Although class A G protein−coupled receptors (GPCRs) can function as monomers, many of them form dimers and oligomers, but the mechanisms and functional relevance of such oligomerization is ill understood. Here, we investigate this problem for the CXC chemokine receptor 4 (CXCR4), a GPCR that regulates immune and hematopoietic cell trafficking, and a major drug target in cancer therapy. We combine single-molecule microscopy and fluorescence fluctuation spectroscopy to investigate CXCR4 membrane organization in living cells at densities ranging from a few molecules to hundreds of molecules per square micrometer of the plasma membrane. We observe that CXCR4 forms dynamic, transient homodimers, and that the monomer−dimer equilibrium is governed by receptor density. CXCR4 inverse agonists that bind to the receptor minor pocket inhibit CXCR4 constitutive activity and abolish receptor dimerization. A mutation in the minor binding pocket reduced the dimer-disrupting ability of these ligands. In addition, mutating critical residues in the sixth transmembrane helix of CXCR4 markedly diminished both basal activity and dimerization, supporting the notion that CXCR4 basal activity is required for dimer formation. Together, these results link CXCR4 dimerization to its density and to its activity. They further suggest that inverse agonists binding to the minor pocket suppress both dimerization and constitutive activity and may represent a specific strategy to target CXCR4.

G protein-coupled receptors (GPCRs) are regulated by complex molecular mechanisms, both in physiologic and pathologic conditions, and their signaling can be intricate. Many factors influence their signaling behavior, including the type of ligand that activates the GPCR, the presence of interacting partners, the kinetics involved, or their location. The two CXC-type chemokine receptors, CXC chemokine receptor 4 (CXCR4) and atypical chemokine receptor 3 (ACKR3), both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, human immunodeficiency virus infection, and inflammatory diseases. Therefore, it is crucial to understand how the signaling of these receptors works to be able to specifically target them. In this review, we discuss how the signaling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signaling of CXCR4 depends on the cellular context, and discrepancies exist depending on the cell lines used. ACKR3, as an atypical chemokine receptor, is generally reported to not activate G proteins but can broaden its signaling spectrum upon heteromerization with other receptors, such as CXCR4, endothelial growth factor receptor, or the a 1-adrenergic receptor (a 1-AR). Also, CXCR4 forms heteromers with CC chemokine receptor (CCR) 2, CCR5, the Na 1 / H 1 exchanger regulatory factor 1, CXCR3, a 1-AR, and the opioid receptors, which results in differential signaling from that of the monomeric subunits. In addition, CXCR4 is present on membrane rafts but can go into the nucleus during cancer progression, probably acquiring different signaling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signaling.

Antinociceptive pathways are activated in the periphery in inflammatory pain, for instance resolvins and opioid peptides. Resolvins are biosynthesized from omega-3 polyunsaturated fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Resolvin D1 (RvD1) and resolvin E1 (RvE1) initiate the resolution of inflammation and control of hypersensitivity via induction of anti-inflammatory signaling cascades. RvD1 binds to lipoxin A4/annexin-A1 receptor/formyl-peptide receptor 2 (ALX/FPR2), RvE1 to chemerin receptor 23 (ChemR23). Antinociception of RvD1 is mediated by interaction with transient receptor potential channels ankyrin 1 (TRPA1). Endogenous opioid peptides are synthesized and released from leukocytes in the tissue and bind to opioid receptors on nociceptor terminals. Here, we further explored peripheral mechanisms of RvD1 and chemerin (Chem), the ligand of ChemR23, in complete Freund’s adjuvant (CFA)-induced hindpaw inflammation in male Wistar rats. RvD1 and Chem ameliorated CFA-induced hypersensitivity in early and late inflammatory phases. This was prevented by peripheral blockade of the μ-opioid peptide receptor (MOR) using low dose local naloxone or by local injection of anti-β-endorphin and anti-met-enkephalin (anti-ENK) antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not stimulate the release of β-endorphin from macrophages and neutrophils, nor did RvD1 itself activate G-proteins coupled MOR or initiate β-arrestin recruitment to the membrane. TRPA1 blockade by HC-030031 in inflammation in vivo as well as inhibition of the TRPA1-mediated calcium influx in dorsal root ganglia neurons in vitro was hampered by naloxone. Peripheral application of naloxone alone in vivo already lowered mechanical nociceptive thresholds. Therefore, either a perturbation of the balance of endogenous pro- and antinociceptive mechanisms in early and late inflammation, or an interaction of TRPA1 and opioid receptors weaken the antinociceptive potency of RvD1 and TRPA1 blockers.

The serine/threonine protein kinase AKT1 is a downstream target of the chemokine receptor 4 (CXCR4), and both proteins play a central role in the modulation of diverse cellular processes, including proliferation and cell survival. While in chronic myeloid leukemia (CML) the CXCR4 is downregulated, thereby promoting the mobilization of progenitor cells into blood, the receptor is highly expressed in breast cancer cells, favoring the migratory capacity of these cells. Recently, the LIM and SH3 domain protein 1 (LASP1) has been described as a novel CXCR4 binding partner and as a promoter of the PI3K/AKT pathway. In this study, we uncovered a direct binding of LASP1, phosphorylated at S146, to both CXCR4 and AKT1, as shown by immunoprecipitation assays, pull-down experiments, and immunohistochemistry data. In contrast, phosphorylation of LASP1 at Y171 abrogated these interactions, suggesting that both LASP1 phospho-forms interact. Finally, findings demonstrating different phosphorylation patterns of LASP1 in breast cancer and chronic myeloid leukemia may have implications for CXCR4 function and tyrosine kinase inhibitor treatment.

Using the available structural information of the chemokine receptor CXCR4, we present hit finding and hit exploration studies that make use of virtual fragment screening, design, synthesis and structureactivity relationship (SAR) studies. Fragment 2 was identified as virtual screening hit and used as a starting point for the exploration of 31 N-substituted piperidin-4-yl-methanamine derivatives to investigate and improve the interactions with the CXCR4 binding site. Additionally, subtle structural ligand changes lead to distinct interactions with CXCR4 resulting in a full to partial displacement of CXCL12 binding and competitive and/or non-competitive antagonism. Three-dimensional quantitative structure-activity relationship (3D-QSAR) and binding model studies were used to identify important hydrophobic interactions that determine binding affinity and indicate key ligand-receptor interactions.

G protein-coupled receptors (GPCRs) are biologic switches that transduce extracellular stimuli into intracellular responses in the cell. Temporally resolving GPCR transduction pathways is key to understanding how cell signaling occurs. Here, we investigate the kinetics and dynamics of the activation and early signaling steps of the CXC chemokine receptor (CXCR) 4 in response to its natural ligands CXC chemokine ligand (CXCL) 12 and macrophage migration inhibitory factor (MIF), using Förster resonance energy transfer-based approaches. We show that CXCR4 presents a multifaceted response to CXCL12, with receptor activation (%0.6 seconds) followed by a rearrangement in the receptor/G protein complex (%1 seconds), a slower dimer rearrangement (%1.7 seconds), and prolonged G protein activation (%4 seconds). In comparison, MIF distinctly modulates every step of the transduction pathway, indicating distinct activation mechanisms and reflecting the different pharmacological properties of these two ligands. Our study also indicates that CXCR4 exhibits some degree of ligandindependent activity, a relevant feature for drug development. SIGNIFICANCE STATEMENT The CXC chemokine ligand (CXCL) 12/CXC chemokine receptor (CXCR) 4 axis represents a well-established therapeutic target for cancer treatment. We demonstrate that CXCR4 exhibits a multifaceted response that involves dynamic receptor dimer rearrangements and that is kinetically embedded between receptor-G protein complex rearrangements and G protein activation. The alternative endogenous ligand macrophage migration inhibitory factor behaves opposite to CXCL12 in each assay studied and does not lead to G protein activation. This detailed understanding of the receptor activation may aid in the development of more specific drugs against this target.

The CXCL12 chemokine receptor CXCR4 belongs to the GPCR superfamily and is often overexpressed in cancer, being involved in tumor progression and metastasis. How CXCR4 signaling integrates with other relevant oncogenic transduction pathways and the role of GPCR regulatory mechanisms in such contexts are not well-understood. Recent data indicate concurrent upregulation in certain tumors of CXCR4, EGF receptor (EGFR), and G protein-coupled receptor kinase 2 (GRK2), a signaling node functionally linked to both receptor types. We have investigated in a model system the effect of the EGFR and GRK2 status on CXCL12/CXCR4-mediated activation of Gi, the earliest step downstream of receptor activation. We find that overexpressed and activated EGFR reduces CXCR4-mediated Gi1 activation and that GRK2 phosphorylation at tyrosine residues is required to exert its inhibitory actions on CXCR4–Gi stimulation, suggesting a shared path of modulation. Our data point to a role for GRK2 in the crosstalk of the CXCR4 and EGFR signal transduction pathways in pathological contexts characterized by concurrent overactivation of these proteins.

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