Light-driven activation of β2-adrenergic receptor signaling by a chimeric rhodopsin containing the β2-adrenergic receptor cytoplasmic loops
Light-driven activation of β2-adrenergic receptor signaling by a chimeric rhodopsin containing the β2-adrenergic receptor cytoplasmic loops
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DOI：
10.1021/bi048328i
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发表时间：
2005-02-22
期刊：
影响因子：
2.9
通讯作者：
Khorana, HG
中科院分区：
文献类型：
--
作者：
Kim, JM;Hwa, J;Khorana, HG
视紫红质的结构-功能研究表明，椎间盘内和跨膜（TM）结构域都需要视网膜结合和随后的光诱导的结构变化的胞质结构域。此外，已经提出了涉及G蛋白偶联受体（GPCR）激活的共同机制的假设。为了检验这一假设，需要嵌合受体，其中视紫红质的胞质结构域被β（2）-肾上腺素能受体（β（2）-AR）的胞质结构域取代。它们的制备需要识别视紫红质TM结构域和β（2）-AR细胞质结构域之间的边界，这是形成视紫红质发色团及其被光激活以及随后β（2）-AR信号传导的最佳激活所必需的。构建嵌合受体，其中视紫红质的细胞质环一次一个地和组合地被替换。在这些替换中，第三胞质（EF）环的大小决定了发色团形成的程度，其稳定性和随后的信号转导特异性。所有的EF环置换均显示转导蛋白活化显著降低，而CD和AB环置换仅观察到轻微影响。仅在EF 2嵌合体中观察到导致Gas信号传导的β（2）-AR的光依赖性激活，并且其激活通过替换其他环而进一步增强。结果表明，光诱导的构象变化之间的耦合发生在视紫红质的跨膜结构域和β（2）-AR的胞质结构域。
Structure-function studies of rhodopsin indicate that both intradiscal and transmembrane (TM) domains are required for retinal binding and subsequent light-induced structural changes in the cytoplasmic domain. Further, a hypothesis involving a common mechanism for activation of G-protein-coupled receptor (GPCR) has been proposed. To test this hypothesis, chimeric receptors were required in which the cytoplasmic domains of rhodopsin were replaced with those of the beta(2)-adrenergic receptor (beta(2)-AR). Their preparation required identification of the boundaries between the TM domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR necessary for formation of the rhodopsin chromophore and its activation by light and subsequent optimal activation of beta(2)-AR signaling. Chimeric receptors were constructed in which the cytoplasmic loops of rhodopsin were replaced one at a time and in combination. In these replacements, size of the third cytoplasmic (EF) loop critically determined the extent of chromophore formation, its stability, and subsequent signal transduction specificity. All the EF loop replacements showed significant decreases in transducin activation, while only minor effects were observed by replacements of the CD and AB loops. Light-dependent activation of beta(2)-AR leading to Gas signaling was observed only for the EF2 chimera, and its activation was further enhanced by replacements of the other loops. The results demonstrate coupling between light-induced conformational changes occurring in the transmembrane domain of rhodopsin and the cytoplasmic domain of the beta(2)-AR.