Abstract
Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors. Signalling is fine-tuned by the exo–endocytic cycling of these receptors to control cellular responses such as FA dynamics, which determine cell motility. How precisely endocytosis regulates turnover of the various cell surface receptors remains unclear. Here we identify Stonin1, an endocytic adaptor of unknown function, as a regulator of FA dynamics and cell motility, and demonstrate that it facilitates the internalization of the oncogenic proteoglycan NG2, a co-receptor of integrins and platelet-derived growth factor receptor. Embryonic fibroblasts obtained from Stonin1-deficient mice display a marked surface accumulation of NG2, increased cellular signalling and defective FA disassembly as well as altered cellular motility. These data establish Stonin1 as a specific adaptor for the endocytosis of NG2 and as an important factor for FA dynamics and cell migration.
Highlights
Cellular functions, ranging from focal adhesion (FA) dynamics and cell motility to tumour growth, are orchestrated by signals cells receive from outside via cell surface receptors
We show that Stonin[1] is crucial for the efficient internalization of the proteoglycan NG2, an FA-associated transmembrane protein serving as a coreceptor for integrins and the platelet-derived growth factor receptor (PDGFR)[10], and as a promoter of cellular motility[11,12,13,14,15,16,17] and tumour growth[18,19,20]
Immunofluorescence stainings revealed a high degree of co-localization of Stonin[1] with endocytic proteins such as AP-2, clathrin, dynamin[2] and intersectin at clathrin-coated pits (CCPs), but not with the endosomal adaptor AP-1 (Fig. 1e,f; Supplementary Fig. 2a,b)
Summary
Stonin[1] is an endocytic adaptor localizing close to FAs. Stonin[1] and Stonin[2] share WVxF motifs for interacting with AP-2, a central Stonin homology domain and a cargo-binding C-terminal m-homology domain (mHD) (Fig. 1a). Stonin1-positive CCPs localize predominantly to the leading edge (Fig. 1g) and are found adjacent to FAs enriched in vinculin and phosphotyrosinated proteins (Fig. 1i). This characteristic staining pattern is shared by Numb, a specific adaptor for b1-integrin endocytosis at disassembling FAs21. The reduced overall recovery of EGFP-Paxillin indicates that Paxillin molecules are not efficiently exchanged, suggesting that FAs in Stonin[1] À / À MEFs contain a large proportion of immobile molecules.
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