14-3-3 Dependent targeting of K2P channels: A fundamental pathway for the intracellular vesicular trafficking of cell surface membrane proteins

Cells respond to and communicate with their surroundings through the proteins (receptors, ion channels and transporters) they present on their surface. Receptors are embedded in the cell membrane and allow the cell to recognise and respond to specific signals which result in a cellular response. Ion channels and transporters also play a role in controlling the response of cells to stimuli by controlling the flow of ions into and out of the cell. The number and location of these membrane proteins on the cell surface can greatly affect the response of cells to external signals while mis-location of these critical cell surface proteins can give rise to conditions such as diabetes and cystic fibrosis. This project will investigate one mechanism by which cells control the number of membrane proteins on the cell surface. Cells have a number of ways of monitoring and controlling the number and type of membrane proteins on their surface. Proteins carry signal motifs (similar to postal codes) which determine their end location within the cell. Cell surface membrane proteins are synthesised in the endoplasmic reticulum (ER). Many of these proteins carry a set of signals which results in them being held within the cell (primarily the ER) if they fail to form correctly folded proteins. Some membrane proteins also carry another set of signals which aid their exit from the ER and delivery to the cell surface. It is thought that when a protein is folded correctly it has the ability to hide signals that result in it staying within the ER and expose signals that will aid its transport to the correct location within the cell. A general strategy is proposed, proteins synthesised in the ER are transported to the Golgi apparatus by transport vesicles (called COP II vesicles) which bud off from the ER and fuse with the Golgi apparatus. If the proteins carried in these vesicles are incorrectly folded and have an uncovered ER retention signal / motif they are then placed in different transport vesicles (COPI vesicles) and returned to the ER. If the proteins are destined to reach the cell surface they must conceal their retention motifs. Masking of retention motifs occurs through correct folding of the membrane proteins and interaction with additional proteins. We have previously demonstrated that ER retention motifs can be masked by a protein called 14-3-3. To date a group of more than a dozen proteins have been shown to require 14 3 3 to allow them move out of the ER. This project will examine the mechanism by which 14-3-3 helps proteins reach their correct location on the cell surface. Many questions remain to be answered, including does 14-3-3 bind to and mask the ER retention signals on membrane proteins only at the ER or does it travel with the membrane protein all the way to the cell surface. If 14-3-3 does travel all the way to the surface does it have an effect there? Or is the role of 14-3-3 to make it easier or faster for cell surface membrane proteins to reach thier destinations? Do other proteins also help membrane proteins make their way to the cell surface? As 14-3-3 appears to help membrane proteins get to the cell surface, if we can determine conditions which help 14-3-3 interact with these proteins we can increase the number of proteins on the cell surface. Similarly we could also prevent the interaction and reduce the number of membrane proteins reaching the surface. As membrane proteins are critical to the functioning of cells a mechanism by which we can control their number on the cell surface would provide a means to alter cell function in health and disease.

Cell surface expression of integral membrane proteins proceeds under tight control. This regulation enables the cell to monitor the quality of multimeric complexes produced, maintain stable expression levels of critical membrane proteins and as a result respond to stimuli in an appropriate manner. Intrinsic signal sequences are among the quality control mechanisms which mediate the transit of proteins through the secretory pathway. Arginine-based signals (RR or RXR) found in the cytoplasmic domains of an array of membrane proteins are recognised by coatomer (COPI) vesicle coat proteins which are implicated in ER retention / retrieval. Masking of the retention motifs is the probable means of overcoming ER retention. We demonstrate that 14-3-3 (a ubiquitously expressed cytosolic family of adaptor proteins) has the ability to overcome ER retention. By interacting with target membrane proteins 14-3-3 displaces COPI interaction enabling forward transport. Many of the retained membrane proteins demonstrate a phosphorylation dependence on 14-3-3 interaction. Whilst the basic mechanism of 14-3-3-dependent forward transport has been uncovered, our knowledge of the exact processes which occur is limited. Using K2P3 as a model protein (a background K+ channel used in our initial studies) this proposal will elucidate fundamental aspects of this 14-3-3 dependent forward transport pathway. We will (i) determine the mechanism of 14-3-3-dependent forward transport and elucidate if the role of 14-3-3 is limited to ER release or is involved in the kinetics of membrane protein turnover (ii) determine if 14-3-3 plays a role in recruitment of additional modulatory proteins that aid in the forward transport of membrane proteins and (iii) determine the protein kinase(s) and phosphatase(s) responsible for phosphorylation dependent modification of 14-3-3 interaction and in so doing determine a physiologically relevant control of cell surface expression of key membrane proteins.