Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA.
J Cell Biol. 2021 Nov 1;220(11). doi: 10.1083/jcb.202107103. Epub 2021 Sep 13.
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.
RAS GTPases 的亚细胞定位决定了 EGFR-ERK1/2 信号通路在细胞内的作用区室。因此，我们使用活细胞成像技术证明，内源性 KRAS 和 NRAS 被 mNeonGreen 标记后主要定位于质膜。NRAS 也存在于高尔基体和管状的、富含再循环内体标志物（TERC）的质膜衍生的内再循环隔室中。在 EGF 刺激的细胞中，mNeonGreen-KRAS 或 mNeonGreen-NRAS 与内体 EGFR 几乎没有共定位，而 EGFR 仍然与内源性 Grb2-mNeonGreen 结合，Grb2-mNeonGreen 是 RAS 的上游受体接头。用西妥昔单抗阻断细胞表面 EGFR，即使大部分配体结合的、与 Grb2 相关的 EGFR 被内化后，ERK1/2 活性也会降低。RAS 的效应物内源性 mCherry 标记的 RAF1 被募集到质膜，随后在含有 mNG-NRAS 的 TERC 中积累。我们提出，一小部分表面 EGFR 维持着 RAS-ERK1/2 通路中的信号转导，而 RAS 的激活在 TERC 中持续存在，而内体 EGFR 对 ERK1/2 活性没有显著贡献。
