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

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
Authors: Schulein, Ralf; Oksche, Alexander; Rosenthal, Walter; Hermosilla, Ricardo; Dehe, Marcel; Wiesner, Burkhard; Krause, Gerd
Publisher: Molecular Pharmacology
Date Published: September 01, 1998
Reference Number: 232
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.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
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For the vasopressin-2 receptor (V2R) to be able to receive the antidiuretic hormone, arginine vasopressin, it must first transport itself from the endoplasmic reticulum (ER) inside the cell to the surface of the cell membrane. The V2R is a string of 371 amino acid residues. (An amino acid loses a water molecule when it links to another amino acid. Since all the amino acids are linked in the V2R protein, they are referred to as residues.) These residues interact with themselves and with other molecular structures to let the V2R perform its functions. The last 44 amino acid residues form a part of the V2R called the carboxy-terminus. Within this sequence of 41 residues there are two leucine residues next to each other; a leucine that is the 339th residue in the V2R sequence, and one that is the 340th. They are preceded by a glutamate residue at position 335.

Schulein, et al., examined these three residues to see if they played a role in transporting the V2R from the ER to the cell surface. They did this by creating mutated V2Rs: one missing both leucine residues and the three residues that preceded them; one that replaced the glutamate residue with glutamine; one that exchanged both leucine residues for isoleucines; one that exchanged an isoleucine for leucine 339, and one that exchanged an isoleucine for leucine 340, and three other mutants that exchanged polar threonine for the leucine as they did for the isoleucine exchange.

The authors injected each mutant V2R into a separate laboratory cell culture. Then they examined the cell cultures to see if the mutant V2Rs bound with the AVP they had introduced into the cell cultures. If they did, it meant the mutant V2Rs could get to the cell surface without the two leucine residues and the glutamate residue.

The results of their tests showed that leucine residue 339 and glutamate residue 335 are both necessary for the V2R to get from the ER to the cell surface. Leucine residue 340 has a minor but significant influence. The authors suggest that glutamate 335 and leucine 339 help fold the V2R into a shape that allows it to escape from the ER.