Identification of an Insulin-responsive, Slow Endocytic Recycling Mechanism in Chinese Hamster Ovary Cells*
- Amy O. Johnson‡,
- Agathe Subtil‡,
- Rebecca Petrush‡,
- Keith Kobylarz‡,
- Susanna R. Keller§ and
- Timothy E. McGraw‡¶
- From the ‡Department of Biochemistry, Cornell University Medical College, New York, New York 10021 and the§Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755
Abstract
In adipocytes, the insulin-regulated aminopeptidase (IRAP) is trafficked through the same insulin-regulated recycling pathway as the GLUT4 glucose transporter. We find that a chimera, containing the cytoplasmic domain of IRAP fused to transmembrane and extracellular domains of the transferrin receptor, is slowly recycled and rapidly internalized in Chinese hamster ovary cells. Morphological studies indicate that the chimera is slowly trafficked through the general endosomal recycling compartment rather than being sorted to a specialized recycling pathway. A chimera in which a di-leucine sequence within the cytoplasmic domain of IRAP has been mutated to alanines is rapidly internalized and rapidly recycled, indicating that this di-leucine is required for the slow recycling but not for the rapid internalization. Insulin stimulates a 2–3-fold increase in the recycling of the chimera and only a 1.2-fold increase in the recycling of the transferrin receptor. The effect of insulin on the recycling of the chimera is blocked by wortmannin, a phosphatidylinositol 3′-kinase inhibitor. GTPγS (guanosine 5′-3-O-(thio)triphosphate) increases the recycling of the chimera by 50% but has no effect on the recycling of the transferrin receptor. In these studies, we have identified in Chinese hamster ovary cells a novel, slow endocytic recycling mechanism that is regulated by insulin.
Footnotes
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↵* This work was supported by National Institutes of Health Grant RO1-DK52852 (to T. E. M.), a research grant from Metabolix, Inc. (to T. E. M.), National Institutes of Health Grant RO1-DK25336 (to S. R. K.), and a fellowship from the Charles H. Revson Foundation (to A. S.).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: Dept. of Biochemistry, Cornell University Medical College, 1300 York Ave., New York, NY 10021. Tel.: 212-746-4982; Fax: 212-746-8875; E-mail:temcgraw{at}mail.med.cornell.edu.
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↵1 The abbreviations used are: TR, transferrin receptor; Tf, transferrin; CHO, Chinese hamster ovary; TGN, trans-Golgi network; PCR, polymerase chain reaction; SM, sphingomyelin; TM, transmembrane; GTPγS, guanosine 5′-3-O-(thio)triphosphate; MES, 4-morpholineethanesulfonic acid.
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- Received March 27, 1998.
- Revision received May 7, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
