A single phenylalanine residue in β-arrestin2 critically regulates its binding to G protein-coupled receptors.
A single phenylalanine residue in β-arrestin2 critically regulates its binding to G protein-coupled receptors.
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DOI：
10.1016/j.jbc.2022.101837
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发表时间：
2022-05
影响因子：
4.8
通讯作者：
Shenoy, Sudha K.
中科院分区：
文献类型：
--
作者：
Jean-Charles, Pierre-Yves;Rajiv, Vishwaesh;Sarker, Subhodeep;Han, Sangoh;Bai, Yushi;Masoudi, Ali;Shenoy, Sudha K.
抑制蛋白和它们的酵母同系物，抑制蛋白相关的运输衔接子（ART），共有一段29个氨基酸，称为ART基序。然而，该基序的功能是未知的。我们现在报道，删除这个基序可以阻止激动剂诱导的β-arrestin 2（β-arr 2）泛素化，并阻断其与活化的G蛋白偶联受体（GPCR）的结合。在ART基序内，我们已经鉴定了保守的苯丙氨酸残基Phe 116，其对于β-arr 2-GPCR复合物的形成至关重要。β-arr 2 Phe 116 Ala突变体对β2-肾上腺素能受体诱导的cAMP产生的影响可以忽略不计，而β-arr 2则促进快速脱敏。此外，牛β-arr 2的无活性和肌醇六磷酸6-活化形式的可用结构显示，Phe 116位于疏水口袋中，而相邻的Phe 117和Phe 118残基则不是。Phe 117和Phe 118的突变，而不是Phe 116，保留了β-arr 2的GPCR相互作用。令人惊讶的是，Phe 116不适合β-arr 2与其非GPCR配偶体的缔合。与β-arr 2相比，β-arr 2 Phe 116 Ala突变体的蛋白质半衰期显著缩短，并经历组成型Lys-48连接的多聚泛素化，其标记蛋白质用于蛋白酶体降解。我们还发现Phe 116对于激动剂依赖性β-arr 2泛素化与Lys-63-聚泛素连接是至关重要的，所述连接是蛋白质支架和信号转导的已知介质。最后，我们已经证明，β-arr 2 Phe 116 Ala与活化的β2-肾上腺素能受体的相互作用可以通过泛素的框内融合来拯救。综上所述，我们得出结论，Phe 116保留了β-arr 2的结构稳定性，调节抑制G蛋白信号传导的β-arr 2-GPCR复合物的形成，并促进随后的泛素依赖性β-arr 2定位和运输。
Arrestins and their yeast homologs, arrestin-related trafficking adaptors (ARTs), share a stretch of 29 amino acids called the ART motif. However, the functionality of that motif is unknown. We now report that deleting this motif prevents agonist-induced ubiquitination of β-arrestin2 (β-arr2) and blocks its association with activated G protein–coupled receptors (GPCRs). Within the ART motif, we have identified a conserved phenylalanine residue, Phe116, that is critical for the formation of β-arr2–GPCR complexes. β-arr2 Phe116Ala mutant has negligible effect on blunting β2-adrenergic receptor–induced cAMP generation unlike β-arr2, which promotes rapid desensitization. Furthermore, available structures for inactive and inositol hexakisphosphate 6–activated forms of bovine β-arr2 revealed that Phe116 is ensconced in a hydrophobic pocket, whereas the adjacent Phe117 and Phe118 residues are not. Mutagenesis of Phe117 and Phe118, but not Phe116, preserves GPCR interaction of β-arr2. Surprisingly, Phe116 is dispensable for the association of β-arr2 with its non-GPCR partners. β-arr2 Phe116Ala mutant presents a significantly reduced protein half-life compared with β-arr2 and undergoes constitutive Lys-48-linked polyubiquitination, which tags proteins for proteasomal degradation. We also found that Phe116 is critical for agonist-dependent β-arr2 ubiquitination with Lys-63-polyubiquitin linkages that are known mediators of protein scaffolding and signal transduction. Finally, we have shown that β-arr2 Phe116Ala interaction with activated β2-adrenergic receptor can be rescued with an in-frame fusion of ubiquitin. Taken together, we conclude that Phe116 preserves structural stability of β-arr2, regulates the formation of β-arr2–GPCR complexes that inhibit G protein signaling, and promotes subsequent ubiquitin-dependent β-arr2 localization and trafficking.
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DOI：
10.1073/pnas.0705550104
发表时间：
2007-11-13
影响因子：
11.1
作者：
Boularan, Cedric;Scott, Mark G. H.;Marullo, Stefano
通讯作者：
Marullo, Stefano
影响因子：
4
作者：
Binkowski, Brock F.;Butler, Braeden L.;Wood, Keith V.
通讯作者：
Wood, Keith V.
影响因子：
2.6
作者：
Aubry L;Guetta D;Klein G
通讯作者：
Klein G
影响因子：
3.3
作者：
King, SJ;Brown, CL;Schroer, TA
通讯作者：
Schroer, TA
影响因子：
4.8
作者：
Jean-Charles, Pierre-Yves;Zhang, Lisheng;Shenoy, Sudha K.
通讯作者：
Shenoy, Sudha K.