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Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse

A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the tw...

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Autores principales: Felce, James H., Parolini, Lucia, Sezgin, Erdinc, Céspedes, Pablo F., Korobchevskaya, Kseniya, Jones, Mathew, Peng, Yanchun, Dong, Tao, Fritzsche, Marco, Aarts, Dirk, Frater, John, Dustin, Michael L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848080/
https://www.ncbi.nlm.nih.gov/pubmed/33537301
http://dx.doi.org/10.3389/fcell.2020.608484
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author Felce, James H.
Parolini, Lucia
Sezgin, Erdinc
Céspedes, Pablo F.
Korobchevskaya, Kseniya
Jones, Mathew
Peng, Yanchun
Dong, Tao
Fritzsche, Marco
Aarts, Dirk
Frater, John
Dustin, Michael L.
author_facet Felce, James H.
Parolini, Lucia
Sezgin, Erdinc
Céspedes, Pablo F.
Korobchevskaya, Kseniya
Jones, Mathew
Peng, Yanchun
Dong, Tao
Fritzsche, Marco
Aarts, Dirk
Frater, John
Dustin, Michael L.
author_sort Felce, James H.
collection PubMed
description A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4(+) T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC.
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spelling pubmed-78480802021-02-02 Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse Felce, James H. Parolini, Lucia Sezgin, Erdinc Céspedes, Pablo F. Korobchevskaya, Kseniya Jones, Mathew Peng, Yanchun Dong, Tao Fritzsche, Marco Aarts, Dirk Frater, John Dustin, Michael L. Front Cell Dev Biol Cell and Developmental Biology A central process in immunity is the activation of T cells through interaction of T cell receptors (TCRs) with agonistic peptide-major histocompatibility complexes (pMHC) on the surface of antigen presenting cells (APCs). TCR-pMHC binding triggers the formation of an extensive contact between the two cells termed the immunological synapse, which acts as a platform for integration of multiple signals determining cellular outcomes, including those from multiple co-stimulatory/inhibitory receptors. Contributors to this include a number of chemokine receptors, notably CXC-chemokine receptor 4 (CXCR4), and other members of the G protein-coupled receptor (GPCR) family. Although best characterized as mediators of ligand-dependent chemotaxis, some chemokine receptors are also recruited to the synapse and contribute to signaling in the absence of ligation. How these and other GPCRs integrate within the dynamic structure of the synapse is unknown, as is how their normally migratory Gαi-coupled signaling is terminated upon recruitment. Here, we report the spatiotemporal organization of several GPCRs, focusing on CXCR4, and the G protein Gαi2 within the synapse of primary human CD4(+) T cells on supported lipid bilayers, using standard- and super-resolution fluorescence microscopy. We find that CXCR4 undergoes orchestrated phases of reorganization, culminating in recruitment to the TCR-enriched center. This appears to be dependent on CXCR4 ubiquitination, and does not involve stable interactions with TCR microclusters, as viewed at the nanoscale. Disruption of this process by mutation impairs CXCR4 contributions to cellular activation. Gαi2 undergoes active exclusion from the synapse, partitioning from centrally-accumulated CXCR4. Using a CRISPR-Cas9 knockout screen, we identify several diverse GPCRs with contributions to T cell activation, most significantly the sphingosine-1-phosphate receptor S1PR1, and the oxysterol receptor GPR183. These, and other GPCRs, undergo organization similar to CXCR4; including initial exclusion, centripetal transport, and lack of receptor-TCR interactions. These constitute the first observations of GPCR dynamics within the synapse, and give insights into how these receptors may contribute to T cell activation. The observation of broad GPCR contributions to T cell activation also opens the possibility that modulating GPCR expression in response to cell status or environment may directly regulate responsiveness to pMHC. Frontiers Media S.A. 2021-01-18 /pmc/articles/PMC7848080/ /pubmed/33537301 http://dx.doi.org/10.3389/fcell.2020.608484 Text en Copyright © 2021 Felce, Parolini, Sezgin, Céspedes, Korobchevskaya, Jones, Peng, Dong, Fritzsche, Aarts, Frater and Dustin. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Cell and Developmental Biology
Felce, James H.
Parolini, Lucia
Sezgin, Erdinc
Céspedes, Pablo F.
Korobchevskaya, Kseniya
Jones, Mathew
Peng, Yanchun
Dong, Tao
Fritzsche, Marco
Aarts, Dirk
Frater, John
Dustin, Michael L.
Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse
title Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse
title_full Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse
title_fullStr Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse
title_full_unstemmed Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse
title_short Single-Molecule, Super-Resolution, and Functional Analysis of G Protein-Coupled Receptor Behavior Within the T Cell Immunological Synapse
title_sort single-molecule, super-resolution, and functional analysis of g protein-coupled receptor behavior within the t cell immunological synapse
topic Cell and Developmental Biology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848080/
https://www.ncbi.nlm.nih.gov/pubmed/33537301
http://dx.doi.org/10.3389/fcell.2020.608484
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