Abstract
Despite playing a central role in tolerance, little is known regarding the mechanism by which intracellular CTLA-4 is shuttled from the trans-Golgi network to the surfaces of T cells. In this context, Ras-related GTPase Rab8 plays an important role in the intracellular transport, while we have previously shown that CTLA-4 binds to the immune cell adaptor TRIM in T cells. In this study, we demonstrate that CTLA-4 forms a multimeric complex comprised of TRIM and related LAX that in turn binds to GTP bound Rab8 for post-Golgi transport to the cell surface. LAX bound via its N terminus to active GTP-Rab8, as well as the cytoplasmic tail of CTLA-4. TRIM required LAX for binding to Rab8 in a complex. Wild-type LAX or its N terminus (residues 1 to 77) increased CTLA-4 surface expression, whereas small interfering RNAs of Rab8 or LAX or disruption of LAX/Rab8 binding reduced numbers of CTLA-4-containing vesicles and its coreceptor surface expression. LAX also promoted the polarization of CTLA-4 and the reorientation of the microtubule-organizing center to the site of T-cell receptor engagement. Our results identify a novel CTLA-4/TRIM/LAX/Rab8 effector complex in the transport of CTLA-4 to the surfaces of T cells.
Highlights
Despite playing a central role in tolerance, little is known regarding the mechanism by which intracellular CTLA-4 is shuttled from the trans-Golgi network to the surfaces of T cells
Anti-myc and alpha-tubulin antibodies were purchased from Millipore, antihemagglutinin from Covance, anti-SIT from BioLegend, anti-LAT from Upstate Biotechnology, anti-TRIM from Abcam, anti-syntaxin-6 and Rab8 from BD Bioscience, anti-CD3 (1452C11) from BioXCell (West Lebanon, NH), anti-human CTLA-4 conjugated to phycoerythrin (PE) and mouse IgG2a conjugated to PE from BD Pharmingen, anti-V5 and Alexa-conjugated secondary antibodies from Invitrogen, and horseradish peroxidase (HRP)-conjugated secondary antibodies from Jackson Immuno-Research
We previously found that immune cell adaptor TRIM associates with CTLA-4 and is needed for its transport to the surfaces of T cells [22]
Summary
Despite playing a central role in tolerance, little is known regarding the mechanism by which intracellular CTLA-4 is shuttled from the trans-Golgi network to the surfaces of T cells In this context, Ras-related GTPase Rab plays an important role in the intracellular transport, while we have previously shown that CTLA-4 binds to the immune cell adaptor TRIM in T cells. Despite its importance for properly controlled immune responses, surface expression of CTLA-4 is tightly regulated with the majority of the coreceptor being localized in intracellular compartments such as the trans-Golgi network (TGN), endosomes, and lysosomes [19,20,21,22,23]. CTLA-4 Surface Transport Mediated by Rab8/LAX face expression of CTLA-4 [22] Together, these observations have begun to implicate transmembrane type I adaptor proteins in the transport of cargo to the cell surface. There has been no evidence to indicate that either of these adaptors is involved in intracellular trafficking
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
