Identification of a Salt Bridge That Is Functionally Important for Chemokine Receptor CXCR1 but not CXCR2.

CXC chemokine receptors 1 (CXCR1) and 2 (CXCR2) have high sequence similarity and overlapping chemokine ligand profiles. Residue positions 3.32 and 7.39 are critical for signal transduction in the related CXCR4, and in these positions CXCR1 and CXCR2 contain oppositely charged residues (Lys3.32 and Glu7.39). Experimental and computed receptor structures reveal the possible formation of a salt bridge between transmembrane (TM) helices 3 and 7 via these two residues. To investigate the functional importance of Lys1173.32 and Glu2917.39 in CXCR1, along with the flanking Glu1183.33, we performed a signaling study on 16 CXCR1 mutants using two different CXCL8 isoforms. While single Ala-mutation (K1173.32A, E2917.39A) and charge reversal (K1173.32E, E2917.39K) resulted in nonfunctional receptors, double (K1173.32E-E2917.39K) and triple (K1173.32E-E1183.33A-E2917.39K) mutants rescued CXCR1 function. In contrast, the corresponding mutations did not affect the CXCR2 function to the same extent. Our findings show that the Lys3.32-Glu7.39 salt bridge between TM3 and -7 is functionally important for CXCR1 but not for CXCR2, meaning that signal transduction for these highly homologous receptors is not conserved.

Migration mediated by the oxysterol receptor GPR183 depends on arrestin coupling but not receptor internalization.

The chemotactic G protein-coupled receptor GPR183 and its most potent endogenous oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-OHC) are important for immune cell positioning in secondary lymphoid tissues. This receptor-ligand pair is associated with various diseases, in some cases contributing favorably and in other cases adversely, making GPR183 an attractive target for therapeutic intervention. We investigated the mechanisms underlying GPR183 internalization and the role of internalization in the main biological function of the receptor, chemotaxis. We found that the C terminus of the receptor was important for ligand-induced internalization but less so for constitutive (ligand-independent) internalization. ß-arrestin potentiated ligand-induced internalization but was not required for ligand-induced or constitutive internalization. Caveolin and dynamin were the main mediators of both constitutive and ligand-induced receptor internalization in a mechanism independent of G protein activation. Clathrin-mediated endocytosis also contributed to constitutive GPR183 internalization in a ß-arrestin-independent manner, suggesting the existence of different pools of surface-localized GPR183. Chemotaxis mediated by GPR183 depended on receptor desensitization by ß-arrestins but could be uncoupled from internalization, highlighting an important biological role for the recruitment of ß-arrestin to GPR183. The role of distinct pathways in internalization and chemotaxis may aid in the development of GPR183-targeting drugs for specific disease contexts.

Rewiring host-microbe interactions and barrier function during gastrointestinal inflammation.

Organismal survival depends on a well-balanced immune system and maintenance of host-microbe mutualism. The fine-tuned relationship between the gut microbiota and host immunity is constantly challenged by opportunistic bacteria testing the integrity of gastrointestinal (GI) barrier defenses. Barrier dysfunction reduces immunological tolerance towards otherwise innocuous microbes; it is a process that may instigate chronic inflammation. Paradoxically, sustained inflammation further diminishes barrier function, enabling bacterial translocation to extra-intestinal tissues. Once translocated, these bacteria stimulate systemic inflammation, thereby compromising organ function. While genetic risk alleles associate with barrier dysfunction, environmental stressors are key triggers of GI inflammation and associated breakdown in immune tolerance towards resident gut microbes. As dietary components dictate substrate availability, they also orchestrate microbiota composition and function, including migratory and pro-inflammatory potential, thus holding the capacity to fuel both GI and extra-intestinal inflammation. Additionally, Western diet consumption may weaken barrier defenses via curbed Paneth cell function and diminished host-defense peptide secretion. This review focuses on intervenable niches of host-microbe interactions and mucosal immunity with the ambition to provide a framework of plausible strategies to improve barrier function and regain tolerance in the inflamed mucosa via nutritional intervention.

Biased action of the CXCR4-targeting drug plerixafor is essential for its superior hematopoietic stem cell mobilization.

Following the FDA-approval of the hematopoietic stem cell (HSC) mobilizer plerixafor, orally available and potent CXCR4 antagonists were pursued. One such proposition was AMD11070, which was orally active and had superior antagonism in vitro; however, it did not appear as effective for HSC mobilization in vivo. Here we show that while AMD11070 acts as a full antagonist, plerixafor acts biased by stimulating ß-arrestin recruitment while fully antagonizing G protein. Consequently, while AMD11070 prevents the constitutive receptor internalization, plerixafor allows it and thereby decreases receptor expression. These findings are confirmed by the successful transfer of both ligands' binding sites and action to the related CXCR3 receptor. In vivo, plerixafor exhibits superior HSC mobilization associated with a dramatic reversal of the CXCL12 gradient across the bone marrow endothelium, which is not seen for AMD11070. We propose that the biased action of plerixafor is central for its superior therapeutic effect in HSC mobilization.

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 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 ß-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 ß-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.

CCL19 with CCL21-tail displays enhanced glycosaminoglycan binding with retained chemotactic potency in dendritic cells.

CCL19 is more potent than CCL21 in inducing chemotaxis of human dendritic cells (DC). This difference is attributed to 1) a stronger interaction of the basic C-terminal tail of CCL21 with acidic glycosaminoglycans (GAGs) in the environment and 2) an autoinhibitory function of this C-terminal tail. Moreover, different receptor docking modes and tissue expression patterns of CCL19 and CCL21 contribute to fine-tuned control of CCR7 signaling. Here, we investigate the effect of the tail of CCL21 on chemokine binding to GAGs and on CCR7 activation. We show that transfer of CCL21-tail to CCL19 (CCL19CCL21-tail ) markedly increases binding of CCL19 to human dendritic cell surfaces, without impairing CCL19-induced intracellular calcium release or DC chemotaxis, although it causes reduced CCR7 internalization. The more potent chemotaxis induced by CCL19 and CCL19CCL21-tail compared to CCL21 is not transferred to CCL21 by replacing its N-terminus with that of CCL19 (CCL21CCL19-N-term ). Measurements of cAMP production in CHO cells uncover that CCL21-tail transfer (CCL19CCL21-tail ) negatively affects CCL19 potency, whereas removal of CCL21-tail (CCL21tailless ) increases signaling compared to full-length CCL21, indicating that the tail negatively affects signaling via cAMP. Similar to chemokine-driven calcium mobilization and chemotaxis, the potency of CCL21 in cAMP is not improved by transfer of the CCL19 N-terminus to CCL21 (CCL21CCL19-N-term ). Together these results indicate that ligands containing CCL21 core and C-terminal tail (CCL21 and CCL21CCL19-N-term ) are most restricted in their cAMP signaling; a phenotype attributed to a stronger GAG binding of CCL21 and defined structural differences between CCL19 and CCL21.