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

Abstract

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.

Systematic Assessment of Chemokine Signaling at Chemokine Receptors CCR4, CCR7 and CCR10

“…Additionally, consistent with our data, Corbisier et al found that CCL19 was more potent than CCL21 in both G protein activation and β-arrestin recruitment (in HEK293 cells), with no significant bias between these ligands and pathways [9]. Two recent studies by Rosenkilde and colleagues have identified features of both the chemokines and CCR7 that contribute to the higher potency of CCL19 [8,21]. In particular, the higher potency of CCL19 was suggested to result from differential interactions of the core domains of the two chemokines with extracellular loop 2 of the receptor [8].…”

“…Two recent studies by Rosenkilde and colleagues have identified features of both the chemokines and CCR7 that contribute to the higher potency of CCL19 [8,21]. In particular, the higher potency of CCL19 was suggested to result from differential interactions of the core domains of the two chemokines with extracellular loop 2 of the receptor [8].…”

“…Similarly, for CCR7, several studies have found that CCL19 was more effective than CCL21 at inducing receptor internalization and β-arrestin recruitment [7][8][9][18][19][20][21]. However, these studies came to different conclusions regarding whether the two ligands stimulate equivalent G protein-mediated responses via CCR7 [7][8][9]21]. In the only study investigating differential agonism at CCR10, Rajagopal et al [4] reported that both CCL27 and CCL28 effectively stimulated G protein-mediated signaling, whereas only CCL27 was able to simulate β-arrestin recruitment.…”

“…Such differences are classified as biased agonism if the ligands differ in their relative efficiencies of signaling via two alternative pathways, e.g., G protein versus β-arrestin pathways, or pathways mediated by different G protein subtypes. Biased agonism has been reported for several chemokine receptors, including CCR1, CCR2, CCR4, CCR5, CCR7, CCR10, CXCR2 and CXCR3 [4][5][6][7][8][9]. Biased agonism is distinct from differences in intrinsic efficacy (or partial agonism), which applies to ligands that activate the same pathways but differ in the relative magnitude of the signaling outcomes.…”

“…In the case of CCR4, CCL22 was observed to be more effective than CCL17 at inducing receptor internalization [15,16] and activation by the two ligands was affected differently by receptor mutations or inhibitory antibodies [17]. Similarly, for CCR7, several studies have found that CCL19 was more effective than CCL21 at inducing receptor internalization and β-arrestin recruitment [7][8][9][18][19][20][21]. However, these studies came to different conclusions regarding whether the two ligands stimulate equivalent G protein-mediated responses via CCR7 [7][8][9]21].…”