We thus conclude that the DRF motif in CXCR6 is an adaptation of the receptor that preserves its adhesion capacity in leukocyte subsets while diminishing its chemotactic function

We thus conclude that the DRF motif in CXCR6 is an adaptation of the receptor that preserves its adhesion capacity in leukocyte subsets while diminishing its chemotactic function. Materials and methods Antibodies, chemokines and Fc-constructs Recombinant human CXCL16 was purchased from PeproTech GmbH (Hamburg, Germany). for 72 h by automated real time cell imaging in B and BrdU assay Sal003 in C (n = 3). Data in C were expressed in relation to cells in the absence of soluble CXCL16. D: AKT activation was investigated by Western blot analysis. Representative blots are shown. E: Adhesion to immobilized anti-human-Fc was investigated as control experiment (n = 4). F: Random migration was investigated in a Boyden chamber assay (n 4). No statistic differences were observed in B to E.(TIF) pone.0173486.s002.tif (2.9M) GUID:?A4A0DFCC-5F8B-4F37-AD26-040AE0D42352 S3 Fig: Supporting information THP-1 cells expressing CX3CR1. THP-1 cells were transduced with lentivirus encoding murine CX3CR1 variants or EV control. Ligand binding was analyzed by incubation with CX3CL1-Fc fusion protein and FACS Sal003 analysis. Representative histograms are shown.(TIF) pone.0173486.s003.tif (498K) GUID:?7846F9D1-9BE8-4449-8E67-97AEA57892FA Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The CXC-chemokine receptor 6 (CXCR6) is a class A GTP-binding protein-coupled receptor (GPCRs) that mediates adhesion of leukocytes by interacting with the transmembrane cell surface-expressed chemokine ligand 16 (CXCL16), and also regulates leukocyte migration by interacting with the soluble shed variant of CXCL16. In contrast to virtually all other chemokine receptors with chemotactic activity, CXCR6 carries a DRF motif instead of the typical DRY motif as a key element in receptor activation and G protein coupling. In this work, modeling analyses revealed that the phenylalanine F3.51 in CXCR6 might have impact on intramolecular interactions including hydrogen bonds by this possibly changing receptor function. Initial investigations with embryonic kidney HEK293 cells and further studies with monocytic THP-1 cells showed that mutation of DRF into DRY does not influence ligand binding, receptor internalization, receptor recycling, and protein kinase B (AKT) signaling. Adhesion was slightly decreased in a time-dependent manner. However, CXCL16-induced calcium signaling and migration were increased. em Vice versa /em , when the DRY motif of the related receptor CX3CR1 was mutated into DRF the migratory response towards CX3CL1 was diminished, indicating that the presence of a DRF motif generally impairs chemotaxis in chemokine receptors. Transmembrane and soluble CXCL16 play divergent roles in homeostasis, inflammation, and cancer, which can be beneficial or detrimental. Therefore, the DRF motif of CXCR6 may display a receptor adaptation allowing adhesion and cell retention by transmembrane CXCL16 but reducing the chemotactic response to soluble CXCL16. This adaptation may avoid permanent or uncontrolled recruitment of inflammatory cells as well as cancer metastasis. Introduction Specific interactions between chemokines and their receptors regulate the sequential steps of diapedesis including adhesion and directional cell migration during inflammatory processes, tissue development, homeostasis, and cancer progression [1, 2]. CXCR6, first described as STRL33/BONZO [3], is expressed on different T cell subsets, macrophages, natural killer T (NK T) cells, fibroblasts and smooth muscle cells and is one of the T cell Rabbit Polyclonal to IL18R entry coreceptor used by HIV-1 [4C7]. The chemokine CXCL16, also referred to as scavenger receptor for phosphatidylserine and low-density lipoprotein (SR-PSOX), is the only known ligand of CXCR6 and is mainly expressed on endothelial cells [8, 9]. Together with CX3CL1, which binds to CX3CR1, CXCL16 is unique within the family of chemokines as it exists as a transmembrane and a soluble form [10C12], possibly acting as both adhesion and chemotactic molecule [4, 8, 13C17]. As a chemokine receptor, CXCR6 belongs to the class A of GPCRs. Upon activation, the receptor catalyzes the exchange of GDP to GTP in intracellular Gi proteins leading to the activation of phospholipase C, increase in inositol triphosphate concentration, and transient changes in intracellular calcium levels. In addition, activation of CXCR6 also results in the phosphorylation of signaling kinases such as protein kinase B (AKT). Activation of these signaling cascades induces cell migration, adhesion, proliferation, and survival [18]. The highly conserved aspartate-arginine-tyrosine (DRY) motif, located at the cytoplasmic side of transmembrane helix 3 (TM3) of most class A GPCRs, is a key motif for stabilizing the active state of Sal003 the receptor and to activate G proteins, thereby regulating receptor activity [19C21]. Specifically, the negatively charged D3.49 (the number in Sal003 superscript represents the position of the residue in the sequence according to the generic GPCRdb numbering [22]) forms a salt bridge with the positively charged R3.50 which keeps this arginine warped in an inactive conformation. Therefore, D3.49 has been shown to be involved in regulating the activity of many GPCRs including the chemokine receptors CXCR1, CXCR2, and chemokine (C-C motif) receptor 5 CCR5 [19C21]. Upon receptor activation, R3.50 is released from its interaction with D3.49 and extends to interact with Y5.58 and Y7.53, stabilizing the active state and building the binding pocket for.