Abstract
Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.
Highlights
Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors
Constitutive phosphorylation occurs at Ser348, while ligand-induced phosphorylation is found at Ser339 and Ser346/Ser348 that could be executed by several G protein-coupled receptor kinases
Reagents—[3H]Bradykinin (1.48 – 4.07 TBq/mmol), [32P]orthophosphate (360 MBq/ml), and myo-[3H]inositol (2.96 – 4.44 TBq/mmol) were from Amersham; bradykinin was from Bachem; aprotinin (TrasylolTM) was from Bayer; AG-X8 anion exchanger resin was from Bio-Rad; GF109203X and PMA were from Calbiochem; bacitracin and PefablocTM were from FLUKA; FR190997 was a kind gift from Fujisawa Pharmaceutical Co., LipofectAMINETM and protein markers were from Life Technologies; cellulose thin layer chromatography (TLC) plates were from Merck; sequencing grade trypsin was from Promega; leupeptin was from Roche Molecular Bioscience; nitrocellulose membranes were from Schleicher & Schuell; phosphoamino acid standards were from Sigma; and protein A-agarose was from Zymed Laboratories Inc
Summary
Reagents—[3H]Bradykinin (1.48 – 4.07 TBq/mmol), [32P]orthophosphate (360 MBq/ml), and myo-[3H]inositol (2.96 – 4.44 TBq/mmol) were from Amersham; bradykinin was from Bachem; aprotinin (TrasylolTM) was from Bayer; AG-X8 anion exchanger resin was from Bio-Rad; GF109203X and PMA were from Calbiochem; bacitracin and PefablocTM were from FLUKA; FR190997 was a kind gift from Fujisawa Pharmaceutical Co., LipofectAMINETM and protein markers were from Life Technologies; cellulose thin layer chromatography (TLC) plates were from Merck; sequencing grade trypsin was from Promega; leupeptin was from Roche Molecular Bioscience; nitrocellulose membranes were from Schleicher & Schuell; phosphoamino acid standards were from Sigma; and protein A-agarose was from Zymed Laboratories Inc. Cells were washed twice to remove free ligand, and the cell-bound [3H]bradykinin was extracted with 0.2 M acetic acid, pH 2.8, 0.5 M NaCl, 0.2% bovine serum albumin, and radioactivity of the extract was measured (“surface-associated bradykinin”). Cells grown on 24-well plates were labeled with 1 Ci/ml myo-[3H]inositol for 24 h in inositolfree Ham’s F-12 including 0.1% (w/v) bovine serum albumin, treated for 5 min with 10 mM LiCl and challenged with 0.01–1 M bradykinin for 10 min in the presence of 10 mM LiCl. Reactions were stopped by addition of 1 ml of ice-cold 10 mM formic acid. Following removal of excess of ligand by washing cells three times with medium, 10 mM LiCl was added 10 or 15 min after the initial stimulation and inositol phosphate levels were measured as described above. The prediction was verified by in vitro mutagenesis of corresponding phosphoacceptor sites followed by two-dimensional phosphopeptide mapping
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