Activation-dependent Conformational Changes in β-Arrestin 2*
Activation-dependent Conformational Changes in β-Arrestin 2*
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DOI：
10.1074/jbc.m409785200
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发表时间：
2004-12
影响因子：
4.8
通讯作者：
K. Xiao;S. Shenoy;K. Nobles;R. Lefkowitz
中科院分区：
文献类型：
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作者：
K. Xiao;S. Shenoy;K. Nobles;R. Lefkowitz
β-Arrestins是一种多功能的衔接蛋白，介导7种跨膜受体（7 MSRs）的脱敏、内吞和交替信号通路。视觉抑制蛋白（抑制蛋白1）和β-抑制蛋白1（抑制蛋白2）的基础非活性状态的晶体结构已经解析。然而，很少有人知道β-arrestins结合激活的磷酸化受体后发生的构象变化。本文通过比较β-arrestin 2在加压素II型受体（V2 R）C端衍生的磷酸肽（V2 R-pp）或相应的非磷酸肽（V2 R-np）存在下的有限胰蛋白酶蛋白水解模式和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）谱来表征β-arrestin 2（arrestin 3）的构象变化。V2 R-pp特异性结合β-arrestin 2，而V2 R-np不结合。β-抑制蛋白2在V2 R-pp结合后的活化涉及其C末端的释放，如通过暴露先前不可接近的切割位点（极性核心残基之一Arg 394）所指示的，以及其N末端的重排，如通过屏蔽先前可接近的切割位点（残基Arg 8）所指示的。有趣的是，聚阴离子肝素的结合也导致β-抑制蛋白2的C末端释放;然而，肝素和V2 R-pp具有不同的结合位点和/或诱导β-抑制蛋白2的不同构象变化。从β-抑制蛋白2的其余部分释放C末端具有功能性后果，因为其增加网格蛋白结合位点（先前证明位于残基371和379之间）的可及性，从而将网格蛋白与β-抑制蛋白2的结合增强10倍。因此，V2 R-pp可以在体外激活β-抑制蛋白2，最有可能模拟激活的磷酸化7 MSR的作用。这些结果提供了与β-arrestin 2从其基本无活性构象转变为其生物活性构象相关的构象变化的第一个直接证据，并建立了一个可以在体外模拟受体-β-arrestin相互作用的系统。
β-Arrestins are multifunctional adaptor proteins, which mediate desensitization, endocytosis, and alternate signaling pathways of seven membrane-spanning receptors (7MSRs). Crystal structures of the basal inactive state of visual arrestin (arrestin 1) and β-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in β-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in β-arrestin 2 (arrestin 3) by comparing the limited tryptic proteolysis patterns and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) profiles of β-arrestin 2 in the presence of a phosphopeptide (V2R-pp) derived from the C terminus of the vasopressin type II receptor (V2R) or the corresponding nonphosphopeptide (V2R-np). V2R-pp binds to β-arrestin 2 specifically, whereas V2R-np does not. Activation of β-arrestin 2 upon V2R-pp binding involves the release of its C terminus, as indicated by exposure of a previously inaccessible cleavage site, one of the polar core residues Arg394, and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg8. Interestingly, binding of the polyanion heparin also leads to release of the C terminus of β-arrestin 2; however, heparin and V2R-pp have different binding site(s) and/or induce different conformational changes in β-arrestin 2. Release of the C terminus from the rest of β-arrestin 2 has functional consequences in that it increases the accessibility of a clathrin binding site (previously demonstrated to lie between residues 371 and 379) thereby enhancing clathrin binding to β-arrestin 2 by 10-fold. Thus, the V2R-pp can activate β-arrestin 2 in vitro, most likely mimicking the effects of an activated phosphorylated 7MSR. These results provide the first direct evidence of conformational changes associated with the transition of β-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-β-arrestin interactions can be modeled in vitro.