Department of Chemistry and Chemical Biology, Indiana University, Indianapolis, Indiana, USA.
Biophys J. 2011 Oct 5;101(7):1642-50. doi: 10.1016/j.bpj.2011.08.040.
Distinct lipid environments, including lipid rafts, are increasingly recognized as a crucial factor affecting membrane protein function in plasma membranes. Unfortunately, an understanding of their role in membrane protein activation and oligomerization has remained elusive due to the challenge of characterizing these often small and transient plasma membrane heterogeneities in live cells. To address this difficulty, we present an experimental model membrane platform based on polymer-supported lipid bilayers containing stable raft-mimicking domains (type I) and homogeneous cholesterol-lipid mixtures (type II) into which transmembrane proteins are incorporated (α(v)β(3) and α(5)β(1) integrins). These flexible lipid platforms enable the use of confocal fluorescence spectroscopy, including the photon counting histogram method, in tandem with epifluorescence microscopy to quantitatively probe the effect of the binding of native ligands from the extracellular matrix ligands (vitronectin and fibronectin for α(v)β(3) and α(5)β(1), respectively) on domain-specific protein sequestration and on protein oligomerization state. We found that both α(v)β(3) and α(5)β(1) sequester preferentially to nonraft domains in the absence of extracellular matrix ligands, but upon ligand addition, α(v)β(3) sequesters strongly into raft-like domains and α(5)β(1) loses preference for either raft-like or nonraft-like domains. A corresponding photon counting histogram analysis showed that integrins exist predominantly in a monomeric state. No change was detected in oligomerization state upon ligand binding in either type I or type II bilayers, but a moderate increase in oligomerization state was observed for increasing concentrations of cholesterol. The combined findings suggest a mechanism in which changes in integrin sequestering are caused by ligand-induced changes in integrin conformation and/or dynamics that affect integrin-lipid interactions without altering the integrin oligomerization state.
不同的脂质环境，包括脂筏，越来越被认为是影响质膜中膜蛋白功能的关键因素。不幸的是，由于难以在活细胞中对这些通常较小且短暂的质膜异质性进行特征描述，因此对其在膜蛋白激活和寡聚化中的作用仍难以理解。为了解决这个难题，我们提出了一种基于聚合物支撑的双层脂膜的实验模型膜平台，其中包含稳定的筏模拟结构域（I 型）和均匀的胆固醇脂质混合物（II 型），可将跨膜蛋白掺入其中（α(v)β(3)和α(5)β(1)整合素）。这些灵活的脂质平台使我们能够使用共聚焦荧光光谱学，包括光子计数直方图方法，与荧光显微镜联用，定量探测细胞外基质配体（分别为 vitronectin 和 fibronectin 用于α(v)β(3)和α(5)β(1)）与天然配体结合对特定结构域的蛋白质隔离以及对蛋白质寡聚状态的影响。我们发现，在没有细胞外基质配体的情况下，α(v)β(3)和α(5)β(1)都优先隔离到非筏结构域，但在配体添加后，α(v)β(3)强烈隔离到类似筏结构域，而α(5)β(1)不再优先隔离到类似筏结构域或非筏结构域。相应的光子计数直方图分析表明，整合素主要以单体状态存在。在 I 型或 II 型双层膜中，配体结合后，寡聚状态没有变化，但随着胆固醇浓度的增加，寡聚状态略有增加。综合研究结果表明，整合素隔离的变化是由配体诱导的整合素构象和/或动力学变化引起的，这些变化影响整合素-脂质相互作用，而不改变整合素寡聚状态。
