophy
Funding type	Research
Value (£)	362,507
Status	Completed
Type	Research Grant
Start date	01/07/2008
End date	30/06/2011
Duration	36 months

Abstract

Receptor-mediated endocytosis is indispensable to tightly control the duration and strength of signalling. This is required for many physiologically essential processes including control of cell proliferation, wound healing, and organogenesis. Increased signalling from growth factor receptors is one of the hallmarks of many cancers and deregulation of endocytosis might contribute to increased signalling and hence uncontrolled cell proliferation. Growth factor receptor ligation results in Ras activation leading to increased cell proliferation. Receptor activation also induces endocytosis of the receptor. While signalling can persist on the endosome, finally degradation of the receptor terminates signalling. Actin polymerization is essential for the fission step in receptor-mediated endocytosis but how receptor activation and internalization is coupled to the regulation of the actin cytoskeleton is not clear. We have identified Lamellipodin (Lpd), a Ras effector that localizes to the tips of lamellipodia, filopodia and interestingly also to vesicles. Lpd is thought to recruit the actin regulator Ena/VASP to the cell membrane and is necessary for lamellipodium formation. Thus, by binding to both RasGTP and Ena/VASP, Lpd is a good candidate to link activated growth factor receptors to the regulation of the actin cytoskeleton. We propose to dissect by biochemistry how Lpd may link the EGF receptor with regulators of endocytosis and actin effectors. Furthermore, we will analyze the dynamic recruitment of Lpd, regulators of endocytosis and actin effectors to sites of receptor-mediated endocytosis in vivo using multi-color, live cell TIRF microscopy. Several functional assays will be employed to test a requirement for Lpd in receptor-mediated endocytosis. Taken together, Lpd is well positioned to act as a key convergence point linking activated Ras GTPases downstream of growth factor receptors to regulate the actin cytoskeleton during receptor-mediated endocytosis.

Summary

The human body is composed of millions of cells. In order for the body to function properly these cells have to communicate with each other. They communicate by sending out signals that can be recognized by other cells through the activation of receptors on their cell surface. In turn, these activated receptors stimulate cells to react and change their behavior. The signal is stopped by removal of the activated receptors from the cell surface by uptake into small vesicles. This process is called 'endocytosis'. Endocytosis is important for the normal functioning of our body but if it 'goes wrong' it can lead to disease. For example, in the case of many cancers activated receptors might not be taken up efficiently and hence stimulation from these receptors might increase causing cells to grow uncontrolled. We propose to study the basic mechanisms of endocytosis of growth factor receptors using single cells cultured in the laboratory. We will analyze how cells normally control the uptake of receptors using biochemical approaches. We will also visualize individual proteins recruited to the vesicles by labeling them with a fluorescent dye. This will allow us to analyze the recruitment in living cells in real time using a microscope. Our study might permit us to uncover the role of the different proteins for the uptake of the receptors. The outcome of the project will give novel insights into the normal function of individual cells within our body and in the long run may provide the knowledge for the development of targeted therapies for diseases with altered receptor signaling such as cancer.

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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