Abstract
Ganglioside GM1-bound cholera toxin-B sub-unit (CT-b) enters the cell via clathrin-coated pits and dynamin-independent non-caveolar raft-dependent endocytosis. Caveolin-1 (Cav1), associated with caveolae formation, is a negative regulator of non-caveolar raft-dependent endocytosis. In mammary epithelial tumour cells deficient for Mgat5, Cav1 is stably expressed at levels below the threshold for caveolae formation, forming stable oligomerized Cav1 microdomains or scaffolds that were shown to suppress EGFR signalling and reduce the plasma membrane diffusion rate of both EGFR and CT-b. Below threshold levels of Cav1 also inhibit the dynamin-dependent raft-mediated endocytosis of CT-b to the Golgi indicating that Cav1-negative regulation of raft-dependent endocytosis is caveolae independent. Inhibition of CT-b internalization does not require Cav1 phosphorylation but does require an intact Cav1 scaffolding domain. By flow cytometry, both over-expression of Cav1 and the dynamin K44A mutant block CT-b internalization from the plasma membrane defining a dynamin-dependent raft pathway for CT-b endocytosis in these cells. However, only minimal co-localization between CT-b and Cav1 is observed. These results suggest that Cav1 regulates raft-dependent internalization of CT-b indirectly via a mechanism that requires the Cav1 scaffolding domain and the formation of oligomerized Cav1 microdomains but not caveolae.