ESCRTs: from Cell Biology to Pathogenesis 173 Down-regulation of epidermal growth factor receptor signalling within multivesicular bodies Emily R. Eden*, Ian J. White† and Clare E. Futter* 1 *UCL Institute of Ophthalmology, University College London, 11–43 Bath Street, London EC1V 9EL, U.K., and †MRC Laboratory for Molecular Cell Biology, University College London, Gower Street, London WC1E 6BT, U.K. Abstract Activated EGFR (epidermal growth factor receptor) undergoes ESCRT (endosomal sorting complex required for transport)-mediated sorting on to the intraluminal vesicles of MVBs (multivesicular bodies) before degradation in the lysosome. Sorting of endocytosed EGFR on to the intraluminal vesicles of MVBs removes the catalytic domain of the EGFR from the cytoplasm, resulting in termination of receptor signalling. The formation of intraluminal vesicles that contain EGFR is promoted by EGF stimulation in a mechanism that depends on the EGFR substrate, annexin 1. Signalling from endocytosed EGFR is also subject to down-regulation through receptor dephosphorylation by PTPs (protein tyrosine phosphatases), such as PTP1B, an enzyme thought to reside on the ER (endoplasmic reticulum). In the present paper, we review how the phosphorylation state of components of the MVB sorting machinery, as well as the EGFR, may play a critical role in regulating EGFR sorting and signalling. Introduction EGF (epidermal growth factor) binding to the EGFR (EGF receptor) promotes increased RTK (receptor tyrosine kinase) activity, receptor dimerization and receptor trans- autophosphorylation. The latter generates multiple sites for the recruitment of signalling proteins and targets of the EGFR kinase and the consequent activation of signalling pathways that can lead to cell proliferation, differentiation, motility or survival, depending upon the cellular context. The activated EGFRs are endocytosed through clathrin- coated pits, although other internalization mechanisms can also operate [1]. The EGFR kinase remains active after endocytosis, and some, but not all, signalling pathways from the EGFR require endocytosis for their maximal activation [2,3]. Endocytosis of EGFR and/or associated proteins may allow access to downstream targets and specific signals from EGFR may be generated in specific subcompartments of the endosome [4,5]. Given the importance of the spatial regulation of signalling, it is not surprising that EGFR signalling from the endosome is subject to multiple regulatory mechanisms. In the present paper, we review two of those potential mechanisms: EGF-stimulated intraluminal vesicle formation and PTP (protein tyrosine phosphate) 1B-mediated EGFR dephosphorylation (Figure 1). Key words: epidermal growth factor receptor (EGFR), endosomal sorting complex required for transport (ESCRT), multivesicular body (MVB), phosphoinositide 3-kinase (PI3K), protein tyrosine phosphatase 1B (PTP1B). Abbreviations used: CHMP, charged multivesicular body protein; EGF, epidermal growth factor; EGFR, EGF receptor; ER, endoplasmic reticulum; ESCRT, endosomal sorting complex required for transport; Hrs, hepatocyte growth factor-regulated tyrosine kinase substrate; MVB, multivesicular body; PI3K, phosphoinositide 3-kinase; PTP, protein tyrosine phosphatase; RTK, receptor tyrosine kinase; Vps, vacuolar protein sorting. 1 To whom correspondence should be addressed (email c.futter@ucl.ac.uk). EGF-stimulated intraluminal vesicle formation within MVBs (multivesicular bodies) With time, endocytosed activated EGFRs that are destined for lysosomal degradation accumulate on the intraluminal vesicle of MVBs. Receptors that are to be recycled remain on the perimeter membrane of MVBs from where they are returned to the plasma membrane. When all the recycling proteins have been removed, MVBs containing EGFRs fuse directly with the lysosome and the EGFRs are degraded. The activated EGFR is one of the best characterized and most frequently used cargo markers of MVBs. In order to drive EGFRs into MVBs and on to the intraluminal vesicles of MVBs, the cell must be stimulated with EGF. It has only recently been documented that EGF stimulation has profound effects on MVB biogenesis [6]. EGF stimulation causes an increase in the number of MVBs per cell and the number of intraluminal vesicles per MVB [6]. Furthermore, MVB maturation is slowed in EGF-stimulated cells, as shown by the longer time taken for conversion of Rab5 into Rab7 in EGF-stimulated cells [7,8]. The sequestration of EGFRs on the intraluminal vesicles of MVB removes the catalytic domain of the receptor and any signalling proteins associated with the receptor from the cytoplasm. Thus direct signalling from that receptor is terminated. It is therefore likely that EGF-stimulated intralu- minal vesicle formation has evolved as a means of regulating signal transduction from the EGFR. An as yet unanswered question is whether or not sequestration of EGFR on internal vesicles of MVB is a ‘point of no return’ or whether, under certain circumstances, the intraluminal vesicles of MVBs can fuse back with the perimeter membrane and potentially re- sume signalling. Such backfusion of intraluminal vesicles may be involved in lipid transport, as has been demonstrated for Biochem. Soc. Trans. (2009) 37, 173–177; doi:10.1042/BST0370173 C  The Authors Journal compilation C  2009 Biochemical Society