The integration of distinct internalization routes is crucial to determine the fate of plasma membrane (PM) receptorsand the output of their signalling pathways. Contact sites between cellular organelles adds a further layer of regulationby creating microdomains that favour different signalling and metabolic pathways. These regulatory mechanisms arerelevant to the epidermal growth factor receptor (EGFR). We found that EGFR internalization through non-clathrinendocytosis (NCE) leads primarily to receptor degradation and signal extinction, while clathrin-mediated endocytosis(CME) is mainly involved in EGFR recycling and sustaining signalling. Notably, internalization via NCE involvesthe formation of contact sites between the PM, the endoplasmic reticulum (ER) and the mitochondria, where EGF-dependent localized Ca2+ signalling occurs.The founding hypothesis of this proposal is that the PM-ER-mitochondrial interface could represent a functionalunit where direct cross-communication between EGFR signalling and mitochondria takes place. To investigate thishypothesis, we will use a three-tiered strategy aimed at elucidating:1. the EGFR-dependent signalling that leads to NCE-ER-mitochondrial contact site formation and to local Ca2+release, and the role of these contacts in EGFR endocytosis, signalling and fate;2. the crosstalk between EGFR signalling and mitochondrial function at the PM-ER-mitochondrial interface byanalysing the impact of EGFR-NCE on mitochondrial physiology and metabolism;3. the relevance of NCE-ER-mitochondrial crosstalk to EGF-dependent cell physiological responses, e.g., migration,proliferation and differentiation, by exploiting isogenic cell derivatives from embryonic/pluripotent stem cells or exvivo organoid cultures.The verification of this hypothesis will expand our understanding of the impact of EGFR signalling on cellularfunctions not previously linked to this pathway and possibly impinging on cellular energetics and metabolism.