Significance: Class A G protein−coupled receptors (GPCRs) can form dimers and oligomers via poorly understood mechanisms. We show here that the chemokine receptor CXCR4, which is a major pharmacological target, has an oligomerization behavior modulated by its active conformation. Combining advanced, single-molecule, and single-cell optical tools with functional assays and computational approaches, we unveil three key features of CXCR4 quaternary organization: CXCR4 dimerization 1) is dynamic, 2) increases with receptor expression level, and 3) can be disrupted by stabilizing an inactive receptor conformation. Ligand binding motifs reveal a ligand binding subpocket essential to modulate both CXCR4 basal activity and dimerization. This is relevant to develop new strategies to design CXCR4-targeting drugs.

G protein-coupled receptors (GPCRs) are regulated by complex molecular mechanisms, both in physiological and pathological conditions, and their signalling can be intricate. Many factors influence their signalling behaviour, 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 CXCR4 and ACKR3, both members of the GPCR superfamily, are important and established therapeutic targets in relation to cancer, HIV infection and inflammatory diseases. Therefore, it is crucial to understand how the signalling of these receptors works to be able to specifically target them. In this review, we discuss how the signalling pathways activated by CXCR4 and ACKR3 can vary in different situations. G protein signalling 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 signalling spectrum upon heteromerisation with other receptors, such as CXCR4, endothelial growth factor receptor (EGFR) or the α1-adrenergic receptor (α1-AR). Also, CXCR4 forms heteromers with CCR2, CCR5, the Na+/H+ exchanger regulatory factor 1 (NHERF1), CXCR3, α1-AR and the opioid receptors, which results in differential signalling to 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 signalling properties. In this review, we also provide an overview of the currently known critical amino acids involved in CXCR4 and ACKR3 signalling.

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 antibodies. Naloxone also hindered antinociception by the TRPA1 inhibitor HC-030031. RvD1 did not simulate 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. Thus, we hypothesize a dysequillibrium in the pro- and antinociceptive machinery in inflammatory pain.

PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg²⁺ or Mn²⁺, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPol-mediated reactions, and the individual contribution of the putative carboxylate residues (Asp¹¹⁴, Glu¹¹⁶ and Asp²⁸⁰) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu¹¹⁶ as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn²⁺. Herein, we evidenced that PrimPol Glu¹¹⁶ contributes to error-prone tolerance of 8oxodG more markedly when both Mg²⁺ and Mn²⁺ ions are present. Moreover, Glu¹¹⁶ was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn²⁺ is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu¹¹⁶ was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn²⁺ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.

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.