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Roles of the N- and C-termini of GLUT4 in endocytosis

¹ Center for Molecular Medicine, Institute of Biochemistry, University of Cologne, Otto-Fischer-Str. 12-14, 50674 Cologne, Germany
² Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA

View larger version (1K): [in a new window]   Fig. 1. Amino acid sequences of the N- and C-termini of wild-type GLUT4 and GLUT4 mutants. (A) Predicted topology of GLUT4. CHO, carbohydrate; TAG, position of the HA-epitope tag. (B) Predicted cytoplasmic amino acids of the N-terminus (M1-L24) of human GLUT4 (Fukumoto et al., 1989). (C) Predicted cytoplasmic amino acids of the C-terminus (L466-D509). TM I, TM XII, position of the first and twelfth transmembrane domains. The regions with high homology to the GLUT1 C-terminus (R466-F483; 61% identity, 89% similarity), and the locations of the phenylalanine-5 (F5) and the dileucine-489/90 (LL498/90) motifs are indicated. (D) C-terminal amino acid sequences of the C-terminal truncation mutants (37, 28).

View larger version (26K): [in a new window]   Fig. 2. Relative protein expression levels of HA-GLUT4 constructs. Isolated rat adipose cells were transfected with 1.25 µg/ml plasmid DNA (12.5 µg/ml for the 37 mutant) and cultured for 24 hours. After harvesting, total cellular membranes were prepared and the expression of HA-GLUT4 was determined by western blot using anti-HA antibodies as described in Materials and Methods (inset). The HA-GLUT4 proteins migrated according to their expected molecular masses in SDS-PAGE and appeared as single bands on western blots. Results are the means±s.e.m. of at least three independent experiments.

View larger version (58K): [in a new window]   Fig. 3. Subcellular distributions of IRAP, HA-GLUT4 and HA-GLUT4 mutants in rat adipose cells. Isolated cells were transfected with 5 µg/ml plasmid DNA and cultured for 24 hours. After harvesting, the cells were incubated without (basal) or with 67 nM insulin for 30 minutes. Then, the cells were fixed, permeabilized, and stained as described in Materials and Methods. Images were collected in single optical sections. (A) Colocalization of IRAP (red) and HA-GLUT4 (green). Squares indicate regions that are magnified in the next row of images. (B) Localization of HA-GLUT4, F5A, 37 and F5A37. Bars, 10 µm.

View larger version (19K): [in a new window]   Fig. 4. Basal and insulin-stimulated cell-surface distributions of wild-type HA-GLUT4, dileucine (LL489/90AA) mutants and F5A mutants in rat adipose cells. Isolated cells were transfected with 1.25 µg/ml plasmid DNA and cultured for (A) 4 hours or (B) 24 hours. After harvesting, the cells were incubated without (basal, solid bars) or with (open bars) 67 nM insulin for 30 minutes, and the cell-surface levels of HA-GLUT4 were determined using an antibody binding assay as described in Materials and Methods. The cell surface-associated radioactivity was normalized to the relative protein expression level of each respective mutant (Fig. 2). Results are the means±s.e.m. of at least three replicate experiments performed in duplicates to quadruplicates.

View larger version (15K): [in a new window]   Fig. 5. Basal and insulin-stimulated cell-surface distributions of wild-type HA-GLUT4 and C-terminal deletion mutants in rat adipose cells. Isolated cells were transfected with 1.25 µg/ml plasmid DNA and cultured for 4 hours. In the case of the 37 mutant, 12.5 µg/ml plasmid DNA were used. After harvesting, the cells were incubated without (basal, solid bars) or with (open bars) 67 nM insulin for 30 minutes, and the cell-surface levels of HA-GLUT4 were determined using an antibody binding assay as described in Materials and Methods. The cell surface-associated radioactivity was normalized to the relative protein expression level of each respective mutant (Fig. 2). Results are the means±s.e.m. of at least three replicate experiments performed in duplicates to quadruplicates.

View larger version (14K): [in a new window]   Fig. 6. Clearance of insulin-stimulated cell-surface HA-GLUT4 and HA-GLUT4 mutants in rat adipose cells. Isolated cells were transfected with 1.25 µg/ml plasmid DNA (except for 37 where 12.5 µg/ml plasmid DNA were used) and cultured for 4 hours. After harvesting, the cells were stimulated with 67 nM insulin for 30 minutes. Wortmannin (100 nM) was then added at time 0 and the cell-surface levels of HA-GLUT4 were determined at the indicated time points using an antibody binding assay as described in Materials and Methods. , HA-GLUT4 wild-type; , F5A; , 37. Results are the means±s.d. of 2-4 replicate experiments performed in duplicates to quadruplicates.

View larger version (19K): [in a new window]   Fig. 7. Coexpression of HA-GLUT4s and dominant negative dynamin. Isolated cells were cotransfected with 0 (–) or 12.5 (+) µg/ml dynamin-K44A plasmid/cuvette and 2.5 µg/ml of expression plasmid for HA-GLUT4s and cultured for 24 hours. After harvesting, the cells were incubated without (basal, solid bars) or with (open bars) 67 nM insulin for 30 minutes, and the cell-surface levels of HA-GLUT4s were determined using an antibody binding assay as described in Materials and Methods. The cell surface-associated radioactivity was normalized to the relative protein expression level of each respective mutant. Results are the means of the mean values (,) obtained from at least duplicate determinations in two independent experiments.

View larger version (18K): [in a new window]   Fig. 8. Yeast two-hybrid interaction of the N-terminus of GLUT4 with µ-adaptins. (A) Yeast cells (strain HF7c) cotransformed with plasmids for GAL4-BD/GLUT4-NTs and GAL4-AD/µ-adaptins were plated onto drop-out agar plates lacking tryptophane and leucine with (+) and without (–) histidine and incubated for 2 days at 30°C. (B) Quantification of ß-galactosidase activity. Yeast cells (strain SFY526) cotransformed with plasmids for GAL4-BD/GLUT4-NTs and GAL4-AD/µ-adaptins were grown into mid-log phase, harvested and assayed for ß-galactosidase activity using CPRG as substrate (see Materials and Methods).

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