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A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells

Item Type:Article
Title:A dileucine sequence and an upstream glutamate residue in the intracellular carboxyl terminus of the vasopressin V2 receptor are essential for cell surface transport in COS.M6 cells
Creators Name:Schuelein, R. and Hermosilla, R. and Oksche, A. and Dehe, M. and Wiesner, B. and Krause, G. and Rosenthal, W.
Abstract:Little is known concerning the intracellular transport of the G protein-coupled receptors (GPCRs). Previous studies suggested a functional role for those residues immediately preceding the conserved palmitoylated cysteine residues in the intracellular carboxyl termini of some GPCRs in cell surface transport. For the human vasopressin V2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A series of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [3H]arginine vasopressin binding assays to intact cells and immunofluorescence studies with intact and permeabilized cells, we show that residues E335 (mutant E335Q) and L339 (mutant L339T) are obligatory for receptor transport to the plasma membrane. Residue L340 has a minor but significant influence. [3H]Arginine vasopressin binding experiments on membranes of lysed cells failed to detect any intracellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor folding. Studies with green fluorescent protein-tagged receptors demonstrate that the bulk of the mutant receptors E335Q and L339T are trapped in the endoplasmic reticulum. Complex glycosylation was absent in these mutant receptors, supporting this conclusion. These data demonstrate that the glutamate/dileucine motif of the vasopressin V2 receptor is critical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent folding state. A databank analysis revealed that these residues are part of a conserved region in the GPCR family.
Keywords:Amino Acid Sequence, Arginine Vasopressin, Binding Sites, Biological Transport, COS Cells, Cell Membrane, Endoplasmic Reticulum, Glutamic Acid, Leucine, Molecular Sequence Data, Site-Directed Mutagenesis, Point Mutation, Protein Folding, Vasopressin Receptors, Amino Acid Sequence Homology, Transfection, Tritium, Animals
Source:Molecular Pharmacology
ISSN:0026-895X
Publisher:American Society for Pharmacology and Experimental Therapeutics (U.S.A.)
Volume:54
Number:3
Page Range:525-535
Date:September 1998
Official Publication:http://molpharm.aspetjournals.org/cgi/content/abstract/54/3/525
PubMed:View item in PubMed

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