V-2

Amino Acid Sequence

3D Structure (/structures/AF-P00533-F1-model_v6.cif)

Function

Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, AREG, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin. Positively regulates cell migration via interaction with CCDC88A/GIV which retains EGFR at the cell membrane following ligand stimulation, promoting EGFR signaling which triggers cell migration. Plays a role in enhancing learning and memory performance. Plays a role in mammalian pain signaling (long-lasting hypersensitivity). Isoform 2 may act as an antagonist of EGF action. (Microbial infection) Acts as a receptor for hepatitis C virus (HCV) in hepatocytes and facilitates its cell entry. Mediates HCV entry by promoting the formation of the CD81-CLDN1 receptor complexes that are essential for HCV entry and by enhancing membrane fusion of cells expressing HCV envelope glycoproteins.

Mechanism

The ubiquitin-proteasome pathway (UPP) constitutes the principal pathway for protein degradation in eukaryotic cells, mediating the breakdown of target proteins through ubiquitin tagging and subsequent proteasomal degradation. In an ATP-dependent manner, the ubiquitin-activating enzyme (E1) activates ubiquitin (Ub) and conjugates it to a ubiquitin-conjugating enzyme (E2). E3 ligase subsequently catalyzes the transfer of Ub to the target protein, leading to the formation of a polyubiquitin chain that completes the ubiquitination mark. The 19S regulatory particle of the 26S proteasome recognizes this ubiquitination signal and, after deubiquitination, mediates the ATP-dependent delivery of the substrate into the 20S proteasome for degradation.

Description

This study investigated the mechanism by which the HyT compound V-2 induces EGFR degradation, focusing on whether it acts through the ubiquitin-proteasome pathway, using Western blot analysis. In H1299 cells, pretreatment with the proteasome inhibitor MG-132 (10 μM) significantly inhibited V-2 mediated degradation of EGFRWT compared to the DMSO control group, whereas pretreatment with the lysosomal inhibitor Bafilomycin A1 (200 nM) had no significant effect. Addition ally, pretreatment with the EGFR competitive ligand JBJ-07-149 (1 μM) effectively attenuated the degradation activity of V-2 (Fig. 12). These results indicate that compound V-2 primarily mediates EGFR degrada tion via the ubiquitin-proteasome pathway.

Description

Compound V-2 demonstrated potent degradation of EGFRWT in H1299 cells, achieving approximately 70% degradation at 1 μM. Compound V-2 induces EGFR degradation via the ubiquitin-proteasome pathway. Note: H1299 cells were pretreated with DMSO, MG-132 (10 μM), Bafilomycin A1 (200 nM), or JBJ-07-149 (1 μM) for 2 h, followed by treatment with compound V-2 (1 μM) for 24 h. Relative EGFR protein expression levels are presented as“mean ± SD”.All data were derived from three independent Western blot experiments.Compared with the DMSO (0.1%)group,▴P < 0.05,▴▴P < 0.01; compared with the Bafilomycin A1 (200 nM) group,▴P < 0.05,▴▴P < 0.01. Degradation rates are expressed as“mean ± SD”, and all data were collected from three independent Western blot experiments.