Molecular and conformational features of a transport-relevant domain in the C-terminal tail of the vasopressin V-2 receptor

We have previously shown a conserved glutamate/dileucine motif ((ELRSLL340) -E-335) in the intracellular C terminus of the vasopressin V-2 receptor (V2 receptor) to be essential for receptor transport from the endoplasmic reti culum (ER) to the Golgi apparatus. The motif may represent a transport sign al that is recognized by a component of ER to Golgi vesicles. Alternatively , it may be necessary for transport-competent receptor folding to pass the quality-control system of the ER. To assess these two possibilities, we con structed a receptor fragment that allows transport studies independent of f ull-length receptor folding. Transmembrane domains II-VII were deleted, the reby fusing the intracellular C terminus to the first cytoplasmic loop. The mutations that impaired transport of the full-length receptor were introdu ced, and receptor fragments were localized in transiently transfected HEK 2 93 cells. All mutant receptor fragments were detectable at the plasma membr ane, demonstrating that the glutamate/dileucine motif does not function as a small, linear vesicular transport signal. Instead, our data strongly sugg est that this motif is required for transport-competent folding of the full -length receptor. To assess the underlying conformational features, a three -dimensional homology model of the V-2 receptor was computed. Our model pre dicts that the glutamate/dileucine motif contributes to a U-like loop withi n the intracellular C terminus. Residue Leu(339) may be required for foldin g back the intracellular C terminus to residue Leu(62) of the first cytopla smic loop. We characterized the naturally occurring L62P and Delta L62-R64 mutations in the first cytoplasmic loop and show that they lead to transpor t-defective full-length V-2 receptors that are retained in the ER, consiste nt with the structure model.