A specific dileucine motif is required for the GGA-dependent entry of newly synthesized insulin-responsive aminopeptidase into the insulin-responsive compartment.
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/AA(76,77)), 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 LL(76,77) 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/AA(76,77). In addition, the EGFP-IRAP/WT and EGFP-IRAP/AA(76,77) constructs occupied morphologically distinct tubulovesicular compartments in the perinuclear region. Taken together, these data indicate that LL(76,77) functions during the GGA-dependent sorting of newly made IRAP into the insulin-responsive storage compartment.
Hou
June Chunqiu
JC
Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794-8651, USA.
Suzuki
Naoko
N
Pessin
Jeffrey E
JE
Watson
Robert T
RT
D041721:3T3-L1 Cells; D020727:ADP-Ribosylation Factors / Q000235:genetics / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism; D017667:Adipocytes / Q000166:cytology / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D048430:Cell Shape; D010122:Cystinyl Aminopeptidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D017930:Genes, Reporter / Q000235:genetics; D007328:Insulin / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D021381:Protein Transport
D033942:Adaptor Proteins, Vesicular Transport; C407306:GGA adaptor proteins; D007328:Insulin; D010122:Cystinyl Aminopeptidase; C105964:leucyl-cystinyl aminopeptidase; D020727:ADP-Ribosylation Factors; D007930:Leucine; D000409:Alanine