Nobles Kelly N, Guan Ziqiang, Xiao Kunhong, Oas Terrence G, Lefkowitz Robert J
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2007 Jul 20;282(29):21370-81. doi: 10.1074/jbc.M611483200. Epub 2007 May 18.
beta-Arrestins are multifunctional adaptor proteins that regulate seven transmembrane-spanning receptor (7TMR) desensitization and internalization and also initiate alternative signaling pathways. Studies have shown that beta-arrestins undergo a conformational change upon interaction with agonist-occupied, phosphorylated 7TMRs. Although conformational changes have been reported for visual arrestin and beta-arrestin2, these studies are not representative of conformational changes in beta-arrestin1. Accordingly, in this study, we determine conformational changes in beta-arrestin1 using limited tryptic proteolysis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis in the presence of a phosphopeptide derived from the C terminus of the V(2) vasopressin receptor (V(2)Rpp) or the corresponding unphosphorylated peptide (V(2)Rnp). V(2)Rpp binds specifically to beta-arrestin1 causing significant conformational changes, whereas V(2)Rnp does not alter the conformation of beta-arrestin1. Upon V(2)Rpp binding, we show that the previously shielded Arg(393) becomes accessible, which indicates release of the C terminus. Moreover, we show that Arg(285) becomes more accessible, and this residue is located in a region of beta-arrestin1 responsible for stabilization of its polar core. These two findings demonstrate "activation" of beta-arrestin1, and we also show a functional consequence of the release of the C terminus of beta-arrestin1 by enhanced clathrin binding. In addition, we show marked protection of the N-domain of beta-arrestin1 in the presence of V(2)Rpp, which is consistent with previous studies suggesting the N-domain is responsible for recognizing phosphates in 7TMRs. A striking difference in conformational changes is observed in beta-arrestin1 when compared with beta-arrestin2, namely the flexibility of the interdomain hinge region. This study represents the first direct evidence that the "receptor-bound" conformations of beta-arrestins1 and 2 are different.
β-抑制蛋白是多功能衔接蛋白，可调节七次跨膜受体（7TMR）的脱敏和内化，还能启动替代信号通路。研究表明，β-抑制蛋白在与激动剂占据的、磷酸化的7TMR相互作用时会发生构象变化。尽管已报道视觉抑制蛋白和β-抑制蛋白2会发生构象变化，但这些研究并不代表β-抑制蛋白1的构象变化。因此，在本研究中，我们在存在源自血管加压素2型受体（V2R）C末端的磷酸肽（V2Rpp）或相应的未磷酸化肽（V2Rnp）的情况下，使用有限胰蛋白酶解和基质辅助激光解吸电离飞行时间质谱分析来确定β-抑制蛋白1的构象变化。V2Rpp特异性结合β-抑制蛋白1，导致显著的构象变化，而V2Rnp不会改变β-抑制蛋白1的构象。在V2Rpp结合后，我们发现先前被屏蔽的精氨酸393变得可及，这表明C末端被释放。此外，我们发现精氨酸285变得更易接近，该残基位于β-抑制蛋白1中负责稳定其极性核心的区域。这两个发现证明了β-抑制蛋白1的“激活”，并且我们还通过增强网格蛋白结合展示了β-抑制蛋白1的C末端释放的功能后果。此外，我们发现在V2Rpp存在的情况下，β-抑制蛋白1的N结构域受到显著保护，这与先前的研究一致，表明N结构域负责识别7TMR中的磷酸基团。与β-抑制蛋白2相比，β-抑制蛋白1的构象变化存在显著差异，即结构域间铰链区的灵活性。本研究首次直接证明了β-抑制蛋白1和2的“受体结合”构象不同。
