Involvement of Gβγ subunits of Gi protein coupled with S1P receptor on multivesicular endosomes in F-actin formation and cargo sorting into exosomes

Abstract

Exosomes play a critical role in cell-to-cell communication by delivering cargo molecules to recipient cells. However, the mechanism underlying the generation of the exosomal multivesicular endosome (MVE) is one of the mysteries in the field of endosome research. Although sphingolipid metabolites such as ceramide and sphingosine 1-phosphate (S1P) are known to play important roles in MVE formation and maturation, the detailed molecular mechanisms are still unclear. Here, we show that Rho family GTPases, including Cdc42 and Rac1, are constitutively activated on exosomal MVEs and are regulated by S1P signaling as measured by fluorescence resonance energy transfer (FRET)-based conformational changes. Moreover, we detected S1P signaling-induced filamentous actin (F-actin) formation. A selective inhibitor of Gβγ subunits, M119, strongly inhibited both F-actin formation on MVEs and cargo sorting into exosomal intralumenal vesicles of MVEs, both of which were fully rescued by the simultaneous expression of constitutively active Cdc42 and Rac1. Our results shed light on the mechanism underlying exosomal MVE maturation and inform the understanding of the physiological relevance of continuous activation of the S1P receptor and subsequent downstream G protein signaling to Gβγ subunits/Rho family GTPases-regulated F-actin formation on MVEs for cargo sorting into exosomal intralumenal vesicles.

Molecular mechanisms of biogenesis of apoptotic exosome-like vesicles and their roles as damage-associated molecular patterns

“…These results are entirely consistent with the data from the cellular model. Recent research on newly identified roles of the G βγ subunits has shown that the G βγ subunit of S1PR can function in the maturation of MVEs for exosomal cargo sorting via the regulation of the actin cytoskeleton through Rho family small GTPase (22). Therefore, we evaluated the changes in the actin cytoskeleton after apoptotic stimuli.…”

Ceramide and Exosomes: A Novel Target in Cancer Biology and Therapy

“…Secretion of sphingosine-1-phosphate (S1P), a ceramide derivative, is instrumental in promoting angiogenesis during tumor growth (Mizugishi et al, 2005; Nagahashi et al, 2012; Ogretmen, 2018; Pyne, El Buri, Adams, & Pyne, 2017; Spiegel & Kolesnick, 2002; Spiegel & Milstien, 2003; Takabe, Paugh, Milstien, & Spiegel, 2008; Takabe & Spiegel, 2014). It was found that stable complexes of S1P with G-protein coupled receptors (GPCRs) are critical for formation of exosomes in MVEs through constitutive activation of Rho GTPases and stabilization of F-actin (Kajimoto et al, 2018). However, it has not been determined if these stable complexes are formed on exosomes that could then function as extracellular signalosomes.…”

Endo‐lysosomal sorting of G‐protein‐coupled receptors by ubiquitin: Diverse pathways for G‐protein‐coupled receptor destruction and beyond

“…Research into the exosomal sorting of the related S1P 1 receptor has revealed a mechanism for how GPCRs are sorted away from degradative MVBs and into MVBs that contain exosomes. Continuous stimulation of the S1P 1 receptor following internalization activates Rac1 and Cdc42 on MVBs, a process that requires the Gβγ subunits of the heterotrimeric G i protein. Inhibition of Rho GTPase activity or the inhibition of Gβγ prevents the release of S1P 1 in exosomes, suggesting that actin polymerization and GPCR signaling near the limiting membrane of MVBs is critical for exosome release.…”

“…Continuous stimulation of the S1P 1 receptor following internalization activates Rac1 and Cdc42 on MVBs, a process that requires the Gβγ subunits of the heterotrimeric G i protein. Inhibition of Rho GTPase activity or the inhibition of Gβγ prevents the release of S1P 1 in exosomes, suggesting that actin polymerization and GPCR signaling near the limiting membrane of MVBs is critical for exosome release. These findings have highlighted the importance of GPCRs in exosomal signaling, and provide critical mechanistic insight into how GPCRs are sorted into exosomes.…”