Regulation of Rab8 in Toll-Like Receptor Signalling Pathways

Abstract

Small GTPases of the Rab family have wide ranging and essential roles in recruiting effectors to mediate membrane trafficking and receptor signalling events in mammalian cells. Nucleotide loading and activation/deactivation of the Rabs themselves are regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs) and other accessory proteins. While Rabs have important roles in innate immune cells, for host-pathogen interactions, receptor signalling and membrane trafficking, in many cases their GEFs and other accessory proteins have not been elucidated.Pathogen-activated, Toll-like receptors (TLRs) initiate and modulate transcription of inflammatory cytokines and other innate immune responses which are important for immune defence but can also contribute to chronic disease. Macrophages are key cells of the innate immune system and previous work from our laboratory established a role for promiscuous Rab family member, Rab8a, in TLR signalling. Rab8a is activated in TLR pathways to recruit phosphoinositide 3-kinase gamma (PI3Kg) as an effector which upregulates TLR-induced Akt/mTOR signalling to drive a biased program of anti-inflammatory cytokines. TLR-associated PI3Kg has now emerged as a key mediator of macrophage programming (or polarisation) in inflammation and cancer and its cognate GTPase, Rab8a, has a prominent role in immunity and disease. Rab8a is itself recruited to macrophage ruffles and macropinosomes through its association with the TLR crosstalk-activated endocytic receptor LRP1. However, it is not yet known how Rab8a is activated in TLR pathways. This project set out to identify the essential Rab8a GEF(s) needed to activate Rab8a as part of the LRP1 complex.Two well-known Rab8 GEFs, GRAB and Rabin8, which previously were uncharacterised in macrophages, were investigated in this project. GRAB was identified as part of the low density lipoprotein receptor-related protein 1 (LRP1) complex in pull-downs analysed by mass spectrometry. Co-immunoprecipitation and fluorescence microscopy showed that both GRAB and the structurally similar GEF, Rabin8, undergo LPS-inducible binding to Rab8a and are localised at sites of Rab8a enrichment. To carry out functional studies, stable knockouts (KOs) of Rabin8, GRAB, as well as a double KO were produced via CRISPR-Cas9 gene editing in macrophage cell lines. Nucleotide activation assays were developed for this project and live-cell imaging with KO cell lines showed that both GEFs contribute additively to TLR4-induced Rab8a GTP-loading, but they are not needed for Rab8a membrane recruitment. Analysis of TLR signalling after double KO of both GEFs suggested redundant roles for Rabin8 and GRAB in activating Rab8a for PI3Kg-dependent Akt/mTOR signalling. Live cell imaging utilising a fluorescent Akt1 reporter confirmed that LPS/TLR-induced Akt signalling is generated on macropinosomes and requires GRAB and Rabin8 GEF function.Next, to investigate possible regulators of the Rab8 GEFs, pull-downs and mass spectrometry were performed and identified a known multi-Rab effector, oculocerebrorenal syndrome of Lowe (OCRL) as a Rabin8 binding protein in LPS activated macrophages. Follow-up experiments provided initial characterisation of a novel OCRL-Rabin8 interaction and indicate a possible recruitment mechanism for the Rab8 GEF during TLR signalling.In conclusion, these results identified both GRAB and Rabin8 as essential activators of Rab8 downstream of TLR4 for inflammatory signalling in mouse macrophages. The results contribute these GEFs and other possible Rab recruiters to an expanding molecular complex that drives PI3Kg/Akt/mTOR signalling for control of inflammation and innate immune responses.