Endocytosis of the glucose transporter GLUT8 is mediated by interaction of a dileucine motif with the β2-adaptin subunit of the AP-2 adaptor complex

Abstract

The glucose transporter GLUT8 cycles between intracellular vesicles and the plasma membrane. Like the insulin-responsive glucose transporter GLUT4, GLUT8 is primarily located in intracellular compartments under basal conditions. Whereas translocation of GLUT4 to the plasma membrane is stimulated by insulin, the distribution of GLUT8 is not affected by insulin treatment in adipose cells. However, blocking endocytosis by co-expression of a dominant-negative dynamin GTPase (K44A) or mutation of the N-terminal dileucine (LL12/13) motif in GLUT8 leads to accumulation of the glucose transporter at the cell surface in a variety of different cell types. Yeast two-hybrid analyses and GST pulldown assays reveal that the LL signal constitutes a binding site for the β2-adaptin subunit of the heterotetrameric AP-2 adaptor complex, implicating this motif in targeting of GLUT8 to clathrin-coated vesicles. Moreover, yeast two-hybrid assays provide evidence that the binding site for the LL motif maps to the appendage domain of β2-adaptin. To analyze the biological significance of the LL/β2 interaction, we utilized RNA interference to specifically knockdown AP-2. Our results show that RNAi-mediated targeting of the μ2 subunit leads to cellular depletion of AP-2, but not AP-1 adaptor complexes in HeLa cells. As a consequence, GLUT8 accumulates at the plasma membrane at comparable levels to those observed in K44A-transfected cells. Conversely, the intracellular localization of mutant GLUT8-LL/AA is restored by replacing the LL motif in GLUT8 with the transferrin receptor-derived μ2-adaptin binding motif YTRF, indicating that for endocytosis both AP-2 binding motifs can substitute for each other. Thus, our data demonstrate that recruitment of GLUT8 to the endocytic machinery occurs via direct interaction of the dileucine motif with β2-adaptin, and that endocytosis might be the main site at which GLUT8 is likely to be regulated.

Identification of a pivotal endocytosis motif in c-Met and selective modulation of HGF-dependent aggressiveness of cancer using the 16-mer endocytic peptide

“…An adaptin b2, a subunit of AP2 complex, which regulates the plasma membrane to the trans-golgi trafficking, is an example that recognizes C-terminal dileucine motif of GLUT8 during its endocytic-vesicle formation. 12 We were curious regarding the interaction of adaptin proteins with the C-terminal dileucine motif of c-Met. In co-immunoprecipitation experiment, interaction of adaptin b1/2 with IL2RA/3S was observed, but binding was interrupted when dileucine motif was replaced by dialanine (Figure 2d), suggesting that dileucine motif is crucial for interaction with adaptin b1/2.…”

Coxiella burnetii effector protein subverts clathrin-mediated vesicular trafficking for pathogen vacuole biogenesis

“…Each AP complex recognizes a subset of sorting motifs, and the variant amino acids within [DERQ]XXXL [LI] and YXXΦ sequences define the specificity of signal recognition (47)(48)(49). Disruption of AP complex interactions with sorting motifs can lead to missorting of cargo proteins back to the plasma membrane (50)(51)(52)(53). The AP2 complex recognizes proteins with more divergent dileucine motifs and binds tyrosine motifs with higher affinity than AP1 and AP3, a property thought to promote retrieval of proteins missorted to the plasma membrane (54,55).…”

“…Knockdown of clathrin or AP2 subunits with siRNA was conducted to better understand their roles in C. burnetii growth (41,53,57). Growth of C. burnetii at 1 and 3 d post infection is similar in cells depleted of AP2 or clathrin relative to cells treated with nontargeting or AP1 siRNAs.…”

Hrs Recognizes a Hydrophobic Amino Acid Cluster in Cytokine Receptors during Ubiquitin-independent Endosomal Sorting

“…(D/E)XXXL(L/I) signal sequences are found in type I, type II, and multispanning transmembrane proteins, which are widely distributed from the plasma membrane to late endosomes, lysosomes, and specialized antigen-process- ing compartments, similar to YXX signal sequences. For example, the (D/E)XXXL(L/I) signal sequence in the CD3-␥ chain mediates its rapid internalization and lysosomal targeting (43), and internalization of glucose transporter 8 is mediated by the interaction of (D/E)XXXL(L/I) signal sequence with AP2 complex (44), indicating that (D/E)XXXL(L/I) signal sequence plays a essential role for the internalization in the membrane proteins. In contrast, the hydrophobic amino acid clusters found in this study were involved in the interaction with Hrs and not needed for the internalization of the receptors.…”