Author
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HANEY, PETER - BAYLOR COLL MEDICINE |
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Submitted to: Pediatric Research
Publication Type: Abstract Only Publication Acceptance Date: 12/1/2003 Publication Date: 5/3/2003 Citation: HANEY, P. A LACTOGENIC HORMONE-SENSITIVE TARGETING DETERMINANT OF THE GLUT1 GLUCOSE TRANSPORTER IS CONTAINED IN A SEVEN AMINO ACID SEQUENCE OF ITS CYTOPLASMIC LOOP. PEDIATRIC RESEARCH. 2003. v. 53. p. 166A. Interpretive Summary: Interpretive Summary not needed for this 115. Technical Abstract: Background: The GLUT1 glucose transporter colocalizes with alpha-lactalbumin in low-density, exquisitely Brefeldin A-sensitive vesicles of lactating mammary epithelial cells. Lactogenic hormones appear to mediate the redistribution of GLUT1 from the plasma membrane to this unique intracellular compartment, thereby supplying substrate to the site of lactose synthesis. Objective: We hypothesized that the structural basis for this targeting behavior resides in an intracellular portion of GLUT1. Design/Methods: Wild-type and mutant GLUT1 cDNAs were subcloned into phrGFP-N1 so that fusion proteins consisting of wild-type or mutant GLUT1 and the enhanced green fluorescent protein (EGFP) could be expressed and their targeting defined by confocal microscopy. Eighteen mutant GLUT1-EGFPs were prepared by site-directed mutagenesis, producing systematic deletion of 6-8 consecutive amino acids at a time, beginning with the intracellular N-terminal tail and proceeding to the cytoplasmic loop, and finally the intracellular C-terminal tail. Mutations of the extracellular loop or transmembrane domains were not studied. Results: In primary mouse mammary epithelial cells exposed to insulin, prolactin and hydrocortisone, wild-type GLUT1-EGFP colocalized with endogenous GLUT1, validating the use of this system to visualize targeting of GLUT1 mutants. Of the 18 mutants examined, 13 colocalized with GLUT1. Four mutants showed altered intracellular targeting. One mutant showed a shift to predominant plasma membrane distribution, with little intracellular targeting. This mutant lacks the amino acids QMMREKK in the cytoplasmic loop. Swapping the corresponding amino acids from GLUT3, a plasma membrane protein, into the deletion site did not restore intracellular targeting, suggesting that the QMMREKK deletion does not alter GLUT1 targeting by gross structural changes, but rather by removal of a specific targeting signal. Conclusions: A seven amino acid sequence of the cytoplasmic loop of GLUT1, QMMREKK, contains targeting determinants essential to the lactogenic hormone-regulated redistribution of GLUT1 during lactation. This seven amino acid sequence is found in no other protein. Further definition of the minimum requirements for authentic targeting and identification of the mechanism by which this targeting signal is recognized should increase our understanding of the regulation of lactose synthesis and milk production. |
