GLUT-4 NH2 terminus contains a phenylalanine-based targeting motif that regulates intracellular sequestration.

Abstract

Abstract. Expression of chimeras, composed of portions of two different glucose transporter isoforms (GLUT-1 and GLUT-4), in CHO cells had indicated that the cytoplasmic NH: terminus of GLUT-4 contains important targeting information that mediates intracellular sequestration of this isoform (Piper, R. C., C. Tai, J. W. Slot, C. S. Hahn, C. M. Rice, H. Huang, D. E. James. 1992. J. Cell Biol. 117:729-743). In the present studies, the amino acid constituents of the GLUT-4 NH2-terminal targeting domain have been identified. GLUT-4 constructs containing NH2-terminal deletions or alanine substitutions within the NH2 terminus were expressed in CHO cells using a Sindbis virus expression system. Deletion of eight amino acids from the GLUT-4 NH2 terminus or substituting alanine for phenylalanine at position 5 in GLUT-4 resulted in a marked accumulation of the transporter at the plasma membrane. Mutations at other amino acids surrounding Phe5 also caused increased cell surface expression of GLUT-4 but not to the same extent as the Phe5 mutation. GLUT-4 was also localized to clathrin lattices and this colocalization was abolished when either the first 13 amino acids were deleted or when Phe5 was changed to alanine. To ascertain whether the targeting information within the GLUT-4 NH2-terminal targeting domain could function independently of the glucose transporter structure this domain was inserted into the cytoplasmic tail of the HI subunit of the asialoglycoprotein receptor. H1 with the GLUT-4 NH2 terminus was predominantly localized to an intracellular compartment similar to GLUT-4 and was sequestered more from the cell surface than was the wildtype HI protein. It is concluded that the NH2 terminus of GLUT-4 contains a phenylalanine-based targeting motif that mediates intracellular sequestration at least in part by facilitating interaction of the transporter with endocytic machinery located at the cell surface.

Dynamics of insulin‐stimulated translocation of GLUT4 in single living cells visualised using green fluorescent protein

“…Our data now suggest that the N-terminal domain of full-length GLUT4 possesses a critical internalisation motif. This motif is likely to be the phenylalanine-based (FQQI) sequence which is important for GLUT4 sequestration in CHO cells [25] and 3T3-L1 cells [27]. However, we cannot completely exclude the possibility that N-terminal GFP also sterically hinders the C-terminal dileucine motif; however, this is rather unlikely in view of the fact that GFP placed at the Cterminus of GLUT4 does not hinder internalisation.…”

“…Previous studies have concluded that GLUT4 possesses at least two signals that may be responsible for targeting the transporter to this intracellular location in fibroblasts. One, a phenylalanine-based (FQQI) motif, is close to the N-terminus [25,27] and the other, a dileucine motif, is found in the Cterminal tail [29,30]; however, other motifs may be involved [13]. Mutagenesis of either motif causes GLUT4 to be targeted to the plasma membrane of cells (reviewed in [3]).…”

“…Control Insulin by fluorescence microscopy, to heterologously expressed native GLUT4 in CHO cells [24,25], PC12 cells [26] and 3T3-L1 cells [27,28], or endogenous GLUT4 in 3T3-L1 cells [27]. Previous studies have concluded that GLUT4 possesses at least two signals that may be responsible for targeting the transporter to this intracellular location in fibroblasts.…”

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

“…This recycling apparently occurs via the clathrin-coated pit pathway as GLUT4 labelling of cell-surface clathrin-coated pits and lattices has been observed in both non-stimulated and insulin-stimulated adipocytes [10]. It is also not surprising that an investigation of the targeting motifs that regulate the intracellular sequestration of GLUT4 has revealed motifs, one in the N-terminus [39] and one in the C-terminus [40], that appear to modulate internalization from the cell surface.…”

Arrest of endosome acidification by bafilomycin A1 mimics insulin action on GLUT4 translocation in 3T3-L1 adipocytes

“…According to the currently accepted translocation model, originally proposed by Cushman and Wardzala [5] and Suzuki and Kono [6], insulin evokes a redistribution of both GLUT4 and GLUT1 from the interior of the cell to the plasma membrane, but the increased capacity of the cell to transport glucose is due mainly to GLUT4. Studies using chimaeric GLUT1-GLUT4 proteins expressed in both insulin-sensitive and insulinunresponsive cell models in a transient or stable manner helped to identify structural motifs among the transporter molecules responsible for their differential sorting within the cell [7][8][9][10][11]. Thus both the N-and C-termini of GLUT4 contain sequence information responsible for sequestering the transporter into the cell interior, but to different subcompartments ; the insulinsensitive endosomal compartment recognizes the C-terminal 30 amino acids of GLUT4 [11].…”