p115 Interacts with the GLUT4 vesicle protein, IRAP, and plays a critical role in insulin-stimulated GLUT4 translocation.
Insulin-regulated aminopeptidase (IRAP) is an abundant cargo protein of Glut4 storage vesicles (GSVs) that traffics to and from the plasma membrane in response to insulin. We used the amino terminus cytoplasmic domain of IRAP, residues 1-109, as an affinity reagent to identify cytosolic proteins that might be involved in GSV trafficking. In this way, we identified p115, a peripheral membrane protein known to be involved in membrane trafficking. In murine adipocytes, we determined that p115 was localized to the perinuclear region by immunofluorescence and throughout the cell by fractionation. By immunofluorescence, p115 partially colocalizes with GLUT4 and IRAP in the perinuclear region of cultured fat cells. The amino terminus of p115 binds to IRAP and overexpression of a N-terminal construct results in its colocalization with GLUT4 throughout the cell. Insulin-stimulated GLUT4 translocation is completely inhibited under these conditions. Overexpression of p115 C-terminus has no significant effect on GLUT4 distribution and translocation. Finally, expression of the p115 N-terminus construct has no effect on the distribution and trafficking of GLUT1. These data suggest that p115 has an important and specific role in insulin-stimulated Glut4 translocation, probably by way of tethering insulin-sensitive Glut4 vesicles at an as yet unknown intracellular site.
Hosaka
Toshio
T
Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA.
Brooks
Cydney C
CC
Presman
Eleonora
E
Kim
Suk-Kyeong
SK
Zhang
Zidong
Z
Breen
Michael
M
Gross
Danielle N
DN
Sztul
Elizabeth
E
Pilch
Paul F
PF
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000378:metabolism; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D019556:COS Cells; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002522:Chlorocebus aethiops; D004926:Escherichia coli / Q000235:genetics; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D017434:Protein Structure, Tertiary; D013347:Subcellular Fractions / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism*
D007328:Insulin; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D000626:Aminopeptidases