Role of dynamin and clathrin in the cellular trafficking of flotillins.

Flotillin-1 and flotillin-2 are highly conserved, membrane-microdomain-associated proteins that have been shown to be involved in signal transduction, membrane trafficking and cell adhesion. Upon growth factor stimulation, flotillins are tyrosine phosphorylated and become endocytosed from the plasma membrane into endosomes from which they are recycled back to the plasma membrane. Although a role for flotillin-1 in the endocytosis of certain cargo proteins has been suggested, it is not known how the growth-factor-induced endocytosis of flotillins is regulated and which endocytosis pathway is used. However, this is likely to be different from the pathway used by flotillin-dependent cargo. In this study, we have addressed the mechanistic details of flotillin trafficking during growth factor signaling. We show that dynamin-2 activity is required for the uptake of flotillins from the plasma membrane upon epidermal growth factor stimulation, and inhibition of dynamin-2 GTPase activity impairs flotillin endocytosis. Surprisingly, recycling of flotillins from endosomes to the plasma membrane appears to require both dynamin-2 and clathrin. Upon overexpression of dynamin-2 mutants or depletion of clathrin heavy chain, flotillins are permanently trapped in endosomes. These data show that clathrin and dynamin are required for the endosomal sorting of flotillins, and the study provides a mechanistic dissection of the thus far poorly characterized endosomal trafficking of flotillins.