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 |
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| 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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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.