Single-molecule analysis of IL-2 receptor reveals the importance of its clustering for endocytosis and signaling in lymphocytes

Signaling by the interleukin-2 receptor (IL-2R) is regulated by its clathrin-independent endocytosis (CIE) and subsequent degradation, while playing a critical role in immunity. Interestingly, CIE lacks a coat protein that drives pit formation, raising the question of how the CIE vesicle is initiated. Protein clustering generates forces that can induce membrane conformational changes. Notably, IL-2R has been shown to accumulate at the base of membrane protrusions, where receptors might cluster and thereby initiate the pit.To study the relevance of IL-2R clustering in its endocytosis, we generated a CRISPR-edited T cell line expressing GFP-IL-2Rᵧ and analyzed its stoichiometry at the plasma membrane, by TIRF microscopy coupled to a single-molecule endocytic tracking method. We identified distinct IL-2Rᵧ cluster populations. IL-2Rᵧ seems to reach the cell surface as a preassembled cluster to which further molecules are added, reaching an optimal cluster size that is key for its internalization. Binding of IL-2 promotes the formation of endocytic clusters and receptor uptake, highlighting the importance of clustering for CIE internalization.Moreover, we found that cholesterol depletion increases the proportion of large, non-endocytic clusters as well as IL-2R signaling. Disruption of the actin meshwork also promotes the formation of large clusters, yet it decreases IL-2R signaling. Thus, both factors regulate IL-2R endocytosis and signaling in a distinct manner. Our results provide new insights into the mechanisms regulating receptor signaling and CIE.