The F-BAR protein CIP4 promotes GLUT4 endocytosis through bidirectional interactions with N-WASp and Dynamin-2.

F-BAR proteins are a newly described family of proteins with unknown physiological significance. Because F-BAR proteins, including Cdc42 interacting protein-4 (CIP4), drive membrane deformation and affect endocytosis, we investigated the role of CIP4 in GLUT4 traffic by flow cytometry in GLUT4myc-expressing L6 myoblasts (L6 GLUT4myc). L6 GLUT4myc cells express CIP4a as the predominant F-BAR protein. siRNA knockdown of CIP4 increased insulin-stimulated (14)C-deoxyglucose uptake by elevating cell-surface GLUT4. Enhanced surface GLUT4 was due to decreased endocytosis, which correlated with lower transferrin internalization. Immunoprecipitation of endogenous CIP4 revealed that CIP4 interacted with N-WASp and Dynamin-2 in an insulin-dependent manner. FRET confirmed the insulin-dependent, subcellular properties of these interactions. Insulin exposure stimulated specific interactions in plasma membrane and cytosolic compartments, followed by a steady-state response that underlies the coordination of proteins needed for GLUT4 traffic. Our findings reveal a physiological function for F-BAR proteins, supporting a previously unrecognized role for the F-BAR protein CIP4 in GLUT4 endocytosis, and show that interactions between CIP4 and Dynamin-2 and between CIP4 and NWASp are spatially coordinated to promote function.