Study on the Molecular Mechanism of ERK Activation Regulated by conformational change of Gαs protein

Abstract

G protein-coupled receptors (GPCRs regulate diverse cellular processes via downstream signaling pathways including the extracellular signal-regulated kinase (ERK) cascade1,2.Dysregulation of ERK signaling is frequently linked to diseases including such as cancer3, often involving mutations in G protein α subunits4. G proteins function as heterotrimers to transmit signals from activated GPCRs to effectors; their α subunits contain conserved domains—Ras-like domain (RD), and α-helical domain (AHD)5,6—which undergo conformational changes essential for signaling7,8. Upon activation, the AHD is physically displaced relative to the RD7,8 (ref), facilitating interactions with downstream effectors. However, the detailed molecular mechanism connecting G protein conformational dynamics to ERK activation remains unclear. In this study, we elucidated the mechanistic relationship between extracellular signal-regulated kinase (ERK) activation and the conformational dynamics of Gαs protein by employing long (Gαs-L) and short (Gαs-S) splice variants9. The extended linker region between the alpha-helical domain (AHD) and Ras-like domain (RD) in Gαs-L endowed this variant with a markedly greater capacity to stimulate ERK activity at endosomal compartments compared to Gαs-S9, as determined by an endosome-targeted Förster resonance energy transfer (FRET)-based ERK activity reporter (EKAR4)10,11 . Importantly, this enhancement was specific to ERK signaling and was not observed for canonical cAMP or protein kinase A (PKA) signaling pathways, which are well- established downstream effectors of Gαs. Stabilization of the open conformation of Gαs at endosomes using a Chanwei Moon (문찬위). Study on the Molecular Mechanism of ERK Activation Regulated by conformational change of Gαs protein (Gαs 단 백질의 구조적 변화에 의해 매개되는 ERK 활성의 분자적 기 전 연구). College of Life Sciences and Medical Engineering / Department of Life Sciences (생명과학과). 2026. 34p. Advisor Prof. Yonghoon Kwon. MS/BS 20241093 conformation-specific nanobody (Nb37)12further increased basal ERK activity, underscoring the pivotal role of Gαs conformational rearrangement in compartmentalized ERK pathway regulation. To further investigate this mechanism, we engineered constructs targeting either the AHD or RD to endosomes to mimic the AHD displacement associated with Gαs protein activation. FRET imaging confirmed that the endosome targeted AHD is responsible for the activation of both endosomal and nuclear ERK through FRET imaging. Furthermore, direct interactions between the AHD and Raf1 were detected via proximity ligation assay (PLA), indicating that AHD supports the assembly of the MAPK signaling complex at the endosome. Raf1 activation was subsequently validated by FRET-based kinase activity assays. Finally, cell proliferation assays demonstrated that ERK activity generated by the AHD promotes proliferation through the translocation of activated endosomal ERK to the nucleus. Collectively, these findings clarify the intra- and intermolecular mechanisms by which GPCR- induced G protein conformational changes drive AHD-mediated ERK activation, advancing our understanding of cellular signaling and offering insights for targeted therapy development in diseases characterized by aberrant GPCR-ERK signaling.