The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS–containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.

中文翻译：

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EGFR-RAS-MAPK 信号传导仅限于质膜和相关的内循环突起

RAS GTPases 的亚细胞定位定义了细胞内 EGFR-ERK1/2 信号通路的操作区室。因此，我们使用活细胞成像来证明用 mNeonGreen 标记的内源性 KRAS 和 NRAS 主要定位于质膜。NRAS 还存在于高尔基体和管状质膜衍生的内循环室中，富含再循环内体标记物 (TERC)。在 EGF 刺激的细胞中，mNeonGreen-KRAS 或 mNeonGreen-NRAS 与内体 EGFR 基本上没有共定位，相反，它仍然与内源性 Grb2-mNeonGreen（RAS 上游的受体适配器）相关。即使在大多数配体结合、Grb2 相关 EGFR 被内化后，用西妥昔单抗阻断细胞表面 EGFR 也会降低 ERK1/2 活性。内源性 mCherry 标记的 RAF1（RAS 效应子）被募集至质膜，随后在含有 mNG-NRAS 的 TERC 中积累。我们提出，一小部分表面 EGFR 维持 RAS-ERK1/2 通路内的信号传导，并且 TERC 中持续存在 RAS 激活，而内体 EGFR 对 ERK1/2 活性没有显着贡献。