The regulation of endocytic sorting and cholesterol transport by PTP1B-mediated ESCRT dephosphorylation at ER-endosome membrane contact sites

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The transport of proteins and lipids to the right cellular location is critical for the function of all cells. Endocytosis is a mechanism of uptake of extracellular molecules and cell surface membrane proteins into intracellular membrane compartments, called endosomes. Within the endosome, proteins and lipids are sorted. Unwanted cell membrane proteins and lipids can then be removed by delivering them to lysosomes where they are degraded. This degradative pathway is balanced by new synthesis of membrane proteins and lipids in the endoplasmic reticulum (ER). Overproduction or impaired degradation of membrane proteins or lipids can lead to multiple diseases, including cancer and lipid storage disorders. The degradative pathway is important for regulating signaling from growth factor receptors, such as the EGF receptor (EGFR), a cell surface signaling protein that regulates cell division. Dysregulated signaling from the EGFR occurs in about 50% of human cancers and is a target for cancer therapies. It activates signaling pathways by chemically modifying both itself and other signaling proteins. This modification, called phosphorylation, is reversible by dephosphorylation. The EGFR can be dephosphorylated by a phosphatase on the ER, PTP1B. Proteins and lipids can shuttle from cell membrane to membrane via small transport vesicles. Different cellular membranes can also form membrane contacts where they come very close together to form sites where proteins on the apposing membranes can interact and lipids can be flipped directly from one membrane to the other. We recently found that the ER and the endocytic pathway form membrane contacts and have identified molecules that tether the apposing membranes at the contact. We showed that ER:endosome membrane contacts are necessary for PTP1B to dephosphorylate endocytosed EGFR, and that PTP1B activity promotes delivery of EGFR to the lysosome for degradation. Some of the molecules that regulate lysosomal delivery of EGFR are themselves regulated by PTP1B-mediated dephosphorylation and one of those molecules, Hrs, has also been implicated in cholesterol exchange between endosomes and the ER.Cholesterol is a critical component of cell membranes obtained by endocytosis of dietary LDL, or by new synthesis in the ER. Excess endocytosed cholesterol is transferred to the ER and packaged into lipid droplets for storage. However, the endocytic pathway needs a certain amount of cholesterol to function. So when LDL-derived cholesterol is low, newly synthesized cholesterol is transferred from the ER to endosomes. We showed that ER:endosome membrane contacts regulate exchange of cholesterol between the ER and endosomes. Regulation of endosomal cholesterol is important for multiple processes. Endosomal cholesterol is required for normal function of the degradative pathway, but impaired egress of endosomal LDL-cholesterol is associated with lipid storage diseases, such as the childhood neurological disorder, Neimann Pick Type C disease. We hypothesise that PTP1B-mediated dephosphorylation of Hrs at membrane contact sites co-ordinates lysosomal targeting of EGFR and cholesterol exchange between endosomes and the ER. We will test this hypothesis using tools we've developed to manipulate membrane contacts, together with high-resolution electron microscopy (the only way to unequivocally identify membrane contacts) and newly developed super resolution microscopy techniques. Together these studies will provide a better understanding of how PTP1B activity at membrane contact sites regulates EGFR signaling and cholesterol exchange. This will determine how the relationship between PTP1B and EGFR can be exploited for the design of better combination cancer therapies and may yield novel therapeutic targets for the treatment of lipid storage diseases.