Gonzalez-Magaldi Monica, Gullapalli Anuradha, Papoulas Ophelia, Liu Chang, Leung Adelaide Y-H, Guo Luqiang, Brilot Axel F, Marcotte Edward M, Ke Zunlong, Leahy Daniel J
Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712.
Center for Biomedical Research Support, The University of Texas at Austin, Austin, TX 78712.
Proc Natl Acad Sci U S A. 2025 Jun 10;122(23):e2424678122. doi: 10.1073/pnas.2424678122. Epub 2025 Jun 2.
We report here transport of full-length 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 are the most abundant in EV membranes, and the presence of many receptor-interacting proteins in EVs demonstrates their utility for characterizing membrane protein interactomes. Cryo-electron tomography of EGFR-containing EVs reveals that EGFR forms clusters in both the presence and absence of EGF with a ~3 nm gap between the inner membrane and cytoplasmic density. EGFR extracellular region (ECR) dimers do not form regular arrays in these clusters. Subtomogram averaging of the 150 kDa EGF-bound EGFR ECR dimer 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 establish EVs as a versatile platform for structural and functional characterization of human membrane proteins in cell-derived membranes.
我们在此报告，为进行结构和功能研究，将全长表皮生长因子受体（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）中。质谱分析证实，转运的蛋白质在 EV 膜中最为丰富，且 EVs 中存在许多受体相互作用蛋白，这表明它们在表征膜蛋白相互作用组方面具有实用性。对含 EGFR 的 EVs 进行冷冻电子断层扫描显示，无论有无表皮生长因子（EGF），EGFR 均形成簇，内膜与细胞质密度之间存在约 3 纳米的间隙。在这些簇中，EGFR 细胞外区域（ECR）二聚体不形成规则阵列。对 150 kDa EGF 结合的 EGFR ECR 二聚体进行亚断层平均，得到了一个 15 Å 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、聚集和信号传导的理解，并将 EVs 确立为用于细胞衍生膜中人类膜蛋白结构和功能表征的通用平台。
