A double leucine within the GLUT4 glucose transporter COOH-terminal domain functions as an endocytosis signal.

Abstract

Abstract. The unique COOH-terminal 30-arnino acid region of the adipocyte/skeletal muscle glucose transporter (GLUT4) appears to be a major structural determinant of this protein's perinuclear localization, from where it is redistributed to the cell surface in response to insulin. To test whether an underlying mechanism of this domain's function involves glucose transporter endocytosis rates, transfected cells were generated expressing exofacial hemagglutinin epitope (HA)-tagged erythrocyte/brain glucose transporter (GLUTI) or a chimera containing the COOH-terminal 30 amino acids of GLUT4 substituted onto this GLUT1 construct. Incubation of COS-7 or CHO cells expressing the HA-tagged chimera with anti-HA antibody at 37 ° resulted in an increased rate of antibody internalization compared to cells expressing similar levels of HA-tagged GLUT1, which displays a cell surface disposition. Colocalization of the internalized anti-HA antibody in vesicular structures with internalized transfenin and with total transporters was established by digital imaging microscopy, suggesting the total cellular pool of transporters are continuously recycling through the coated pit endocytosis pathway. Mutation of the unique double leucines 489 and 490 in the rat GLUT4 COOH-terminal domain to alanines caused the HA-tagged chimera to revert to the slow endocytosis rate and steady-state cell surface display characteristic of GLUT1. These results support the hypothesis that the double leucine motif in the GLUT4 COOH terminus operates as a rapid endocytosis and retention signal in the GLUT4 transporter, causing its localization to intracellular compartments in the absence of insulin.XPERIMENTS reported over the last four decades have clearly defined the stimulatory effect of insulin on membrane transport of glucose as a key aspect of this hormone's physiological function. This acute action of insulin is maximal within a few minutes, leads to over tenfold increases in hexose influx rates, and is largely restricted to muscle and fat cells in vivo (8,32). Using fat cell fractionation techniques, it was shown that insulin-stimulated glucose uptake is associated with a rapid redistribution of functional glucose transporters from an intracellular membrane fraction to plasma membranes (5, 35). Subsequently, direct demonstration of the rapid increase in glucose transporter proteins on the cell surface of intact cells in response to insulin has been accomplished using transporter affinity labeling (16,20,28) and trypsin cleavage (6) techniques, immunoelectron microscopy (33, 34), and an antibody binding assay using an epitope-tagged glucose transporter construct (24).

Constitutive GABAA Receptor Endocytosis Is Dynamin-mediated and Dependent on a Dileucine AP2 Adaptin-binding Motif within the β2 Subunit of the Receptor

“…It is possible that the phosphorylation state of this site could regulate the interaction of the ␥ 2 L subunit with AP2 adaptin. Such regulation occurs with cluster of differentiation antigen 4 (CD4), the GLUT4 glucose transporter and CD3␥, in which the dileucine motif plays a role in clathrin-dependent endocytosis when a nearby serine is phosphorylated (25)(26)(27).…”

Molecular Basis of Insulin-stimulated GLUT4 Vesicle Trafficking

“…However, further mutational studies and kinetic analysis revealed that this motif was, in fact, required for efficient internalization (43,44). Thus the apparent lack of intracellular sequestration in these mutants resulted from an accumulation of GLUT4 at the plasma membrane because of a reduction in the rate of endocytosis.…”

Aldolase Mediates the Association of F-actin with the Insulin-responsive Glucose Transporter GLUT4

“…Steady-state and kinetic analysis of various expressed GLUT4 chimeric proteins have indicated that both the aminoand carboxyl-terminal domains are important in GLUT4 internalization from the plasma membrane (10 -15). In particular, the carboxyl-terminal dileucine motif (SLL) was found to substantially alter GLUT4 trafficking kinetics and steady-state localization (16,17). Although a specific GLUT4 sequence responsible for intracellular localization has yet to be identified, the presence of a carboxyl-terminal retention signal is consistent with the observation that expression of the GLUT4 carboxyl-terminal domain results in the translocation of the endogenous GLUT4 protein to the plasma membrane (18).…”