Endophilin plays key roles during endocytosis of cellular receptors, including generating membrane curvature to drive internalization. Electrostatic interactions between endophilin’s BAR domain and anionic membrane lipids have been considered the major driving force in curvature generation. However, the SH3 domain of endophilin also interacts with the proline-rich third intracellular loop (TIL) of various G-protein coupled receptors (GPCRs), and it is unclear whether this interaction has a direct role in generating membrane curvature during endocytosis. To examine this, we designed model membranes with a membrane density of 1400 receptors per µm2 represented by a covalently conjugated TIL region from β1-adrenergic receptor. We observed that TIL recruits endophilin to membranes composed of 95 mol% of zwitterionic lipids via the SH3 domain. More importantly, endophilin recruited via TIL tubulates vesicles and gets sorted onto highly curved membrane tubules. These observations indicate that the cellular membrane bending and curvature sensing activities of endophilin can be facilitated through detection of the TIL of activated GPCRs in addition to binding to anionic lipids. Furthermore, we show that TIL electrostatically interacts with membranes composed of anionic lipids. Therefore, anionic lipids can modulate TIL/SH3 domain binding. Overall, our findings imply that an interplay between TIL, charged membrane lipids, BAR domain, and SH3 domain could exist in the biological system and that these components may act in coordination to regulate the internalization of cellular receptors.

中文翻译：

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内亲素募集通过 GPCR 环相互作用和负脂质电荷的巧合检测来驱动膜曲率生成


内亲素在细胞受体的内吞作用过程中发挥着关键作用，包括产生膜曲率以驱动内化。内亲蛋白的 BAR 结构域和阴离子膜脂质之间的静电相互作用被认为是曲率产生的主要驱动力。然而，内亲素的 SH3 结构域也与各种 G 蛋白偶联受体 (GPCR) 的富含脯氨酸的第三细胞内环 (TIL) 相互作用，目前尚不清楚这种相互作用是否在内吞作用期间产生膜曲率具有直接作用。为了检查这一点，我们设计了膜密度为每 µm2 1400 个受体的模型膜，由来自 β1-肾上腺素受体的共价结合的 TIL 区域表示。我们观察到 TIL 通过 SH3 结构域将内亲素募集到由 95 mol% 两性离子脂质组成的膜上。更重要的是，通过 TIL 招募的内亲蛋白使囊泡形成管状，并被分选到高度弯曲的膜管上。这些观察结果表明，除了与阴离子脂质结合之外，还可以通过检测激活的 GPCR 的 TIL 来促进内亲素的细胞膜弯曲和曲率传感活性。此外，我们还发现 TIL 与阴离子脂质组成的膜发生静电相互作用。因此，阴离子脂质可以调节 TIL/SH3 结构域结合。总体而言，我们的研究结果表明，生物系统中可能存在 TIL、带电膜脂、BAR 结构域和 SH3 结构域之间的相互作用，并且这些成分可能协调作用以调节细胞受体的内化。