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

Abstract

To identify potential proteins interacting with the insulin-responsive glucose transporter (GLUT4), we generated fusion proteins of glutathione S-transferase (GST) and the final 30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of these carboxylterminal fusion proteins with adipocyte cell extracts revealed a specific interaction of GLUT4 with fructose 1,6-bisphosphate aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in intact 3T3L1 adipocytes, which decreased following insulin stimulation. Introduction into permeabilized 3T3L1 adipocytes of fructose 1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents that disrupt the interaction between aldolase and actin, inhibited insulin-stimulated GLUT4 exocytosis without affecting GLUT4 endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These data suggest that aldolase functions as a scaffolding protein for GLUT4 and that glucose metabolism may provide a negative feedback signal for the regulation of glucose transport by insulin.The insulin-responsive glucose transporter GLUT4 is expressed primarily in adipose tissue, skeletal, and cardiac muscle (1-4). Under basal conditions, GLUT4 slowly recycles between poorly defined intracellular compartments and the plasma membrane with the vast majority sequestered in these intracellular storage sites. Insulin stimulates a large increase in the rate of GLUT4 exocytosis concomitant with a smaller decrease in the rate of GLUT4 endocytosis (5-7). The overall insulin-induced changes in GLUT4 trafficking kinetics result in a 10 -20-fold increase in the number of cell surface GLUT4 proteins that accounts for the majority of insulin-stimulated increases in glucose transport activity (8,9).Recently, several laboratories have begun to examine the subcellular distribution of GLUT4 to identify the mechanism responsible for the intracellular sequestration of the GLUT4 protein. 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). In addition, the presence of a GLUT4 carboxyl-terminal binding protein...

NKCC2 Surface Expression in Mammalian Cells

“…More importantly, it has been suggested that aldolase via its interaction with cytoskeleton functions as a scaffolding protein and plays a crucial role in regulated vesicles exocytosis. Indeed, Kao et al (27) provided compelling evidence for a specific role of aldolase in the insulin stimulation of GLUT4 translocation in adipocytes. In this study, we showed evidence for a specific binding of aldolase B to NKCC2.…”

“…Aldolase is also an actin-binding protein that exhibits a dynamic interaction with the cytoskeleton (22,23). Importantly, aldolase interacts with the H ϩ -ATPase (25,26) and GLUT4 (27), two proteins regulated by intracellular trafficking. More importantly, it has been suggested that aldolase via its interaction with cytoskeleton functions as a scaffolding protein and plays a crucial role in regulated vesicles exocytosis.…”

“…This phenomenon is commonly taken as an indication that a region close to the active site of aldolase participates in the interaction (24,27,52,53). Hence, it was of interest to examine the effect of FBP on aldolase interaction with NKCC2.…”

“…Fructose 1,6-Bisphosphate (FBP) Disrupts Aldolase Binding to NKCC2-Aldolase binding can be modulated, in most cases, by the presence of its substrate and products (21,24,27,52,53). This phenomenon is commonly taken as an indication that a region close to the active site of aldolase participates in the interaction (24,27,52,53).…”

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

“…In addition to its roles in metabolism, aldolase has been implicated in several so-called moonlighting functions, which are distinct from its metabolic roles. These non-glycolytic functions of aldolase include roles in signal transduction (38 -41), vesicle trafficking (42,43), and cell motility (15,44). As mentioned previously, many of these moonlighting functions may involve the interaction of aldolase with the F-actin cytoskeleton (18,34,35).…”