The CCR7 ligands CCL19 and CCL21 are increasingly recognized as functionally different (biased). Using mature human dendritic cells (DCs), we show that CCL19 is more potent than CCL21 in inducing 3D chemotaxis. Intriguingly, CCL21 induces prolonged and more efficient ERK1/2 activation compared with CCL19 and a C-terminal truncated (tailless) CCL21 in DCs. In contrast, tailless-CCL21 displays increased potency in DC chemotaxis compared with native CCL21. Using a CCL21-specific antibody, we show that CCL21, but not tailless-CCL21, accumulates at the cell surface. In addition, removal of sialic acid from the cell surface by neuraminidase treatment impairs ERK1/2 activation by CCL21, but not by CCL19 or tailless-CCL21. Using standard laboratory cell lines, we observe low potency of both CCL21 and tailless-CCL21 in G protein activation and β-arrestin recruitment compared with CCL19, indicating that the tail itself does not improve receptor interaction. Chemokines interact with their receptors in a stepwise manner with ultimate docking of their N-terminus into the main binding pocket. Employing site-directed mutagenesis we identify residues in this pocket of selective CCL21 importance. We also identify a molecular switch in the top of TM7 important for keeping CCR7 in an inactive conformation (Tyr312), as introduction of the chemokine receptor-conserved Glu (or Ala) induces high constitutive activity. Summarized, we show that the interaction of the tail of CCL21 with polysialic acid is needed for strong ERK signaling, whereas it impairs CCL21-mediated chemotaxis and has no impact on receptor docking consistent with the current model of chemokine:receptor interaction. This indicates that future selective pharmacological targeting of CCL19 versus CCL21 should focus on a differential targeting of the main receptor pocket, while selective targeting of tailless-CCL21 versus CCL21 and CCL19 requires targeting of the glycosaminoglycan (GAG) interaction.

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The seven transmembrane G protein-coupled receptor Epstein-Barr virus (EBV) induced gene 2 (EBI2; also known as GPR183) was identified in 1993 on the basis of its substantial upregulation in EBV-infected cells. It is primarily expressed in lymphoid cells; most abundantly in B cells. EBI2 is central for the positioning of B cells within the lymphoid organs, a process that is regulated in part by a chemotactic gradient formed by the endogenous lipid agonists, and in part by a fine-tuned regulation of EBI2 cell surface expression. The most potent endogenous EBI2 agonist is 7α, 25-dihydroxyxcholesterol (7α,25-OHC), yet many structurally related oxysterols can bind to an EBI2 pocket that is defined by the upper parts of the transmembrane helices and extracellular receptor regions. EBI2 signals via Gαi, as well as via G protein-independent pathways like β-arrestin recruitment. The concerted action of these pathways leads to cell migration. By genetically interfering with its up- and downregulation, EBI2 was also recently shown to induce cell proliferation, an action that could be inhibited by small molecule antagonists. Here, we focus on the oxysterol–EBI2 axis in immune control, including its role in the EBV life cycle. We also summarize the structural and functional properties of EBI2 interaction with oxysterol agonists and small molecule antagonists and discuss EBI2 as therapeutic target for diseases of the immune system.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. The initial contact of chemokines with their receptors depends on highly specified extracellular receptor features. Here we investigate the importance of conserved extracellular disulfide bridges and aromatic residues in extracellular loop 2 (ECL-2) for ligand binding and activation in the chemokine receptor CCR8. We used inositol 1,4,5-trisphosphate accumulation and radioligand binding experiments to determine the impact of receptor mutagenesis on both chemokine and small molecule agonist and antagonist binding and action in CCR8. We find that the seven-transmembrane (TM) receptor conserved disulfide bridge (7TM bridge) linking transmembrane helix III (TMIII) and ECL-2 is crucial for chemokine and small molecule action, whereas the chemokine receptor conserved disulfide bridge between the N terminus and TMVII is needed only for chemokines. Furthermore, we find that two distinct aromatic residues in ECL-2, Tyr 184 (Cys ؉ 1) and Tyr 187 (Cys ؉ 4), are crucial for binding of the CC chemokines CCL1 (agonist) and MC148 (antagonist), respectively, but not for small molecule binding. Finally, using in silico modeling, we predict an aromatic cluster of interaction partners for Tyr 187 in TMIV (Phe 171 ) and TMV (Trp 194 ). We show in vitro that these residues are crucial for the binding and action of MC148, thus supporting their participation in an aromatic cluster with Tyr 187 . This aromatic cluster appears to be present in a large number of CC chemokine receptors and thereby could play a more general role to be exploited in future drug development targeting these receptors.Chemokines (chemotactic cytokines) regulate the differentiation, activation, and recruitment of leukocytes. They also play important roles in several physiological mechanisms outside the immune system such as organogenesis and angiogenesis (1, 2). With ϳ50 members, these cytokines exert their effects through chemokine receptors (23 members), which belong to class A of the family of seven-transmembrane (7TM) 2 G protein-coupled receptors (3). The implications of the chemokine system in a vast number of human diseases (3) have increased the interest in developing potent, selective, and clinically useful chemokine receptor antagonists.The binding of a chemokine to its cognate receptor is initially driven by electrostatic interactions between the overall positively charged chemokine and the negatively charged extracellular surface of the receptor. Then interactions between the chemokine N terminus and residues in the main binding pocket of the receptor trigger receptor activation (4 -6). In contrast, small molecule ligands bind deeper in the main binding pocket and constrain the receptors in either active or inactive conformations (7,8). Whereas most mapping studies of small molecules have focused on the transmembrane areas, newer studies as well as crystal structures of class A receptors suggest that extracellular receptor regions, in particular ex...

The orphan receptor GPR125 (ADGRA3) belongs to subgroup III of the adhesion G protein−coupled receptor (aGPCR) family. aGPCRs, also known as class B2 GPCRs, share basic structural and functional properties with other GPCRs. Many of them couple to G proteins and activate G protein−dependent and −independent signaling pathways, but little is known about aGPCR internalization and β-arrestin recruitment. GPR125 was originally described as a spermatogonial stem cell marker and studied for its role in Wnt signaling and cell polarity. Here, using cell-based assays and confocal microscopy, we show that GPR125 is expressed on the cell surface and undergoes constitutive endocytosis in a β-arrestin−independent, but clathrin-dependent manner, as indicated by colocalization with transferrin receptor 1, an early endosome marker. These data support that the constitutive internalization of GPR125 contributes to its biological functions by controlling receptor surface expression and accessibility for ligands. Our study sheds light on a new property of aGPCRs, namely internalization; a property described to be important for signal propagation, signal termination, and desensitization of class A (rhodopsin-like) and B1 (VIP/secretin) GPCRs.