Recent studies have revealed that the Rab GAP proteins TBC1D1 and AS160 (TBC1D4) have important roles in the metabolic pathways relevant to obesity and type 2 diabetes. A mutant form of TBC1D1 occurs in a rare form of human obesity (34
). Furthermore, truncation and loss of TBC1D1 in a mouse strain have revealed that the presence of TBC1D1 confers leanness when crossed with an obese and insulin-resistant mouse strain (36
). In this mouse model, loss of TBC1D1 reverses whole body insulin resistance and obesity in an artificial situation in which mice have high access to nutrients. Interestingly, differences in TBC1D1
gene sequence have been genetically linked to differences in growth and metabolic activity occurring within chicken lines bred for high meat content or egg-laying activity (37
). The mouse model in which a non-phosphorylatable mutant of AS160 (AS160 T649A) has been expressed leads to a glucose-intolerant phenotype with reduced insulin sensitivity and altered GLUT4 traffic (9
). Human insulin resistance has also been found to be associated with AS160 mutations. Families with AS160 variants including N1206S, N655Y, and N785K have been identified and may contribute to varying degrees of insulin resistance. However, complete co-segregation with the phenotype was difficult to achieve in studies on these families (38
). In addition, a human truncated variant of AS160 leads to acanthosis nigricans and postprandial hyperinsulinemia (10
). The effects of this truncation are associated with impaired GLUT4 translocation in insulin target cells. In 3T3-L1 cells, expression of this truncated mutant leads to elevation of basal and reduction of insulin-stimulated GLUT4 traffic (10
). We have confirmed here that the AS160 tandem PTB domain construct inhibits insulin-stimulated GLUT4 translocation to the cell surface in rat adipose cells.
We found inhibition of fusion by the PTB domains of AS160 and by the 14-3-3-quenching inhibitors R18 and fusicoccin, thus identifying the fusion reaction as an important site for action of these proteins, their mutants, and truncated forms. Previous studies have suggested a prefusion role for AS160 in which the delivery of GLUT4 vesicles to the vicinity of the plasma membrane prior to initiation of fusion is one of the means by which translocation is activated by insulin action (3
). In total internal reflection fluorescence studies that allow a distinction between vesicle docking and fusion, it has been suggested that a block in docking of GLUT4 vesicles by the AS160–4P construct indirectly affects downstream fusion (17
). Insulin action on fusion machinery beyond and downstream of AS160 has been proposed (18
), possibly involving the motor protein Myo1c (39
). The inhibition of GLUT4 vesicle docking and fusion with the plasma membrane by the PTB domain constructs of AS160 revealed here provides direct evidence that AS160 itself is required for in vitro
docking and fusion, where release from intracellular compartments is not occurring.
Our studies on the mechanism by which the PTB domain constructs and 14-3-3-quenching compounds inhibit the fusion reaction have led us to the discovery that 14-3-3γ is highly enriched on GLUT4 vesicles and has marked selectivity for interaction with phospho-AS160. The combination of AS160 with 14-3-3γ appears to be critical, and the PTB domains of AS160 can act as inhibitors of fusion by inhibiting this interaction. This inhibitory effect of the PTB domains on 14-3-3γ binding occurs on GLUT4 vesicles but can also be reproduced in studies on direct protein-protein interaction.
These data led us to propose a model for regulation of GLUT4 vesicle fusion in which insulin-stimulated 14-3-3γ interaction, but not AS160 phosphorylation alone, generates a fusion-activating GAP-inactive form of AS160 (A
). We suggest that the PTB domain constructs form heterodimers with endogenous AS160 and that this interaction prevents the binding of 14-3-3 to phosphorylated AS160 (B
). Therefore, AS160 remains in its GAP-active form, the associated Rab remains in the GDP form, and fusion activity is therefore low. The basis for the R18 inhibition of fusion is likely to be due to quenching of all available 14-3-3, so, again, although AS160 is phosphorylated, the GAP is active, the associated Rab remains in its GDP form, and fusion activity is therefore low. Our data appear to be inconsistent with a receptor-blocking mode of action of the fusion inhibitory PTB domains. If interaction with a receptor, such as IRAP (Insulin-regulated aminopeptidase) (11
), were blocked by the PTB domain constructs, then AS160 would be unable to be recruited to the vesicles, and the vesicle Rab would be expected to be in the fusion-facilitating and fusion-active form. Fusion would be expected to be high, but this has not been observed. In contrast to a study on GLUT4 vesicles in 3T3-L1 cells (11
), we found that, in rat adipose cells, AS160 is continuously associated with GLUT4 vesicles and that insulin action does not lead to its dissociation. However, our data support the hypothesis that 14-3-3 interaction with AS160 is largely responsible for its functional inactivation as proposed by Ramm et al.
FIGURE 6. Model for inhibitory effects of AS160 N-terminal PTB domain constructs and 14-3-3-quenching reagents on GLUT4 vesicle fusion. A, insulin-stimulated generation of fusion-active GLUT4 vesicles (G4V). Insulin action leads to AS160 phosphorylation and 14-3-3γ (more ...)
In conclusion, this investigation of the inhibitory effects of N-terminal PTB domain constructs of AS160, including a human truncated variant, has revealed that the inhibitory PTB domains stop the loading of vesicles with 14-3-3γ but do not stop AS160 phosphorylation. This suggests that phosphorylation alone, without 14-3-3γ combination, does not generate GLUT4 vesicles that can fuse with the insulin-activated plasma membrane in adipocytes. The emerging roles of the Rab GAPs TBC1D1 and AS160 (TBC1D4) in the control of whole body and muscle glucose metabolism in human subjects with obesity and type 2 diabetes indicate that further studies on the roles of both of these proteins in the process of GLUT4 vesicle fusion in muscle are warranted.