The Carboxyl Terminus of GLUT4 Contains a Serine-Leucine-Leucine Sequence That Functions as a Potent Internalization Motif in Chinese Hamster Ovary Cells*
- From the Department of Pathology and
- ‡ Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, New York 10032
- ¶ Junior Investigator of the American Heart Association, NYC Affiliate. To whom correspondence should be addressed. Tel.: 212-305-8545; Fax: 212-305-5498; E-mail: TEM2{at}Columbia.edu.
Abstract
To characterize the trafficking motifs contained in the carboxyl terminus of GLUT4, a chimera (GTCTR) was constructed in which the carboxyl-terminal 30 amino acids of GLUT4 were substituted for the amino-terminal cytoplasmic domain of the transferrin receptor (TR). The endocytic behavior of this chimera was characterized in Chinese hamster ovary cells. The GTCTR chimera had a more predominant intracellular distribution compared to the TR. Only 20% of the GTCTR chimera is on the surface at steady-state compared to 35% of the TR. The GTCTR chimera is internalized 50% more rapidly and recycled 20% more slowly than the TR. Acidification of the cytosol inhibited internalization of the GTCTR chimera, indicating that the chimera is internalized through clathrin-coated pits. Mutations of GTCTR were constructed in which a di-leucine sequence of the carboxyl domain of GLUT4 was mutated to a di-alanine sequence (GTCTR-AA) and serine residue 488, immediately preceding the di-leucine sequence, was mutated to either an alanine or aspartate residue. In each case, albeit to varying degrees, the substitutions shifted the distribution of the mutated GTCTR constructs toward the surface. The shift in the distribution of GTCTR-AA resulted from a 10-fold reduction in internalization, and the shift of serine 488 mutants resulted from a 3-fold reduction in the internalization rate compared to GTCTR. None of these mutations affected the recycling rate. These results demonstrate that the carboxyl terminus of GLUT4 contains a serine-leucine-leucine-based motif that, when expressed in non-insulin responsive cells, functions as a potent internalization motif which promotes more rapid internalization than does the native TR internalization motif.
Footnotes
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↵§ Contributed equally to the results in this report.
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↵* This work was supported by research grants from the American Cancer Society and the Council for Tobacco Research. 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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↵1 The abbreviations used are:
- TR
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transferrin receptor
- Tf
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transferrin
- CHO
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Chinese hamster ovary
- GTCTR
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cytoplasmic domain of the TR
- PCR
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polymerase chain reaction
- MES
-
4-morpholineethanesulfonic acid
- wt
-
wild-type.
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- Received November 13, 1995.
- Revision received April 11, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
