Cloning and Characterization of a Novel Insulin-regulated Membrane Aminopeptidase from Glut4 Vesicles

Abstract

The insulin-regulated glucose transporter isotype GluT4 expressed only in muscle and adipose cells is sequestered in a specific secretory vesicle. These vesicles harbor another major protein, referred to as vp165 (for vesicle protein of 165 kDa), that like GluT4 redistributes to the plasma membrane in response to insulin. We describe here the cloning of vp165 and show that it is a novel member of the family of zinc-dependent membrane aminopeptidases, with the typical large extracellular catalytic domain and single transmembrane domain but with a unique extended cytoplasmic domain. The latter contains two dileucine motifs, which may be critical for the specific trafficking of vp165, since this has been shown to be the case for this motif in GluT4.

Sortilin Is a Major Protein Component of Glut4-containing Vesicles

“…The first two are the IGF-II/Man-6-phosphate receptor (8) and IRAP, respectively (6,7,17). To identify gp110, we isolated adipocytes from 60 rats, prepared the light microsomal fraction from these cells (see "Materials and Methods") and immunoadsorbed Glut4-containing vesicles.…”

The Inactivation of RabGAP Function of AS160 Promotes Lysosomal Degradation of GLUT4 and Causes Postprandial Hyperglycemia and Hyperinsulinemia

“…We are currently unable to distinguish these two possibilities, and further experimentation is required to address this question in the future. It is well known that the other protein residing in GSVs, IRAP, traffics together with GLUT4 in response to insulin (40), and its expression is decreased in various GLUT4-null cells (41,42). Interestingly, in contrast to GLUT4, IRAP levels were normal in tissues or primary adipocytes from the AS160 KO mice, suggesting that the degradation of these two proteins might be differentially regulated.…”

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

“…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).…”

“…All this strongly suggests the involvement of IRAP with the retention and sorting machinery of GSVs and its potential association with the targeting and tethering proteins involved in this process. IRAP has a single transmembrane domain with the Nterminus projecting into the cytosol (Keller et al, 1995), whereas GLUT4 has potential sorting and targeting motifs in three cytosolic domains corresponding to N-and C-termini as well as the central loop that connects helices 6 and 7 (Fukumoto et al, 1989). The N-terminus of IRAP has dileucine motifs and an acidic cluster domain similar to that found in the C-terminus of GLUT4, which is thought to be important region for its trafficking (Verhey et al, 1993(Verhey et al, , 1995Corvera et al, 1994;Verhey and Birnbaum, 1994;Haney et al, 1995;Marsh et al, 1995).…”

“…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).…”