The Uncoupled State of the Human Formyl Peptide Receptor

Abstract

The formyl peptide receptor (FPR) has been widely used to study the kinetics of the interaction between ligand, receptor and G protein with real-time fluorescence methods. Because the wild type receptor rapidly signals, and is then desensitized and internalized once occupied by ligand, it has been difficult to study the uncoupled receptor form. We have examined a mutant form of the FPR expressed in U937 cells that does not bind G protein and is thus ideal to study the uncoupled form of the FPR in the intact cell. Using kinetic flow cytometry, we have measured the dissociation kinetics of a fluorescent ligand from this mutant in intact. permeabilized and fixed cells. We observed a novel uncoupled receptor form in the intact cell with a dramatically reduced off-rate (-0.02 s^-1) from LR in a broken cell preparation (～0.2 s^-1). Both receptor forms are retained in the presence of formaldehyde. We also observed this novel receptor form coexisting with the LRG complex when the wild type receptor is fixed in neutrophils or transfectants. These results lead us to suggest that there are distinct receptor structures in cells and membranes and that only a fraction of receptors in intact cells exist in the uncoupled form.     

The Role of Water in Activation Mechanism of Human N-Formyl Peptide Receptor 1 (FPR1) Based on Molecular Dynamics Simulations

The Formyl Peptide Receptor 1 (FPR1) is an important chemotaxis receptor involved in various aspects of host defense and inflammatory processes. We constructed a model of FPR1 using as a novel template the chemokine receptor CXCR4 from the same branch of the phylogenetic tree of G-protein-coupled receptors. The previously employed template of rhodopsin contained a bulge at the extracellular part of TM2 which directly influenced binding of ligands. We also conducted molecular dynamics (MD) simulations of FPR1 in the apo form as well as in a form complexed with the agonist fMLF and the antagonist tBocMLF in the model membrane. During all MD simulation of the fMLF-FPR1 complex a water molecule transiently bridged the hydrogen bond between W254^6.48 and N108^3.35 in the middle of the receptor. We also observed a change in the cytoplasmic part of FPR1 of a rotamer of the Y301^7.53 residue (tyrosine rotamer switch). This effect facilitated movement of more water molecules toward the receptor center. Such rotamer of Y301^7.53 was not observed in any crystal structures of GPCRs which can suggest that this state is temporarily formed to pass the water molecules during the activation process. The presence of a distance between agonist and residues R201^5.38 and R205^5.42 on helix TM5 may suggest that the activation of FPR1 is similar to the activation of β-adrenergic receptors since their agonists are separated from serine residues on helix TM5. The removal of water molecules bridging these interactions in FPR1 can result in shrinking of the binding site during activation similarly to the shrinking observed in β-ARs. The number of GPCR crystal structures with agonists is still scarce so the designing of new ligands with agonistic properties is hampered, therefore homology modeling and docking can provide suitable models. Additionally, the MD simulations can be beneficial to outline the mechanisms of receptor activation and the agonist/antagonist sensing.     

Structural Determinants for the Interaction of Formyl Peptide Receptor 2 with Peptide Ligands

Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-281^7.32 is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-281^7.32 might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D281^7.32G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides.     

Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 10⁸ M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.     

Cathepsin G-regulated Release of Formyl Peptide Receptor Agonists Modulate Neutrophil Effector Functions

Neutrophil serine proteases play an important role in inflammation by modulating neutrophil effector functions. We have previously shown that neutrophils deficient in the serine proteases cathepsin G and neutrophil elastase (CG/NE neutrophils) exhibit severe defects in chemokine CXCL2 release and reactive oxygen species (ROS) production when activated on immobilized immune complex. Exogenously added active CG rescues these defects, but the mechanism remains undefined. Using a protease-based proteomic approach, we found that, in vitro, the addition of exogenous CG to immune complex-stimulated CG/NE neutrophils led to a decrease in the level of cell-associated annexin A1 (AnxA1) and cathelin-related antimicrobial peptide (CRAMP), both known inflammatory mediators. We further confirmed that, in vivo, CG was required for the extracellular release of AnxA1 and CRAMP in a subcutaneous air pouch model. In vitro, CG efficiently cleaved AnxA1, releasing the active N-terminal peptide Ac2-26, and processed CRAMP in limited fashion. Ac2-26 and CRAMP peptides enhanced the release of CXCL2 by CG/NE neutrophils in a dose-dependent manner via formyl peptide receptor (FPR) stimulation. Blockade of FPRs by an antagonist, Boc2 (t-Boc-Phe-d-Leu-Phe-d-Leu-Phe), abrogates CXCL2 release, whereas addition of FPR agonists, fMLF and F2L, relieves Boc2 inhibition. Furthermore, the addition of active CG, but not inactive CG, also relieves Boc2 inhibition. These findings suggest that CG modulates neutrophil effector functions partly by controlling the release (and proteolysis) of FPR agonists. Unexpectedly, we found that mature CRAMP, but not Ac2-26, induced ROS production through an FPR-independent pathway.     

Regulation of Apolipoprotein A-I Gene Expression in Human Macrophages by Oxidized Low-Density Lipoprotein

Biochemistry (Moscow) - Apolipoprotein A-I (ApoA-I) is a key component of reverse cholesterol transport in humans. In the previous studies, we demonstrated expression of the apoA-I gene in human...     

Activated Human Mast Cells Induce LOX-1-Specific Scavenger Receptor Expression in Human Monocyte-Derived Macrophages

Objective

Activated mast cells in atherosclerotic lesions degranulate and release bioactive compounds capable of regulating atherogenesis. Here we examined the ability of activated human primary mast cells to regulate the expression of the major scavenger receptors in cultured human primary monocyte-derived macrophages (HMDMs).

Results

Components released by immunologically activated human primary mast cells induced a transient expression of lectin-like oxidized LDL receptor (LOX-1) mRNA in HMDMs, while the expression of two other scavenger receptors, MSR1 and CD36, remained unaffected. The LOX-1-inducing secretory components were identified as histamine, tumor necrosis factor alpha (TNF-α), and transforming growth factor beta (TGF-β1), which exhibited a synergistic effect on LOX-1 mRNA expression. Histamine induced a transient expression of LOX-1 protein. Mast cell –induced increase in LOX-1 expression was not associated with increased uptake of oxidized LDL by the macrophages.

Conclusions

Mast cell-derived histamine, TNF-α, and TGF-β1 act in concert to induce a transient increase in LOX-1 expression in human primary monocyte-derived macrophages. The LOX-1-inducing activity potentially endows mast cells a hitherto unrecognized role in the regulation of innate immune reactions in atherogenesis.     

Synthesis of a Gene Coding for Human Vasoactive Intestinal Peptide (VIP)

Abstract

A gene coding for human Vasoactive Intestinal Peptide (VIP) was designed as a double-stranded 99 base pair DNA sequence. The sixteen fragments of the gene were chemically synthesized using a solid-phase phosphoramidite triester coupling approach and enzymatically assembled using T4 DNA ligase. The resulting gene was cloned into pBR322 and sequenced using the Maxam-Gilbert sequencing procedure.     

RETRACTED ARTICLE: Astrocytes Regulate Differentiation and Glutamate Uptake of Glioma Stem Cells via Formyl Peptide Receptor

Cellular and Molecular Neurobiology -     

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

Relaxin, an emerging pharmaceutical treatment for acute heart failure, activates the relaxin family peptide receptor (RXFP1), which is a class A G-protein-coupled receptor. In addition to the classic transmembrane (TM) domain, RXFP1 possesses a large extracellular domain consisting of 10 leucine-rich repeats and an N-terminal low density lipoprotein class A (LDLa) module. Relaxin-mediated activation of RXFP1 requires multiple coordinated interactions between the ligand and various receptor domains including a high affinity interaction involving the leucine-rich repeats and a predicted lower affinity interaction involving the extracellular loops (ELs). The LDLa is essential for signal activation; therefore the ELs/TM may additionally present an interaction site to facilitate this LDLa-mediated signaling. To overcome the many challenges of investigating relaxin and the LDLa module interactions with the ELs, we engineered the EL1 and EL2 loops onto a soluble protein scaffold, mapping specific ligand and loop interactions using nuclear magnetic resonance spectroscopy. Key EL residues were subsequently mutated in RXFP1, and changes in function and relaxin binding were assessed alongside the RXFP1 agonist ML290 to monitor the functional integrity of the TM domain of these mutant receptors. The outcomes of this work make an important contribution to understanding the mechanism of RXFP1 activation and will aid future development of small molecule RXFP1 agonists/antagonists.     

Novel Mutations and Deletions of the KIT (Steel Factor Receptor) Gene in Human Piebaldism

Piebaldism is an autosomal dominant genetic disorder of pigmentation characterized by white patches of skin and hair. Melanocytes are lacking in these hypopigmented regions, the result of mutations of the KIT gene, which encodes the cell surface receptor for steel factor (SLF). We describe the analysis of 26 unrelated patients with piebaldism-like hypopigmentation—17 typical patients, 5 with atypical clinical features or family histories, and 4 with other disorders that involve white spotting. We identified novel pathologic mutations or deletions of the KIT gene in 10 (59%) of the typical patients, and in 2 (40%) of the atypical patients. Overall, we have identified pathologic KIT gene mutations in 21 (75%) of 28 unrelated patients with typical piebaldism we have studied. Of the patients without apparent KIT mutations, none have apparent abnormalities of the gene encoding SLF itself (MGF), and genetic linkage analyses in two of these families are suggestive of linkage of the piebald phenotype to KIT. Thus, most patients with typical piebaldism appear to have abnormalities of the KIT gene.     

人成骨蛋白成熟肽基因在毕氏酵母(Pichia Yeast)中的高效表达

采用PCR方法,根据文献报道的人成骨蛋白（osteogenic protein-1,OP-1）成熟肽基因序列,设计并合成一对引物,从构建的含人成骨蛋白基因的质粒中扩增获得大小为420bp的DNA片段,连接到pGEM-T载体进行测序,证明获得人成骨蛋白成熟肽基因片段,并以pPIC9K为表达载体构建重组表达质粒,转化大肠杆菌细胞,经鉴定的阳性重组质粒并线形化,电转化毕氏酵母细胞GS115,于30℃进行甲醇诱导分泌表达,表达产物存在于培养基中,占分泌蛋白的10％.重组表达产物进行Western Blot可以检测到重组表达产物,ELISA检测其具有特异性结合活性.     

Regulation of Phagocytosis in Macrophages by Neuraminidase 1

The differentiation of monocytes into macrophages and dendritic cells is accompanied by induction of cell-surface neuraminidase 1 (Neu1) and cathepsin A (CathA), the latter forming a complex with and activating Neu1. To clarify the biological importance of this phenomenon we have developed the gene-targeted mouse models of a CathA deficiency (CathA^S190A) and a double CathA/Neu1 deficiency (CathA^S190A-Neo). Macrophages of CathA^S190A-Neo mice and their immature dendritic cells showed a significantly reduced capacity to engulf Gram-positive and Gram-negative bacteria and positively and negatively charged polymer beads as well as IgG-opsonized beads and erythrocytes. Properties of the cells derived from CathA^S190A mice were indistinguishable from those of wild-type controls, suggesting that the absence of Neu1, which results in the increased sialylation of the cell surface proteins, probably affects multiple receptors for phagocytosis. Indeed, treatment of the cells with purified mouse Neu1 reduced surface sialylation and restored phagocytosis. Because Neu1-deficient cells showed reduced internalization of IgG-opsonized sheep erythrocytes whereas binding of the erythrocytes to the cells at 4 °C persisted, we speculate that the absence of Neu1 in particular affected transduction of signals from the Fc receptors for immunoglobulin G (FcγR). Indeed the macrophages from the Neu1-deficient mice showed increased sialylation and impaired phosphorylation of FcγR as well as markedly reduced phosphorylation of Syk kinase in response to treatment with IgG-opsonized beads. Altogether our data suggest that the cell surface Neu1 activates the phagocytosis in macrophages and dendritic cells through desialylation of surface receptors, thus, contributing to their functional integrity.