p115 Interacts with the GLUT4 Vesicle Protein, IRAP, and Plays a Critical Role in Insulin-stimulated GLUT4 Translocation

Abstract

Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site. INTRODUCTIONInsulin normalizes blood glucose levels by mobilizing the muscle and adipocyte glucose transporter isoform, GLUT4, from intracellular storage vesicles and moving it to the plasma membrane (Simpson et al., 2001;Bryant et al., 2002). Various models of GLUT4 trafficking suggest that GLUT4 must exist in more than one intracellular compartment and the major insulin sensitive pool is localized to a compartment that is distinct from endosomal markers and is commonly referred to as glucose transporter storage vesicles (GSVs), or insulin responsive vesicle (IRVs). Despite the critical function of glucose transport in glucose homeostasis, many of the details by which adipocytes and muscle form a pathway of insulin-sensitive GLUT4 trafficking remain unknown. However, it is virtually certain that the major cargo proteins of GSVs must interact with a number of cytosolic and membrane proteins, such as adaptors and tethers, in order to be properly sorted and regulated by insulin.Insulin-responsive aminopeptidase (IRAP) was identified as an abundant cargo protein associated with GLUT4 vesicles that translocates in response to insulin in a manner seemingly identical to GLUT4 Mastick et al., 1994;Keller et al., 1995;Malide et al., 1997;Martin et al., 1997;Ross et al., 1997). In fact, it is more abundantly expressed in vesicles than the transporter (Kupriyanova et al., 2002). When the cytoplasmic N-terminus of IRAP was microinjected into 3T3-L1 adipocytes, GLUT4 was localized on the plasma membrane even in the basal state (Waters et al., 1997), suggesting IRAP can play a role in GSV movement/targeting. A chimeric protein containing the intracellular domain of IRAP and ...

Golgin-160 Is Required for the Golgi Membrane Sorting of the Insulin-responsive Glucose Transporter GLUT4 in Adipocytes

“…We therefore wondered whether there could also be a pre-TGN (Golgi cisternae) decision step that directs GLUT4 toward a specific pathway required for appropriate TGN sorting. While our study was in progress Hosaka et al (2005) provided further support for this speculation by demonstrated that the p115 golgin protein could directly bind to IRAP (Hosaka et al, 2005). Regardless, we elected to examine the role of golgin-160 on the trafficking of GLUT4 due to the observations that golgin-160 can directly interact with the protein PIST/GOPC/CAL/ FIG (Hicks and Machamer, 2005).…”

“…In support of this model, the TGN cargo adaptor Golgi-localized, ␥-earcontaining ARF-binding protein (GGA) is required for the initial entry of GLUT4 from the TGN into the insulin-responsive compartment (Watson et al, 2004). More recently, the expression of an amino-terminal deletion mutant of the Golgi tethering protein, p115 was found to inhibit insulinstimulated GLUT4 translocation to the plasma membrane, suggesting a role for p115 in the intracellular tethering of GLUT4 (Hosaka et al, 2005).p115 is one member of a large golgin family of Golgilocalized proteins. These proteins have been implicated in the maintenance of proper Golgi structure and function (Barr et al, 2003).…”

IRAP+ endosomes restrict TLR9 activation and signaling

“…The regulated trafficking of IRAP depends on the cytosolic domain of the enzyme, which has been shown to interact with several proteins involved in vesicle formation or in cytoskeleton remodeling, such as the golgin p115 18 , vimentin 19 and FHOS (formin homologue overexpressed in the spleen, also called FHOD1) 20 . Whether these proteins and their interaction with the cytosolic domain of IRAP play a role in the trafficking of IRAP + endosomes is not known.…”

Cysteine string protein 1 (CSP1) modulates insulin sensitivity by attenuating glucose transporter 4 (GLUT4) vesicle docking with the plasma membrane

“…Akt antibody and pAkt (Ser473) antibody were from Cell Signaling Technology (Beverly, MA). GLUT4 antibody was raised and purified as described previously (19). HRP-conjugated IgG second antibodies were purchased from Life Technologies (Carlsbad, CA).…”

“…3T3-L1 fibroblasts were induced to differentiate as described previously with minor modification (19). Briefly, 3T3-L1 fibroblasts were cultured in 15 cm dishes for electroporation with DMEM containing 4.5 g/l glucose, L-glutamine and 10% calf serum.…”