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. 1998 Mar 3;95(5):2222-6.
doi: 10.1073/pnas.95.5.2222.

A serine cluster prevents recycling of the V2 vasopressin receptor

G Innamorati  1 , H M Sadeghi, N T Tran, M Birnbaumer
Affiliations

A serine cluster prevents recycling of the V2 vasopressin receptor

G Innamorati et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
Figure 1
Time course of receptor recycling. After AVP-promoted sequestration, the hormone was removed by acid wash and the sites on the cell surface were measured before and after incubation at 37°C. In all experiments the expression of the mutant receptors was determined by performing binding assays with a known number of intact cells. The number of receptors expressed by the different constructs as percent of wild type (WT) were 102 ± 10% for the V2–V1a chimera, 33 ± 5% for 345t, 86 ± 11% for 369t, 88 ± 5% for 362t, 102 ± 7% for S362A, 99 ± 12% for S363A, and 94 ± 8% for S362A V2R. (A) Recycling of WT (n = 16), V2–V1a chimera (n = 3), and 345t mutant (n = 6) receptors. (B) Recycling of WT (n = 16), 369t (n = 4), and 362t (n = 3) mutant receptors. (C) Recycling of WT (n = 16) and three mutant V2Rs carrying a single Ala substitution in the Ser cluster at positions 362 (n = 3), 363 (n = 3), or 364 (n = 3). Numbers in parentheses specify number of experiments performed.
Figure 2
Figure 2
Schematic representation of the V2R and amino acid composition of the C terminus. The amino acids downstream of the palmitoylation sites of the V2R, Cys-341 and Cys-342 are identified (10). The residues on a black background indicate the sites substituted by a stop codon to produce the truncated receptors in boldface type. The residues represented by pentagons indicate single amino acid substitutions that were introduced to obtain the constructs in italic type.
Figure 3
Figure 3
Phosphorylation of wild-type and mutant V2Rs. The phosphate incorporated into the proteins in response to 100 nM AVP was quantified in the immunoprecipitated receptors for the WT, 369t, 362t, and S363A mutant receptors. (A) Autoradiographic image of WT, 369t, and 362t phosphorylated V2Rs in a representative experiment. (B) Summary of the quantification of receptor phosphorylation. S363A V2R incorporated 88 ± 12%, 369t incorporated 60 ± 8%, and 362t incorporated 33 ± 7% of the phosphate incorporated by WT V2R. The values for each construct are expressed as percentage (mean ± SEM, n = 4) of the phosphate incorporated into the wild-type V2R after normalization for the number of binding sites detected with each construct.
Figure 4
Figure 4
Time course of receptor dephosphorylation. After 100 nM AVP promoted phosphorylation, excess hormone was removed by acid wash, and the cells were allowed to recover. The amount of radioactive phosphate present in each receptor protein was quantified at the indicated times by immunoprecipitating receptors from detergent cell extracts. Results obtained for wild-type and S363A mutant receptors are shown. The values for each construct are expressed as the mean ± SEM of the percent phosphate incorporated in the immunoprecipitated receptor before the recovery. Number of experiments were n = 7 for −20 min (untreated) and time 0, n = 3 for 30 min, n = 4 for 60 min, and n = 5 for 120 min of recycling.
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