Xiao Kunhong, Shenoy Sudha K, Nobles Kelly, Lefkowitz Robert J
Department of Medicine, Duke University Medical Center, Durham, NC 27710, USA.
J Biol Chem. 2004 Dec 31;279(53):55744-53. doi: 10.1074/jbc.M409785200. Epub 2004 Oct 22.
Beta-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 beta-arrestin 1 (arrestin 2) have been resolved. However, little is known about the conformational changes that occur in beta-arrestins upon binding to the activated phosphorylated receptor. Here we characterize the conformational changes in beta-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 beta-arrestin 2 in the presence of a phosphopeptide (V(2)R-pp) derived from the C terminus of the vasopressin type II receptor (V(2)R) or the corresponding nonphosphopeptide (V(2)R-np). V(2)R-pp binds to beta-arrestin 2 specifically, whereas V(2)R-np does not. Activation of beta-arrestin 2 upon V(2)R-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 Arg(394), and rearrangement of its N terminus, as indicated by the shielding of a previously accessible cleavage site, residue Arg(8). Interestingly, binding of the polyanion heparin also leads to release of the C terminus of beta-arrestin 2; however, heparin and V(2)R-pp have different binding site(s) and/or induce different conformational changes in beta-arrestin 2. Release of the C terminus from the rest of beta-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 beta-arrestin 2 by 10-fold. Thus, the V(2)R-pp can activate beta-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 beta-arrestin 2 from its basal inactive conformation to its biologically active conformation and establish a system in which receptor-beta-arrestin interactions can be modeled in vitro.
β-抑制蛋白是多功能衔接蛋白，介导七跨膜受体（7MSR）的脱敏、内吞作用及其他信号转导途径。视觉抑制蛋白（抑制蛋白1）和β-抑制蛋白1（抑制蛋白2）的基础无活性状态的晶体结构已得到解析。然而，关于β-抑制蛋白与活化的磷酸化受体结合后发生的构象变化却知之甚少。在此，我们通过比较在存在源自血管加压素II型受体（V(2)R）C末端的磷酸肽（V(2)R-pp）或相应非磷酸肽（V(2)R-np）的情况下β-抑制蛋白2的有限胰蛋白酶消化模式和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）图谱，来表征β-抑制蛋白2（抑制蛋白3）的构象变化。V(2)R-pp特异性结合β-抑制蛋白2，而V(2)R-np则不然。V(2)R-pp结合后β-抑制蛋白2的活化涉及其C末端的释放，这表现为一个先前无法接近的切割位点暴露，极性核心残基之一精氨酸394暴露，以及其N末端的重排，这表现为一个先前可接近的切割位点精氨酸8被屏蔽。有趣的是，多聚阴离子肝素的结合也会导致β-抑制蛋白2的C末端释放；然而，肝素和V(2)R-pp在β-抑制蛋白2上具有不同的结合位点和/或诱导不同的构象变化。β-抑制蛋白2的C末端从其余部分释放具有功能后果，即它增加了网格蛋白结合位点（先前证明位于残基371和379之间）的可及性，从而使网格蛋白与β-抑制蛋白2的结合增强了10倍。因此，V(2)R-pp能够在体外激活β-抑制蛋白2，很可能模拟了活化的磷酸化7MSR的作用。这些结果提供了与β-抑制蛋白2从其基础无活性构象转变为其生物活性构象相关的构象变化的首个直接证据，并建立了一个可以在体外模拟受体-β-抑制蛋白相互作用的系统。
