Milligram production and biological activity characterization of the human chemokine receptor CCR3.
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ABSTRACT:
SUBMITTER: Wang M
PROVIDER: S-EPMC3670934 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis.
| S-EPMC2637981 | biostudies-literature
Project description:CC motif chemokine receptor 3 (CCR3) is a Class A G protein-coupled receptor (GPCR) mainly responsible for the cellular trafficking of eosinophils. As such, it plays key roles in inflammatory conditions, such as asthma and arthritis, and the metastasis of many deadly forms of cancer. However, little is known about how CCR3 functionally interacts with its bilayer environment. Here, we investigate cholesterol binding sites in silico through Coarse-Grained Molecular Dynamics (MD) and Pylipid analysis using an extensively validated homology model based on the crystal structure of CCR5. These simulations identified several cholesterol binding sites containing Cholesterol Recognition/Interaction Amino Acid Consensus motif (CRAC) and its inversion CARC motifs in CCR3. One such site, a CARC site in TM1, in conjunction with aliphatic residues in TM7, emerged as a candidate for future investigation based on the cholesterol residency time within the binding pocket. This site forms the core of a cholesterol binding site previously observed in computational studies of CCR2 and CCR5. Most importantly, these cholesterol binding sites are conserved in other chemokine receptors and may provide clues to cholesterol regulation mechanisms in this subfamily of Class A GPCRs.
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Project description:Chemokine receptor CCR3 is highly expressed by eosinophils and signals in response to binding of the eotaxin family of chemokines, which are up-regulated in allergic disorders. Consequently, CCR3 blockade is of interest as a possible therapeutic approach for the treatment of allergic disease. We have described previously a bispecific antagonist of CCR1 and CCR3 named UCB35625 that was proposed to interact with the transmembrane residues Tyr-41, Tyr-113, and Glu-287 of CCR1, all of which are conserved in CCR3. Here, we show that cells expressing the CCR3 constructs Y113A and E287Q are insensitive to antagonism by UCB35625 and also exhibit impaired chemotaxis in response to CCL11/eotaxin, suggesting that these residues are important for antagonist binding and also receptor activation. Furthermore, mutation of the residue Tyr-113 to alanine was found to turn the antagonist UCB35625 into a CCR3 agonist. Screens of small molecule libraries identified a novel specific agonist of CCR3 named CH0076989. This was able to activate eosinophils and transfectants expressing both wild-type CCR3 and a CCR1-CCR3 chimeric receptor lacking the CCR3 amino terminus, indicating that this region of CCR3 is not required for CH0076989 binding. A direct interaction with the transmembrane helices of CCR3 was supported by mutation of the residues Tyr-41, Tyr-113, and Glu-287 that resulted in complete loss of CH0076989 activity, suggesting that the compound mimics activation by CCL11. We conclude that both agonists and antagonists of CCR3 appear to occupy overlapping sites within the transmembrane helical bundle, suggesting a fine line between agonism and antagonism of chemokine receptors.
| S-EPMC2151197 | biostudies-literature
Project description:Human C-C motif ligand 16 (CCL16) is a chemokine that is distinguished by a large cleavable C-terminal extension of unknown significance. Conflicting data have been reported concerning its tissue distribution and modulation of expression, rendering the biological function of CCL16 enigmatic. Here, we report an integrated approach to the characterisation of this chemokine, including a re-assessment of its expression characteristics as well as a biophysical investigation with respect to its structure and dynamics. Our data indicate that CCL16 is chiefly synthesised by hepatocytes, without an appreciable response to mediators of inflammation, and circulates in the blood as a full-length protein. While the crystal structure of CCL16 confirms the presence of a canonical chemokine domain, molecular dynamics simulations support the view that the C-terminal extension impairs the accessibility of the glycosaminoglycan binding sites and may thus serve as an intrinsic modulator of biological activity.
| S-EPMC9687583 | biostudies-literature
Project description:Cloning and sequencing of the upstream region of the gene of the CC chemokine HCC-1 led to the discovery of an adjacent gene coding for a CC chemokine that was named "HCC-2." The two genes are separated by 12-kbp and reside in a head-to-tail orientation on chromosome 17. At variance with the genes for HCC-1 and other human CC chemokines, which have a three-exon-two-intron structure, the HCC-2 gene consists of four exons and three introns. Expression of HCC-2 and HCC-1 as studied by Northern analysis revealed, in addition to the regular, monocistronic mRNAs, a common, bicistronic transcript. In contrast to HCC-1, which is expressed constitutively in numerous human tissues, HCC-2 is expressed only in the gut and the liver. HCC-2 shares significant sequence homology with CKbeta8 and the murine chemokines C10, CCF18/MRP-2, and macrophage inflammatory protein 1gamma, which all contain six instead of four conserved cysteines. The two additional cysteines of HCC-2 form a third disulfide bond, which anchors the COOH-terminal domain to the core of the molecule. Highly purified recombinant HCC-2 was tested on neutrophils, eosinophils, monocytes, and lymphocytes and was found to exhibit marked functional similarities to macrophage inflammatory protein 1alpha. It is a potent chemoattractant and inducer of enzyme release in monocytes and a moderately active attractant for eosinophils. Desensitization studies indicate that HCC-2 acts mainly via CC chemokine receptor CCR1.
| S-EPMC27666 | biostudies-literature
Project description:Chemokine (C-C-motif) receptor 3 (CCR3), playing an important role in endometrium related metabolic pathways, may influence the onset of menarche. To test linkage and/or association between CCR3 polymorphisms with the variation of age at menarche (AAM) in Caucasian females, we recruited a sample of 1,048 females from 354 Caucasian nuclear families and genotyped 16 SNPs spanning the entire CCR3 gene. Linkage disequilibrium and haplotype blocks were inferred by Haploview. Both single-SNP markers and haplotypes were tested for linkage and/or association with AAM using QTDT (quantitative transmission disequilibrium test). We also tested associations between CCR3 polymorphisms and AAM in a selected random sample of daughters using ANOVA (analysis of variance). We identified two haplotype blocks. Only block two showed significant results. After correction for multiple testing, significant total associations of SNP7, SNP9 with AAM were detected (P = 0.009 and 0.006, respectively). We also detected significant within-family association of SNP9 (P = 0.01). SNP14 was linked to AAM (P = 0.02) at the nominal level. In addition, there was evidence of significant total association and nominal significant linkage (P = 0.008 and 0.03, respectively) with AAM for the haplotype AGA reconstructed by SNP7, SNP9 and SNP13. ANOVA confirmed the results by QTDT. For the first time we reported that CCR3 is linked and associated with AAM variation in Caucasian women. However, further studies are necessary to substantiate our conclusions.
| S-EPMC1829487 | biostudies-literature
Project description:Bone metastasis remains the most frequent and the deadliest complication of prostate cancer (PCa). Mechanisms leading to the homing of tumor cells to bone remain poorly characterized. Role of chemokines in providing navigational cues to migrating cancer cells bearing specific receptors is well established. Bone is an adipocyte-rich organ since 50 to 70% of the adult bone marrow (BM) volume comprise bone marrow adipocytes (BM-Ads), which are likely to produce chemokines within the bone microenvironment. Using in vitro migration assays, we demonstrated that soluble factors released by human primary BM-Ads are able to support the directed migration of PCa cells in a CCR3-dependent manner. In addition, we showed that CCL7, a chemokine previously involved in the CCR3-dependent migration of PCa cells outside of the prostate gland, is released by human BM-Ads. These effects are amplified by obesity and ageing, two clinical conditions known to promote aggressive and metastatic PCa. In human tumors, we found an enrichment of CCR3 in bone metastasis vs. primary tumors at mRNA levels using Oncomine microarray database. In addition, immunohistochemistry experiments demonstrated overexpression of CCR3 in bone versus visceral metastases. These results underline the potential importance of BM-Ads in the bone metastatic process and imply a CCR3/CCL7 axis whose pharmacological interest needs to be evaluated.
| S-EPMC7922974 | biostudies-literature
Project description:Human immunodeficiency virus type 1 (HIV-1) infection in mononuclear phagocyte lineage cells (monocytes, macrophages, and microglia) is a critical component in the pathogenesis of viral infection. Viral replication in macrophages serves as a reservoir, a site of dissemination, and an instigator for neurological sequelae during HIV-1 disease. Recent studies demonstrated that chemokine receptors are necessary coreceptors for HIV-1 entry which determine viral tropism for different cell types. To investigate the relative contribution of the beta-chemokine receptors CCR3 and CCR5 to viral infection of mononuclear phagocytes we utilized a panel of macrophage-tropic HIV-1 strains (from blood and brain tissue) to infect highly purified populations of monocytes and microglia. Antibodies to CD4 (OKT4A) abrogated HIV-1 infection. The beta chemokines and antibodies to CCR3 failed to affect viral infection of both macrophage cell types. Antibodies to CCR5 (3A9) prevented monocyte infection but only slowed HIV replication in microglia. Thus, CCR5, not CCR3, is an essential receptor for HIV-1 infection of monocytes. Microglia express both CCR5 and CCR3, but antibodies to them fail to inhibit viral entry, suggesting the presence of other chemokine receptors for infection of these cells. These studies demonstrate the importance of mononuclear phagocyte heterogeneity in establishing HIV-1 infection and persistence.
| S-EPMC109817 | biostudies-literature
Project description:We recently reported the discovery of preclinical CCR5 inhibitor GSK214096, 1 (J. Med. Chem. 2011, 54, 756). Detailed characterization of 1 revealed that it exists as a mixture of four separable atropisomers A-D. The two slow-interconverting pairs of rotamers A + B and C + D were separated and further characterized. HIV and CCR5-mediated chemotaxis data strongly suggest that the antiviral potency of 1 is due to rotamers A + B and not C + D. Furthermore, integrated UV, vibrational circular dichroism VCD and computational approach allowed to determine the M chirality in C + D (and P chirality in A + B). These findings imply additional avenues to be pursued toward new CCR5 antagonists.
| S-EPMC4265823 | biostudies-other
Project description:BackgroundCC chemokine receptor 7 (CCR7) is a member of G-protein-coupled receptor family and mediates chemotactic migration of immune cells and different cancer cells induced via chemokine (C-C motif) ligand 19 (CCL19) or chemokine (C-C motif) ligand 21 (CCL21). Hence, the identification of blockade antibodies against CCR7 could lead to the development of therapeutics targeting metastatic cancer.MethodsCCR7 was purified and stabilized in its active conformation, and antibodies specific to purified CCR7 were screened from the synthetic M13 phage library displaying humanized scFvs. The in vitro characterization of selected scFvs identified two scFvs that exhibited CCL19-competitive binding to CCR7. IgG4's harboring selected scFv sequences were characterized for binding activity in CCR7+ cells, inhibitory activity toward CCR7-dependent cAMP attenuation, and the CCL19 or CCL21-dependent migration of CCR7+ cells.ResultsAntibodies specifically binding to purified CCR7 and CCR7+ cells were isolated and characterized. Two antibodies, IgG4(6RG11) and IgG4(72C7), showed ligand-dependent competitive binding to CCR7 with KD values of 40 nM and 50 nM, respectively. Particularly, IgG4(6RG11) showed antagonistic activity against CCR7, whereas both antibodies significantly blocked the ligand-induced migration and invasion activity of CCR7+ cancer cells.ConclusionsTwo antibody clones were successfully identified from a synthetic scFv-displaying phage library using purified recombinant CCR7 as an antigen. Antibodies specifically bound to the surface of CCR7+ cells and blocked CCR7+ cell migration. Particularly, 6RG11 showed antagonist activity against CCR7-dependent cAMP attenuation.
| S-EPMC9372883 | biostudies-literature