Interactions of netrin-1 through its glycosylation sites immobilize Deleted in Colorectal Cancer (DCC) by favoring its constitutive clustering

Abstract

AbstractNetrin-1 is a secreted protein that attracts neurons expressing the membrane receptor Deleted in Colorectal Cancer DCC in the nervous system. In colon carcinoma, the interaction between netrin-1 and DCC prevents apoptosis in an autocrine manner. Crystallographic data suggest that these processes involve DCC homodimerization and clustering, but the observation of such clusters in cells is missing. Moreover, the molecular determinants of DCC-netrin-1 interactions and their impact on DCC organization and mobility remain unclear.To address these questions, we employed fluorescence photobleaching, single particle tracking and super-resolution techniques to characterize the organization and mobility of DCC at the cell surface. Our findings support that netrin-1 impairs the mobility of DCC in the plasma membrane. Additionally, we show that netrin-1 promotes the growth of constitutive nano-clusters of DCC at the expense of free DCC. All these effects are primarily mediated by the three glycosylation sites in netrin-1 LamVI domain and to a lesser extent by the C-terminal domain and its RGD binding site.Taken together, these findings suggest that DCC mobility results from its constitutive nanoscale clustering, which is primarily facilitated by the interaction between DCC and netrin-1 through netrin-1 glycosylation sites and secondarily its RGD site.