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

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Publications

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Woodman P (2016) ESCRT-III on endosomes: new functions, new activation pathway. in The Biochemical journal

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Ridley C (2016) Biosynthesis of the polymeric gel-forming mucin MUC5B. in American journal of physiology. Lung cellular and molecular physiology

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Granger E (2014) The role of the cytoskeleton and molecular motors in endosomal dynamics in Seminars in Cell & Developmental Biology

 
Description BBSRC Project grant BB/I012109/1
Amount £341,900 (GBP)
Funding ID BB/I012109/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2011 
End 08/2014
 
Description Investigator Award
Amount £990,352 (GBP)
Funding ID 212246/Z/18/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2019 
End 02/2024
 
Description LITAF BBSRC project grant
Amount £660,000 (GBP)
Funding ID BB/M000877/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 08/2018
 
Description Project grant ESCRT-I structure
Amount £498,000 (GBP)
Funding ID BB/K008773/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2013 
End 11/2015
 
Description Project grant HD-PTP structure
Amount £582,000 (GBP)
Funding ID MR/K011049/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2013 
End 12/2015
 
Title FRT HDPTP cell line 
Description We have developed a HeLa cell line, based on the Clontech FRT system, that allows inducible expression of HDPTP under a doxycyclin-sensitive promoter. We have also generated several cell lines expressing a range of HDPTP mutants 
Type Of Material Cell line 
Provided To Others? No  
Impact We have used these cell lines to identify regions within HDPTP that are important for function. These studies are currently being prepared for publication. 
 
Title HDPTP monoclonal 
Description We have generated rat monoclonal antibodies to HDPTP. 
Type Of Material Antibody 
Provided To Others? No  
Impact None to date. 
 
Description Chiara Francavilla 
Organisation University of Manchester
Department School of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution This is a recently established collaboration to use SILAC-based mass spectrometry to dissect the function of HD-PTP during MVB sorting. We have contributed ideas, reagents and experiments to this partnership.
Collaborator Contribution Dr Francavilla is an expert in SILAC-based mass spec, with a particular interest in endosomal trafficking. She has contributed her ideas and expertise.
Impact We have submitted an application to the BBSRC
Start Year 2016
 
Description Robert Layfield 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Understanding how the ubiquitin-binding domain of UBAP1 contributes to endosome function
Collaborator Contribution Quantitative analysis of the ubiquitin-binding properties of UBAP1
Impact None to date
Start Year 2012
 
Description Sabine Hilfiker 
Organisation Spanish National Research Council (CSIC)
Country European Union (EU) 
Sector Public 
PI Contribution My laboratory has hosted 3 PhD students from the Hilfiker lab who have worked on aspects of membrane trafficking and autophagy. A particular interest at the moment is in LRRK2 kinase, which is a major risk factor for Parkinson's and which influences autophagic traffic and interacts with the ESCRT machinery.
Collaborator Contribution Dr Hilfiker has provided expertise and manpower
Impact 2 primary research papers
 
Description Stuart Pickering-Brown 
Organisation University of Manchester
Department School of Medicine Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution Professor Pickering-Brown works on frontotemporal lobular degeneration, and had identified UBAP1 as a risk factor with unknown function. We identified the molecular basis for UBAP1 function
Collaborator Contribution Professor Pickering-Brown has provided expertise and reagents.
Impact One primary research paper
Start Year 2010
 
Description Sylvie Urbé 
Organisation University of Liverpool
Country United Kingdom 
Sector Academic/University 
PI Contribution Investigation of the molecular basis for UBPY function at the endosome
Collaborator Contribution Provision of reagents and expertise
Impact Publication
Start Year 2011
 
Description School Visit Manchester 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact Conducting mock UCAS interviews

Prepared students for University applications
Year(s) Of Engagement Activity 2008,2009
 
Description Science Fair 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Pupils from Manchester attended an event at the Manchester Science Week. This included activity-based learning.

None documented
Year(s) Of Engagement Activity 2011
 
Description What is Endocytosis? 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Schools
Results and Impact 4 page article for lay publication

unknown
Year(s) Of Engagement Activity 2010