A DILEUCINE SEQUENCE AND AN UPSTREAM GLUTAMATE RESIDUE IN THE INTRACELLULAR CARBOXYL-TERMINUS OF THE VASOPRESSIN V-2 RECEPTOR ARE ESSENTIALFOR CELL-SURFACE TRANSPORT IN COS.M6 CELLS

Little is known concerning the intracellular transport of the G protei n-coupled receptors (GPCRs). Previous studies suggested a functional r ole for those residues immediately preceding the conserved palmitoylat ed cysteine residues in the intracellular carboxyl termini of some GPC Rs in cell surface transport. For the human vasopressin V-2 receptor, we assessed the significance of a dileucine sequence with an upstream glutamate residue (ELRSLLCC) in mediating cell surface delivery. A ser ies of deletion and point mutants in this region were constructed, and the mutant receptors were expressed in transiently transfected COS.M6 cells. By using [H-3]arginine vasopressin binding assays to intact ce lls and immunofluorescence studies with intact and permeabilized cells , we show that residues E335 (mutant E335Q) and L339 (mutant L339T) ar e obligatory for receptor transport to the plasma membrane. Residue L3 40 has a minor but significant influence. [H-3]Arginine vasopressin bi nding experiments on membranes of lysed cells failed to detect any int racellular binding sites for the transport-deficient mutant receptors, suggesting that residues E335 and L339 participate in receptor foldin g. 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 m utant receptors, supporting this conclusion. These data demonstrate th at the glutamate/dileucine motif of the vasopressin V-2 receptor is cr itical for the escape of the receptor from the endoplasmic reticulum, most presumably by establishing a functional and transport-competent f olding state. A databank analysis revealed that these residues are par t of a conserved region in the GPCR family.