2000 Global Researcher Conference Proceeding
March 10 - 12, 2000
| Conference: | 2000 Global Researcher Conference |
|---|---|
| Title: | The C-terminus as a determinant for vasopressin receptors sorting |
| Authors: | Innamorati, Giulio; Le Gouill, Christian; Birnbaumer, Mariel |
| Institutions: | Vita Salute University School of Medicine, Universite de Sherbrooke, National Institute of Environmental Health Sciences |
The C-terminus of the V2 vasopressin receptor contains multiple phosphorylation sites; among them, a cluster of phosphorylated amino acids contributes to create a signal that prevents internalized receptor from returning to the cell surface. The retention signal was validated by transferring it to another receptor (V1a) that normally recycles; the resulting chimeric V1a/V2 receptor was blocked intracellularly by the V2 tail. Thus, the entire domain blunting recycling seems confined between the palmytoylation sites and the receptor C-terminus.
In an attempt to define which step decreed the atypical sorting, the intracellular distribution of the internalized V2 and V1a receptors was compared. A physical separation of their endocytic pathways was thus demonstrated. Both receptors co-internalized in small endosomes, but, after one hour in the presence of ligand, the V1a receptor was evenly distributed throughout the cytosol while the V2 receptor accumulated in an aggregation of vesicles in the proximity of the nucleus.
In this large endosomal aggregate, the V2 receptor was co-localized with rab11, a small GTP-binding protein that has been shown to be concentrated in the perinuclear recycling compartment of cultured mammalian cells. Vice versa, no significant co-localization for rab11 and the V1a receptor was observed.
Although at this point no direct correlation could be made between the endosomal distribution and the ability to recycle, the perinuclear recycling compartment could represent the organelle where the V2 receptor is sequestered.
The intracellular tail (C-terminus) of the vasopressin-2 receptor (V2R) contains clusters of amino acids that can be phosphorylated. One such cluster helps create a signal that prevents the V2R from returning to the cell surface once it has been endocytosed. To prove this, the researchers exchanged the C-terminus of the V1a receptor (V1aR), which is normally recycled from the cell interior to the cell surface, with the correspondent portion of the V2 vasopressin receptor. The resulting combination, V1aR with a V2R C-terminus, was retained inside the cell.
The researchers then examined where V1aR and V2R localized when they were internalized within the cell. Both receptors share the same type of vesicles as they leave the surface and move inside the cell. However, after an hour the V1aRs are evenly distributed throughout the cell’s interior whereas the V2Rs accumulated in a cluster of vesicles close to the cell’s nucleus.
The V2R clusters shared this location with Rab 11, a small GTP-binding protein that has been shown to be concentrated in the cell’s recycling compartment located near the nucleus. Rab 11 did not co-localize with the internalized V1aR. The researchers speculate that this recycling compartment is the part of the cell where the V2R is retained once it leaves the cell surface.