His Domain Phosphotyrosine Phosphatase (HDPTP), a key regulator of endocytic trafficking and receptor downregulation

The behaviour of cells within a tissue is controlled by their environment. Amongst the most important signals that cells receive are in the form of circulating small proteins called growth factors. These bind to specific receptors that are found on the surface of cells. Binding of growth factors causes the receptors to alter their pattern of interactions with many molecules inside the cell that control cell growth. In this way growth factor receptors act as essential bridges between the cell exterior and interior to stimulate proliferative, or mitogenic responses. In order to prevent such mitogenic responses continuing endlessly, which would lead to uncontrolled cell division, the growth factor receptor must be inactivated shortly after the growth factor binds. This is achieved by removing the activated receptor from the cell surface and sending it to a specialised compartment within the cell, where it can be degraded. This compartment is called the lysosome. Movement of the receptor from the cell surface to the lysosome involves the receptor being sequestered into regions of the cell surface membrane that invaginate and pinch off to form spherical packages, or vesicles, within the cell interior. These vesicles move in a directed fashion to the lysosome, via a number of intermediate compartments. The situation becomes more complicated when it is realised that the route that growth factor receptors take to the lysosome is also followed part of the way by other types of receptor, which are engaged in taking up nutrients. These receptors are returned to the cell surface from the intermediate compartments so that they can participate in further rounds of nutrient uptake. Hence, at a critical point along the pathway towards the lysosome, these receptors are selected away. The aim of this project is to understand how this diversion takes place.

Downregulation of activated mitogenic receptors such as EGF receptor (EGFR) is crucial to controlling the longevity of mitogenic signalling responses. One essential component in the process of receptor downregulation is the transfer of the activated receptor from the cell surface to the endosome, and its ubiquitination and subsequent transport to the lysosome via the multivesicular body (MVB). This project grant application addresses the molecular basis for the selection of EGFR and other endocytosed cargo to the lysosomal pathway, and the formation of the MVB. Specifically, it examines the function of a novel regulator of this process, His Domain Phosphotyrosine Phosphatase (HDPTP). Preliminary evidence outlined in the application shows that HDPTP is essential for sorting ubiquitinated cargo at the early endosome and the formation of MVB. Loss of HDPTP blocks forward transport from the early endosome of EGFR and other cargo, leads to the accumulation of ubiquitinated proteins on endocytic compartments, and prevents the normal morphogenesis of MVB.

HDPTP is a multidomain protein, and the project aims to investigate how each domain contributes to HDPTP function. This investigation will use a range of strategies, including the transfection of HDPTP mutants into RNAi-depleted cells in order to examine the effects of recessive mutations, the expression of dominant-negative mutants, and the reconstitution of partial HDPTP reactions in vitro. These studies will be combined with detailed phenotypic analysis to test, amongst others, the following hypotheses: that the HDPTP Bro1 domain is essential for regulating the assembly of ESCRT (Endosomal Sorting Complex Required for Transport) complexes; that the HDPTP V domain is important for recruiting deubiquitinating enzymes and other factors to the endosome; that the HDPTP PTP domain is an active PTPase with protein and/or lipid substrates that are important regulators of endocytic transport. The project will also examine in detail the role of HDPTP during the maturation of the MVB from a domain of the early endosome into a late endosome, capable of delivering its content to the lysosome. In particular, the project will investigate whether HDPTP contributes to endosome maturation by regulating the replacement on the endosome membrane of the small, early endosome-specific GTPase, Rab5, with the late endosome-specific GTPase, Rab7.