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 ((335)ELRSLL(340)) in the intracellular C terminus of the vasopressin V(2) receptor (V(2) receptor) to be essential for receptor transport from the endoplasmic reticulum (ER) to the Golgi apparatus. The motif may represent a transport signal 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 constructed a receptor fragment that allows transport studies independent of full-length receptor folding. Transmembrane domains II-VII were deleted, thereby fusing the intracellular C terminus to the first cytoplasmic loop. The mutations that impaired transport of the full-length receptor were introduced, and receptor fragments were localized in transiently transfected HEK 293 cells. All mutant receptor fragments were detectable at the plasma membrane, demonstrating that the glutamate/dileucine motif does not function as a small, linear vesicular transport signal. Instead, our data strongly suggest 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 predicts that the glutamate/dileucine motif contributes to a U-like loop within the intracellular C terminus. Residue Leu(339) may be required for folding back the intracellular C terminus to residue Leu(62) of the first cytoplasmic loop. We characterized the naturally occurring L62P and DeltaL62-R64 mutations in the first cytoplasmic loop and show that they lead to transport-defective full-length V(2) receptors that are retained in the ER, consistent with the structure model.
Krause
G
G
Forschungsinstitut für Molekulare Pharmakologie, Freie Universität Berlin, Berlin, Germany.
Hermosilla
R
R
Oksche
A
A
Rutz
C
C
Rosenthal
W
W
Schülein
R
R
D000595:Amino Acid Sequence; D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017483:Receptors, Vasopressin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
D017483:Receptors, Vasopressin; D018698:Glutamic Acid; D007930:Leucine