| 1. |
- Blaukat, Andree, et al.
(författare)
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Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:44, s. 40431-40440
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Pizard, Anne, et al.
(författare)
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Palmitoylation of the human bradykinin B2 receptor influences ligand efficacy.
- 2001
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Ingår i: Biochemistry. - 0006-2960. ; 40:51, s. 15743-51
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Tidskriftsartikel (refereegranskat)abstract
- To investigate the palmitoylation of the human bradykinin B2 receptor, we have mutated individually or simultaneously into glycine two potential acylation sites (cysteines 324 and 329) located in the carboxyl terminus of the receptor and evaluated the effects of these mutations by transfection in COS-7, CHO-K1, and HEK 293T. The wild-type receptor and the single mutants, but not the double mutant, incorporated [3H]palmitate, indicating that the receptor carboxyl tail can be palmitoylated at both sites. The mutants did not differ from the wild-type receptor for the kinetics of [3H]bradykinin binding, the basal and bradykinin-stimulated coupling to phospholipases C and A2, and agonist-induced phosphorylation. The nonpalmitoylated receptor had a 30% reduced capacity to internalize [3H]bradykinin. This indicates that palmitoylation does not influence the basal activity of the receptor and its agonist-driven activation. However, the mutants triggered phospholipid metabolism and MAP kinase activation in response to B2 receptor antagonists. Pseudopeptide and nonpeptide compounds that behaved as antagonists on the wild-type receptor became agonists on the nonpalmitoylated receptor and produced phospholipases C and A2 responses of 25-50% as compared to that of bradykinin. These results suggest that palmitoylation is required for the stabilization of the receptor-ligand complex in an uncoupled conformation.
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