Characterization of the Insulin-regulated Endocytic Recycling Mechanism in 3T3-L1 Adipocytes Using a Novel Reporter Molecule

The endocytic trafficking of the GLUT4 glucose transporter and the insulin-regulated aminopeptidase (IRAP) are regulated by insulin. We have used a chimera between the intracellular domain of IRAP and the extracellular and transmembrane domains of the transferrin receptor (vpTR) to characterize IRAP-like trafficking in 3T3-L1 adipocytes. Our data demonstrate that the cytoplasmic domain of IRAP is sufficient to target vpTR to the insulin-regulated, slow recycling pathway in adipocytes and that the dynamic retention of vpTR is dependent on a di-leucine motif. Our kinetic analysis demonstrates that vpTR recycles as a single kinetic pool and that vpTR is very efficiently sorted from endosomes to the insulin-regulated recycling pathway. An implication of these findings is that the key step in the dynamic retention of vpTR occurs within the early endosomal system. We have previously shown that vpTR is trafficked by an insulin-regulated pathway in Chinese hamster ovary cells (Johnson, A. O., Subtil, A., Petrush, R., Kobylarz, K., Keller, S., and Mc Graw, T. E. (1998) J. Biol. Chem. 273, 17968–17977). The behavior of vpTR in Chinese hamster ovary cells is similar to its behavior in 3T3-L1 adipocytes. The main difference is that insulin has a larger effect on the trafficking of vpTR in the adipocytes. We concluded that the insulin-regulated slow recycling endocytic mechanism is expressed in many different cell types and therefore is not a unique characteristic of cells that express GLUT4.