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首页> 美国卫生研究院文献>Elsevier Sponsored Documents >Fluorescence correlation spectroscopy combined with bimolecular fluorescence complementation reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors
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Fluorescence correlation spectroscopy combined with bimolecular fluorescence complementation reveals the effects of β-arrestin complexes and endocytic targeting on the membrane mobility of neuropeptide Y receptors

机译：荧光相关光谱结合双分子荧光互补揭示了β-arrestin复合物和内吞靶向对神经肽Y受体膜迁移率的影响

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Fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis are powerful ways to study mobility and stoichiometry of G protein coupled receptor complexes, within microdomains of single living cells. However, relating these properties to molecular mechanisms can be challenging. We investigated the influence of β-arrestin adaptors and endocytosis mechanisms on plasma membrane diffusion and particle brightness of GFP-tagged neuropeptide Y (NPY) receptors. A novel GFP-based bimolecular fluorescence complementation (BiFC) system also identified Y1 receptor-β-arrestin complexes. Diffusion co-efficients (D) for Y1 and Y2-GFP receptors in HEK293 cell plasma membranes were 2.22 and 2.15 × 10− 9 cm2 s− 1 respectively. At a concentration which promoted only Y1 receptor endocytosis, NPY treatment reduced Y1-GFP motility (D 1.48 × 10− 9 cm2 s− 1), but did not alter diffusion characteristics of the Y2-GFP receptor. Agonist induced changes in Y1 receptor motility were inhibited by mutations (6A) which prevented β-arrestin recruitment and internalisation; conversely they became apparent in a Y2 receptor mutant with increased β-arrestin affinity. NPY treatment also increased Y1 receptor-GFP particle brightness, changes which indicated receptor clustering, and which were abolished by the 6A mutation. The importance of β-arrestin recruitment for these effects was illustrated by reduced lateral mobility (D 1.20–1.33 × 10− 9 cm2 s− 1) of Y1 receptor-β-arrestin BiFC complexes. Thus NPY-induced changes in Y receptor motility and brightness reflect early events surrounding arrestin dependent endocytosis at the plasma membrane, results supported by a novel combined BiFC/FCS approach to detect the underlying receptor-β-arrestin signalling complex.
机译：荧光相关光谱（FCS）和光子计数直方图（PCH）分析是研究单个活细胞微域内G蛋白偶联受体复合物的迁移率和化学计量的有效方法。然而，将这些性质与分子机制联系起来可能具有挑战性。我们研究了β-arrestin衔接子和胞吞机制对GFP标记的神经肽Y（NPY）受体的质膜扩散和颗粒亮度的影响。一个新颖的基于GFP的双分子荧光互补（BiFC）系统还确定了Y1受体-β-抑制蛋白复合物。 HEK293细胞质膜中Y1和Y2-GFP受体的扩散系数（D）为2.22和2.15×10 −9 cm 2 s -1 < / sup>。在仅促进Y1受体胞吞作用的浓度下，NPY处理降低了Y1-GFP的运动能力（D 1.48×10 −9 cm 2 s −1 ），但不会改变Y2-GFP受体的扩散特性。激动剂诱导的Y1受体运动性改变被阻止β-arrestin募集和内在化的突变（6A）所抑制；相反，它们在具有增加的β-arrestin亲和力的Y2受体突变体中变得明显。 NPY处理还增加了Y1受体-GFP颗粒的亮度，这种变化表明受体聚集，并且被6A突变所消除。 β-抑制蛋白募集对于这些作用的重要性通过降低的横向活动性来说明（D 1.20–1.33×10 −9 cm 2 s −1 ）Y1受体-β-arrestinBiFC复合物。因此，NPY诱导的Y受体运动性和亮度变化反映了围绕质膜处抑制蛋白依赖性内吞作用的早期事件，这一结果得到了新颖的结合BiFC / FCS方法检测潜在的受体β-arrestin信号复合物的支持。

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