Endophilin Recruitment via GPCR Interactions Enables Membrane Curvature Generation in the Absence of Anionic Lipids

The Bin/Amphiphysin/Rvs (BAR) family protein endophilin plays key roles in membrane curvature generation during endocytosis of cellular receptors. The Src homology 3 (SH3) domain of endophilin interacts with the proline rich third intracellular loop (TIL) of various G-protein coupled receptors (GPCRs). While electrostatic interactions between BAR domain and anionic membrane lipids have been considered to be the major driving force in curvature generation, it is unclear how the direct interaction between TIL and SH3 affects this function and its coupling with receptor internalization. Here we show that TIL mediated interactions alone not only recruit endophilin to the membrane but also facilitate curvature sorting and curvature generating behavior of endophilin. To demonstrate this, we designed model membranes with covalently lipid-conjugated TIL and lipids without net negative charge so that endophilin was recruited exclusively via SH3/TIL interactions. We find curvature generation and curvature sorting under those conditions. Furthermore, we show that TIL interacts electrostatically with membranes in the presence of anionic lipids and that this interaction can interfere with binding of SH3. Overall, our study suggests that an interplay between TIL, charged membranes, BAR domain, and SH3 domain mediate membrane curvature generation to regulate receptor endocytosis following receptor stimulation.