INPROTEOLYS

Receptor Endocytosis and Fate

The main interest of our lab is aimed at elucidating the molecular mechanisms governing ubiquitination and endocytosis of the epidermal growth factor receptor (EGFR), and understanding the functional significance of these processes in normal physiological processes and in cancer. Indeed this kinase receptor has important clinical relevance, as EGFR deregulated signalling is strongly associated with cancer. The EGFR has long known to be internalized by clathrin-mediated endocytosis (CME), upon its activation by ligand. However, we have recently discovered that it can also be internalised via an alternative clathrin-independent/lipid raft-dependent endocytic route: non-clathrin endocytosis (NCE) [1]. Why distinct pathways of internalization have evolved? One possibility is that they might differentially regulate receptors functions and fate. Indeed, we found that internalisation by NCE is regulated by receptor ubiquitination and commits the EGFR to degradation. In contrast, internalisation by CME does not require receptor ubiquitination and results in recycling of EGFR to the cell surface and sustained EGFR signalling [2]. These results helped in clarifying the role of receptor ubiquitination in the initial step of EGFR internalization, still the exact nature and the molecular determinants of NCE are unknown. In order to have a molecular characterization of this pathway, purification of NCE EGFR-containing vesicles and analysis by mass-spectrometry is on going in the lab. One major issue concern how is the EGFR recruited into the two pathways of internalization. Interestingly, data from our lab suggest that cells can react to different doses of EGF in strikingly different ways [1]. At low doses of ligand, the receptor (EGFR) is internalized almost exclusively through CME and it is not ubiquitinated; at higher concentrations of ligand a substantial fraction of the receptor is endocytosed through NCE, as the receptor becomes ubiquitinated. Thus, EGF concentration is critical for the final biological outcome. One of the ongoing projects in the lab is aimed at understanding how cells decode the information stored in stimulus strength. Preliminary results show that the interplay between phosphorylation and ubiquitination of the EGFR plays a major role in this process. Ubiquitination depends on phosphorylation, because three out of the nine tyrosines need to be phosphorylated in order for the EGFR to be ubiquitinated. Interestingly, while phosphorylation increases linearly with EGF doses, ubiquitination does not start before the EGF concentration reaches a critical threshold. To gain insight into this phenomenon we are using a multidisciplinary approach, involving both molecular biology experiments and mathematical modelling. [1] S. Sigismund et al., Proc Natl Acad Sci U S A 102, 2760 (2005). [2] S. Sigismund et al., Dev Cell, 15, 209-19 (2008).

Sara Sigismund

Veronica Algisi

Gilda Nappo

Lisette Verhoef

E-mail

sara.sigismund@ifom-ieo-campus.it

Phone

0039-02-574303302-235

Fax

0039-02-574303231

Address

Via adamello16, 20139 Milano, Italy

Center

IFOM, The FIRC Institute of Molecular Oncology Foundation

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Research Lines

EGFR ubiquitination- EGFR internalization pathways

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Keywords

EGFR, ubiquitin, endocytosis, signalling, degradation