1998 Global Conference Proceeding
March 02 - 04, 1998
| Conference: | 1998 Global Conference |
|---|---|
| Title: | Recycling of the V2 Vasopressin Receptor |
| Authors: | Innamorati, Giulio; Sadeghi, Hamid; Birnbaumer, Mariel |
| Institutions: | Vita Salute University School of Medicine, University of Isfahan, National Institute of Environmental Health Sciences |
Agonist-promoted phosphorylation of G protein coupled receptors (GPCR) has been related to their desensitization, internalization, and sequestration. Recently the dephosphorylation of internalized GPCR by cytoplasmic phosphatases has been shown to be pH dependent, and it has been postulated to be necessary for receptors to recycle to the cell surface. Phosphorylation of the V2 vasopressin receptor (V2R) was shown to be dependent of occupancy by agonist, and catalyzed by G protein coupled receptor kinases but not by other protein kinases. Phosphorylation was not required for V2 receptor internalization, although it modulated this process. Opposite to what has been described for other receptors, the internalized V2R expressed in HEK 293 cells did not recycle to the cell surface after removal of the ligand. Since this receptor is phosphorylated only by GRKs, the relationship between recycling and GRK mediated phosphorylation was examined. Truncation of the V2R upstream of the GRK phosphorylation sites yielded a non-phosphorylated V2R that returned to the cell surface after removal of vasopressin. Less drastic progressive truncations of the protein revealed the presence of multiple phosphorylation sites in the V2R, and suggested a role for a serine cluster present at the carboxy terminus in receptor retention. Substitution by alanine of any one of serines 362, 363 or 364 allowed quantitative recycling of full length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the recycling mutant V2Rs revealed that the recycling of the receptor was associated with dephosphorylation after hormone withdrawal. These experiments provide molecular evidence for the hypothesis that GRK sites must be de-phosphorylated prior to receptor recycling. The lack of recycling of the V2R has been observed in transient and stably transfected COS and MDCK cells suggesting that it is a receptor dependent not a cell dependent phenomenon. Whether the principal cell of the collecting duct handle the ligand-bound V2 receptor in a similar manner is currently under investigation.
This work was supported in part by NIH Grant DK 41-244 to MB.



