A Specific Dileucine Motif Is Required for the GGA-dependent Entry of Newly Synthesized Insulin-responsive Aminopeptidase into the Insulin-responsive Compartment

• Published in: The Journal of biological chemistry 2006-11, Vol.281 (44), p.33457-33466
• Main Authors: Hou, June Chunqiu, Suzuki, Naoko, Pessin, Jeffrey E., Watson, Robert T.
• Format: Article
• Language: English
• Subjects: 3T3-L1 Cells; Adaptor Proteins, Vesicular Transport - genetics; Adaptor Proteins, Vesicular Transport - metabolism; Adipocytes - cytology; Adipocytes - metabolism; ADP-Ribosylation Factors - genetics; ADP-Ribosylation Factors - metabolism; Alanine - genetics; Alanine - metabolism; Amino Acid Sequence; Animals; Cell Shape; Cystinyl Aminopeptidase - chemistry; Cystinyl Aminopeptidase - genetics; Cystinyl Aminopeptidase - metabolism; Cytosol - metabolism; Genes, Reporter - genetics; Insulin - metabolism; Leucine - genetics; Leucine - metabolism; Mice; Molecular Sequence Data; Mutation - genetics; Protein Transport
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M601583200

In muscle and adipose cells, the insulin-responsive aminopeptidase (IRAP) is localized to intracellular storage sites and undergoes insulin-dependent redistribution to the cell surface. Following expression, the newly synthesized IRAP protein traffics to the perinuclear insulin-sensitive compartment and acquires insulin sensitivity 6-9 h following biosynthesis. Knockdown of GGA1 by RNA interference prevented IRAP from entering, but not exiting, the insulin-responsive compartment. Mutation of the dileucine motif at positions 76 and 77 (EGFP-IRAP/AA76,77), but not the dileucine motif at positions 53 and 54, resulted in the rapid default of the reporter to the cell surface beginning at 3 h following biosynthesis. Alanine substitution of 9 residues amino- or carboxyl-terminal to LL76,77 did not perturb basal intracellular sequestration or abrogate insulin-stimulated IRAP translocation. Moreover, a dominant interfering GGA mutant (VHS-GAT) potently inhibited insulin-stimulated translocation of EGFP-IRAP/WT but did not block the constitutive exocytotic trafficking of EGFP-IRAP/AA76,77. In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA76,77 constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL76,77 functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.