Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7
Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its t...
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doaj-adc9d50f5c624485a70b0f829f9929bd2020-11-24T21:23:58ZengFrontiers Media S.A.Frontiers in Immunology1664-32242019-09-011010.3389/fimmu.2019.02156479458Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7Astrid S. Jørgensen0Olav Larsen1Edith Uetz-von Allmen2Michael Lückmann3Daniel F. Legler4Thomas M. Frimurer5Christopher T. Veldkamp6Gertrud M. Hjortø7Mette M. Rosenkilde8Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkDepartment of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkBiotechnology Institute Thurgau (BITg), University of Konstanz, Kreuzlingen, SwitzerlandThe Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, DenmarkBiotechnology Institute Thurgau (BITg), University of Konstanz, Kreuzlingen, SwitzerlandThe Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Copenhagen, DenmarkDepartment of Chemistry, University of Wisconsin-Whitewater, Whitewater, WI, United StatesDepartment of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkDepartment of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, DenmarkChemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of β-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K1303.26, to be important for G protein signaling, but not β-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors.https://www.frontiersin.org/article/10.3389/fimmu.2019.02156/fullligand biased signalingCCR7CCL21CCL19chemokine core domainECL2 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Astrid S. Jørgensen Olav Larsen Edith Uetz-von Allmen Michael Lückmann Daniel F. Legler Thomas M. Frimurer Christopher T. Veldkamp Gertrud M. Hjortø Mette M. Rosenkilde |
spellingShingle |
Astrid S. Jørgensen Olav Larsen Edith Uetz-von Allmen Michael Lückmann Daniel F. Legler Thomas M. Frimurer Christopher T. Veldkamp Gertrud M. Hjortø Mette M. Rosenkilde Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7 Frontiers in Immunology ligand biased signaling CCR7 CCL21 CCL19 chemokine core domain ECL2 |
author_facet |
Astrid S. Jørgensen Olav Larsen Edith Uetz-von Allmen Michael Lückmann Daniel F. Legler Thomas M. Frimurer Christopher T. Veldkamp Gertrud M. Hjortø Mette M. Rosenkilde |
author_sort |
Astrid S. Jørgensen |
title |
Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7 |
title_short |
Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7 |
title_full |
Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7 |
title_fullStr |
Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7 |
title_full_unstemmed |
Biased Signaling of CCL21 and CCL19 Does Not Rely on N-Terminal Differences, but Markedly on the Chemokine Core Domains and Extracellular Loop 2 of CCR7 |
title_sort |
biased signaling of ccl21 and ccl19 does not rely on n-terminal differences, but markedly on the chemokine core domains and extracellular loop 2 of ccr7 |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Immunology |
issn |
1664-3224 |
publishDate |
2019-09-01 |
description |
Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of β-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K1303.26, to be important for G protein signaling, but not β-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors. |
topic |
ligand biased signaling CCR7 CCL21 CCL19 chemokine core domain ECL2 |
url |
https://www.frontiersin.org/article/10.3389/fimmu.2019.02156/full |
work_keys_str_mv |
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