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.
Hartig
Sean M
SM
Division of Pediatrics, University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA.
Ishikura
Shuhei
S
Hicklen
Rachel S
RS
Feng
Yanming
Y
Blanchard
Elisabeth G
EG
Voelker
Kevin A
KA
Pichot
Christina S
CS
Grange
Robert W
RW
Raphael
Robert M
RM
Klip
Amira
A
Corey
Seth J
SJ
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D005434:Flow Cytometry; D031541:Fluorescence Resonance Energy Transfer; D055785:Gene Knockdown Techniques; D051275:Glucose Transporter Type 4 / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D032446:Myoblasts / Q000166:cytology / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000235:genetics / Q000378:metabolism*
D051275:Glucose Transporter Type 4; D007328:Insulin; D008869:Microtubule-Associated Proteins; D020033:Protein Isoforms; D034741:RNA, Small Interfering; C495770:Wasl protein, rat; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D034285:Dynamin II