Gonzalez-Magaldi Monica, Gullapalli Anuradha, Papoulas Ophelia, Liu Chang, Leung Adelaide Y-H, Guo Luqiang, Brilot Axel, Marcotte Edward M, Ke Zunlong, Leahy Daniel J
bioRxiv. 2024 Nov 28:2024.11.25.625301. doi: 10.1101/2024.11.25.625301.
We report here transport of the Epidermal Growth Factor Receptor (EGFR), Insulin Receptor, 7-pass transmembrane receptor Smoothened, and 13-pass Sodium-iodide symporter to extracellular vesicles (EVs) for structural and functional studies. Mass spectrometry confirmed the transported proteins as the most abundant in EV membranes, and the presence of many receptor-interacting proteins demonstrates the utility of EVs for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in the presence of EGF with a ∼3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular regions do not form regular arrays, suggesting that clustering is mediated by the intracellular region. Subtomogram averaging of the EGFR extracellular region (ECR) yielded a 15 Å map into which the crystal structure of the ligand-bound EGFR ECR dimer fits well. These findings refine our understanding of EGFR activation, clustering, and signaling, and they establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in a native-like environment.
Atomic or near-atomic resolution structural studies of proteins embedded in cell membranes have proven challenging. We show that transporting integral membrane proteins to cell-derived extracellular vesicles enables structural and functional studies of human membrane proteins in a native membrane environment. We have used this approach to visualize an active form of full-length Epidermal Growth Factor Receptor (EGFR) and show that it forms clusters in the membrane and projects its cytoplasmic signaling domains ∼3 nm away from the membrane surface. EGFR is essential for normal development, but abnormal EGFR activity is associated with several human cancers and is the target of many anticancer therapies. Our studies refine current models of how ligand binding to EGFR transmits signals across cell membranes.
我们在此报告将表皮生长因子受体（EGFR）、胰岛素受体、7次跨膜受体Smoothened和13次跨膜钠碘同向转运体转运至细胞外囊泡（EVs）用于结构和功能研究。质谱分析证实所转运的蛋白质是EV膜中含量最丰富的，并且许多受体相互作用蛋白的存在证明了EVs在表征膜蛋白相互作用组方面的实用性。含EGFR的EVs的冷冻电子断层扫描显示，在表皮生长因子（EGF）存在的情况下，EGFR形成簇，内膜与细胞质密度之间有~3纳米的间隙。EGFR细胞外区域不形成规则阵列，这表明簇集是由细胞内区域介导的。EGFR细胞外区域（ECR）的亚断层平均产生了一个15埃的图谱，配体结合的EGFR ECR二聚体的晶体结构很好地拟合其中。这些发现完善了我们对EGFR激活、簇集和信号传导的理解，并将EVs确立为在类似天然环境中对人类膜蛋白进行结构和功能表征的通用平台。
对嵌入细胞膜中的蛋白质进行原子或近原子分辨率的结构研究已被证明具有挑战性。我们表明，将整合膜蛋白转运至细胞衍生的细胞外囊泡能够在天然膜环境中对人类膜蛋白进行结构和功能研究。我们已使用这种方法可视化全长表皮生长因子受体（EGFR）的活性形式，并表明它在膜中形成簇，其细胞质信号结构域从膜表面突出约3纳米。EGFR对正常发育至关重要，但EGFR活性异常与多种人类癌症相关，并且是许多抗癌疗法的靶点。我们的研究完善了当前关于配体与EGFR结合如何跨细胞膜传递信号的模型。
