GIULIO INNAMORATI*, HAMID M. SADEGHI*, NATHANIEL T. TRAN*, AND MARIEL BIRNBAUMER†‡
*Department of Anesthesiology and †Molecular Biology Institute, University of California School of Medicine, Los Angeles, CA 90095
Communicated by Lutz Birnbaumer, University of California School of Medicine, Los Angeles, CA, December 31, 1997 (received for review
December 22, 1997)
*Department of Anesthesiology and †Molecular Biology Institute, University of California School of Medicine, Los Angeles, CA 90095
Communicated by Lutz Birnbaumer, University of California School of Medicine, Los Angeles, CA, December 31, 1997 (received for review
December 22, 1997)
ABSTRACT :Receptor recycling plays a critical role in the regulation of cellular responsiveness to environmental stimuli.
Agonist-promoted phosphorylation of G protein-coupled receptors has been related to their desensitization, internalization, and sequestration. Dephosphorylation of internalized
G protein-coupled receptors 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. The
internalized V2 vasopressin receptor (V2R) expressed in HEK 293 cells is an exception to this hypothesis because it does not recycle to the plasma membrane for hours after removal of the
ligand. Because this receptor is phosphorylated only by G protein-coupled receptor kinases (GRKs), the relationship between recycling and GRK-mediated phosphorylation was
examined. A nonphosphorylated V2R, truncated upstream of the GRK phosphorylation sites, rapidly returned to the cell surface after removal of vasopressin. Less-drastic truncations
of V2R revealed the presence of multiple phosphorylation sites and suggested a key role for a serine cluster present at the C terminus. Replacement of any one of Ser-362, Ser-363, or
Ser-364 with Ala allowed quantitative recycling of full-length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the
recycling S363A mutant V2Rs revealed that the recycling receptor was dephosphorylated after hormone withdrawal, whereas the wild-type V2R was not, providing molecular
evidence for the hypothesis that GRK sites must be dephosphorylated prior to receptor recycling. These experiments uncovered a role for GRK phosphorylation in intracellular
sorting and revealed a GRK-dependent anchoring domain that blocks V2R recycling.
Agonist-promoted phosphorylation of G protein-coupled receptors has been related to their desensitization, internalization, and sequestration. Dephosphorylation of internalized
G protein-coupled receptors 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. The
internalized V2 vasopressin receptor (V2R) expressed in HEK 293 cells is an exception to this hypothesis because it does not recycle to the plasma membrane for hours after removal of the
ligand. Because this receptor is phosphorylated only by G protein-coupled receptor kinases (GRKs), the relationship between recycling and GRK-mediated phosphorylation was
examined. A nonphosphorylated V2R, truncated upstream of the GRK phosphorylation sites, rapidly returned to the cell surface after removal of vasopressin. Less-drastic truncations
of V2R revealed the presence of multiple phosphorylation sites and suggested a key role for a serine cluster present at the C terminus. Replacement of any one of Ser-362, Ser-363, or
Ser-364 with Ala allowed quantitative recycling of full-length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the
recycling S363A mutant V2Rs revealed that the recycling receptor was dephosphorylated after hormone withdrawal, whereas the wild-type V2R was not, providing molecular
evidence for the hypothesis that GRK sites must be dephosphorylated prior to receptor recycling. These experiments uncovered a role for GRK phosphorylation in intracellular
sorting and revealed a GRK-dependent anchoring domain that blocks V2R recycling.
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