G protein-coupled receptors (GPCRs) rapidly transmit extracellular signals by activating effector proteins, thereby producing well-characterized second messenger molecules. Free, unanchored polyubiqui Show more
G protein-coupled receptors (GPCRs) rapidly transmit extracellular signals by activating effector proteins, thereby producing well-characterized second messenger molecules. Free, unanchored polyubiquitin chains have been proposed as secondary messengers in immune and inflammatory pathways that regulate cellular responses to invading pathogens and the inflammatory cytokine Interleukin-1 beta (IL-1β). It remains unknown whether these molecules play a role in GPCR signaling. The present study used primary, immortalized, and transformed cellular models, together with loss-of-function approaches, to demonstrate the presence and functions of unanchored polyubiquitin chains in inflammatory signaling pathways that activate the activator protein 1 (AP-1) and nuclear factor-kappa B (NF-κB) transcription factors. In response to inflammatory GPCR agonists Angiotensin II (Ang II), lysophosphatidic acid (LPA), and thrombin (Thr), the E3 ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6) controls early signaling events that lead to T-loop phosphorylation of Transforming Growth Factor β-activated kinase 1 (TAK1), IκB kinase beta (IKKβ), c-Jun N-terminal kinases (JNK1/2), and p38 mitogen-activated protein kinases (p38). In parallel, we document the rapid, transient TRAF6-dependent production of unanchored lysine (K)63-linked polyubiquitin chains that accumulate in TAK1 and IKKβ immunocomplexes. Pull-down assays specifically designed to capture unanchored polyubiquitin chains in cellular extracts from stimulated cells further support their transient nature. Lastly, stable expression of a zinc finger ubiquitin-binding protein (ZnF-UBP) domain, which specifically binds unanchored polyubiquitin chains in immortalized keratinocytes exposed to LPA and Thr, shows that this production occurs in the proximity to the plasma membrane and diminishes the T-loop phosphorylation of TAK1, IKKβ, JNK1/2, and p38, thereby affecting the induction of early transcriptional events. Our results support a novel paradigm in GPCR signal transduction, identifying unanchored K63-linked polyubiquitin chains as second messenger molecules. The online version contains supplementary material available at 10.1186/s12964-025-02646-6. Show less