Regulation of membrane trafficking and signalling by clathrin light chain phosphorylation

The Smythe lab is interested in the way in the way in which cells take up material from the external environment in the process of endocytosis. Material to be taken up is packaged into small spherical vesicles that bud into the cell from the cell surface. A protein coat on the inside of the cell surrounds the incoming vesicle and helps it to bud off. Clathrin is one of the major coat proteins and so the vesicles are named clathrin coated vesicles. These vesicles act as transport carriers and deliver material to another compartment within the cell called an endosome, which acts as a major cellular sorting station and either sends the material to be broken down in yet another intracellular compartment, the lysosome, or recycles it back to the cell surface. Clathrin helps vesicles to bud at a number of locations in the cell.

The Mundell and Kelly labs study G protein-coupled receptors (GPCRs), which are proteins found on the plasma membrane that respond to a whole range of messages received from the environment. These include hormones, sensory stimuli such as odorants, neurotransmitters and drugs such as morphine. Because of the range of their biological roles, these receptors are major drug targets. When these receptors encounter a signal they respond to it by activating a variety of events within the cell. As part of this process they are also taken up or internalised into the cell by clathrin coated vesicles and there they may either be targeted for destruction in the lysosome (desensitization) or sent back to the cell surface where they can respond to further stimuli (resensitization).

The activity of many proteins may be changed if phosphate is added to them in the process of phosphorylation and this is carried out by a group of cellular enzymes called kinases. The aim of this proposal is to understand how the role of clathrin is regulated by phosphorylation. Our recent studies have demonstrated the novel finding that a kinase that adds phosphate to some GPCRs also appears to phosphorylate clathrin. This may act as an address label for these particular GPCRs, sending them on a specific route within the cell. It is becoming increasingly clear that the intracellular routes followed by GPCRs and the rate at which they move along this route affects how they signal because often signals give rise to more than one output. For example in one situation a signal may cause a cell to move and, in another, to divide. We are interested in exploring how modifications of clathrin by phosphorylation may affect the rate of transport of signals. We expect this work to give us important information into how cells respond to signals and in the longer term it is possible that we will identify novel therapeutic targets.

The overall aim of this proposal is to understand how endocytosis regulates intracellular signalling. Increasing evidence supports the idea that the rate of flux of signalling molecules through the endocytic pathway plays a key role in their downstream signalling output, thus providing one possible mechanism for how a single signal can give rise to multiple outcomes depending on cellular context. Clathrin plays a major role in intracellular trafficking, forming clathrin coated vesicles at the cell surface, at the Trans-Golgi Network, on recycling endosomes and also establishing microdomains for sorting material from early to late endosomes. Here we propose to study the role of clathrin light chains (CLCs), which have been highly conserved in evolution but whose function is relatively unknown.

This proposal builds on our novel findings which have revealed that CLCb has a newly-discovered role in endocytic trafficking and that differential phosphorylation of CLCb plays key roles in the endocytosis of physiologically important G protein-coupled receptors (GPCRs) involved in both platelet regulation in the cardiovascular system (P2Y receptors) and pain and reward in the central nervous system (mu opioid receptor). Our working model is that CLC phosphorylation modulates endocytic flux and this may be physiologically relevant for the transit of signalling molecules along the endocytic pathway. We have already identified G-protein coupled receptor kinase2 (GRK2) as a potential kinase to phosphorylate CLCb.

Building on these novel observations we will use a combination of cell biology assays and fluorescence microscopy to explore the role of CLC phosphorylation on the endocytosis and downstream signalling of GPCRs. We will also analyse the role of CLC phosphorylation, and the kinases involved, in other trafficking and signalling pathways.

This proposal addresses how fundamental membrane trafficking mechanisms are regulated and how they cross-talk with intracellular signaling pathways. Much of the focus of the signaling pathways will be on G protein-coupled receptors (GPCRs) and how they may be modulated by clathrin light chain phosphorylation. Comprising approximately 4% of the human genome, GPCRs respond to a large variety of signals including neurotransmitters, sensory stimuli, and hormones amongst others. As a result they are involved in many biological processes and are the targets of approximately 40% of all prescription drugs on the market. There is still considerable interest from the pharmaceutical industry in the development of more selective drugs with fewer side-effects and signalling components downstream of GPCRs also have considerable therapeutic potential. The idea that individual GPCRs can give rise to multiple signals adds importance to the search for new drugs that can selectively activate or inhibit particular aspects of downstream signaling in the cell. This work will address how endocytosis may modulate GPCR activation and downstream targets. There are therefore potential long-term benefits for healthcare and drug development. Dr Susan Smith, the Department of Biomedical Science (University of Sheffield) research translator, will facilitate the exploitation of any potential therapeutic targets by identification of links with appropriate pharmaceutical companies.

Dissemination of the results arising from this proposal will generally be by the traditional routes of high quality publications and conference presentations. The PIs and postdoctoral workers will also seek to publicise their work to a broader public through public engagement. Smythe is an active participant in Science Week activities in South Yorkshire. Furthermore there are opportunities for presentation of research at Café Scientifique in Sheffield (www.sciencecafesheffield.org). To ensure maximum exposure of the outcomes of this research, the media relations teams in the University of Sheffield and Bristol and MRC will also be alerted to conference presentations and journal publications and their skills utilised to engage more mainstream routes to inform other stakeholders.

The RAs employed on the grant will be trained in addressing fundamental biological questions using a range of molecular cell biological approaches using both biochemistry and state-of-the-art microscopy. They will also become aware of the potential translational aspects of basic biology and of how they may be exploited. The skills obtained will be transferable to a range of careers including in industrial research and development.