Insulin-responsive Aminopeptidase Trafficking in 3T3-L1 Adipocytes*
- From the Howard Hughes Medical Institute, The Cox Institute, and the Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Abstract
The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3–5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane “sheets,” GLUT4 and IRAP showed similar internalization rates that are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.
Footnotes
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↵* This work was supported by National Institutes of Health Grants DK39615 (to M. J. B.) and MSTP 5-T32-GM-07170 (to L. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Pennsylvania Medical School, Clinical Research Bldg., 415 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-898-5001; Fax: 215-573-9138; E-mail: birnbaum@hhmi.upenn.edu.
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↵2 L. A. Garza and M. J. Birnbaum, unpublished observations.
- Abbreviations:
- GLUT
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glucose transporter
- IRAP
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insulin responsive aminopeptidase
- BSA
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bovine serum albumin
- HRP
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horseradish peroxidase
- NHS
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N-hydroxysuccinimide
- PM
-
plasma membrane
- LDM
-
low density microsomes
- LC
-
long chain
- ABTS
-
2,2′-azine-di[3-ethylbenzthiazoline sulfonate]
- CHO
-
Chinese hamster ovary
- MES
-
4-morpholineethanesulfonic acid
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- Received October 26, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
