The Cdc42-interacting Protein-4 (CIP4) Gene Knock-out Mouse Reveals Delayed and Decreased Endocytosis

Yanming Feng,Sean M Hartig,John E Bechill,Elisabeth G Blanchard,Eva Caudell,Seth J Corey

doi:10.1074/jbc.m109.041038

Abstract

The newly described F-BAR (Fer/CIP4 and Bin, amphiphysin, Rvs) family of proteins includes Cdc42-interacting protein-4 (CIP4), formin-binding protein-17 (FBP-17) and transactivator of cytoskeletal assembly-1 (Toca-1), and drives membrane deformation and invagination. Membrane remodeling affects endocytosis, vesicle budding, and cargo selection. The F-BAR family presents a novel family of proteins, which little is known about their in vivo function. We investigated the physiological role of CIP4, by creating Cip4-null mice through homologous recombination. Compared with their wild-type littermates, the Cip4-null mice displayed lower early post-prandial glucose levels. Adipocytes isolated from Cip4-null mice exhibited increased [(14)C]2-deoxyglucose uptake compared with cells from wild-type mice. The enhanced insulin sensitivity was not due to higher levels of insulin or phospho-Akt, a critical player in insulin signaling. However, higher glucose transporter 4 (GLUT4) levels were detected in muscle membrane fractions in Cip4-null mice under insulin stimulation. Mouse embryonic fibroblasts from Cip4-null mice demonstrated decreased transferrin uptake, fluorescein isothiocyanate-dextran, and horseradish peroxidase uptake, indicating that CIP4 affects multiple modes of endocytosis. These studies demonstrate a physiological role for CIP4 in endocytosis leading to a whole animal phenotype.

Highlights

The signaling events underlying the translocation of glucose transporter 4 (GLUT4) to the plasma membrane are well established, the molecular events that lead to endocytosis of GLUT4 are less well understood [17]
Based on that observation and the growing recognition that F-BAR domains play a role in regulating biological processes that require membrane curvature deformation, we hypothesized that Cdc42-interacting protein-4 (CIP4) may regulate GLUT4 endocytosis
These physiological observations were substantiated at the cellular level by demonstrating that surface expression of GLUT4 on muscle cells under insulin stimulation is altered in the absence of CIP4

Summary

Introduction

We concluded that greater glucose tolerance of the Cip4-null mice was due to decreased and delayed GLUT4 endocytosis. Glucose Transport in Immortalized Adipocytes—In 6-well plates, fully differentiated wild-type or Cip4-null adipocytes were incubated at 37 °C for 2 h with DMEM containing 1% bovine serum albumin, and washed with Krebs-Ringer buffer (130 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.3 mM MgSO4, 25 mM HEPES, pH 7.4). Statistical Analysis—Statistical analysis of the difference between wild-type and knock-out mice was performed with a Student’s t test for individual glucose tolerance, serum insulin, the area under the curve, and endocytosis assays.

Results

Conclusion