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1
Wang, Jixin. "Bioinformatic analysis of chicken chemokines, chemokine receptors, and Toll-like receptor 21." Texas A&M University, 2006. http://hdl.handle.net/1969.1/4212.
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Chemokines triggered by Toll-like receptors (TLRs) are small chemoattractant
proteins, which mainly regulate leukocyte trafficking in inflammatory reactions via
interaction with G protein-coupled receptors. Forty-two chemokines and 19 cognate
receptors have been found in the human genome. Prior to this study, only 11 chicken
chemokines and 7 receptors had been reported. The objectives of this study were to
identify systematically chicken chemokines and their cognate receptor genes in the
chicken genome and to annotate these genes and ligand-receptor binding by a
comparative genomics approach. Twenty-three chemokine and 14 chemokine receptor
genes were identified in the chicken genome. The number of coding exons in these genes
and the syntenies are highly conserved between human, mouse, and chicken although the
amino acid sequence homologies are generally low between mammalian and chicken
chemokines. Chicken genes were named with the systematic nomenclature used in
humans and mice based on phylogeny, synteny, and sequence homology. The independent nomenclature of chicken chemokines and chemokine receptors suggests that
the chicken may have ligand-receptor pairings similar to mammals.
The TLR family represents evolutionarily conserved components of the patternrecognizing
receptors (PRRs) of the innate immune system that recognize specific
pathogen-associated molecular patterns (PAMPs) through their ectodomains (ECDs).
TLR's ECDs contain 19 to 25 tandem copies of leucine-rich repeat (LRR) motifs. TLRs
play important roles in the activation of pro-inflammatory cytokines, chemokines and
modulation of antigen-specific adaptive immune responses. To date, nine TLRs have
been reported in chicken, along with a non-functional TLR8. Two non-mammalian
TLRs, TLR21 and TLR22, have been identified in pufferfish and zebrafish. The
objectives of this study were to determine if there is the existence of chicken genes
homologous to fish-specific TLRs, and if possible ligands of these receptors exist. After
searching the chicken genome sequence and EST database, a novel chicken TLR
homologous to fish TLR21 was identified. Phylogenetic analysis indicated that the
identified chicken TLR is the orthologue of TLR21 in fish. Bioinformatic analysis of
potential PAMP binding sites within LRR insertions showed that CpG DNA is the
putative ligand of this receptor.
2
Arnatt, Christopher Kent. "DEVELOPMENT OF ANTAGONISTS TARGETING CHEMOKINE RECEPTOR CCR5 AND THE CHEMOKINE RECEPTOR CCR5 – MU OPIOID RECEPTOR HETERODIMER." VCU Scholars Compass, 2013. http://scholarscompass.vcu.edu/etd/517.
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The chemokine receptor CCR5 (CCR5) plays an integral role within the inflammatory network of cells. Importantly, CCR5 is a mediator in several disease states and can be targeted using small molecule antagonists. Within this work, CCR5’s role in prostate cancer and HIV/AIDS has been exploited in order to develop potential therapeutics and probes. First, a series of novel compounds was designed by using pharmacophore-based drug design based upon known CCR5 antagonists and molecular modeling studies of the CCR5 receptor’s three-dimensional conformation. Once synthesized, these compounds were tested for their CCR5 antagonism and their anti-proliferative effects in several prostate cancer cell lines. The data from both the calcium mobilization studies and the anti-proliferation studies suggests that the compounds synthesized have activity as CCR5 antagonists and as anti-proliferative agents in certain prostate cancer cell lines. In addition, a bivalent ligand containing both a mu opioid receptor (MOR) and a CCR5 antagonist pharmacophore was designed and synthesized in order to study the pharmacological profile of the putative CCR5-MOR heterodimer and its relation with NeuroAIDS. The structural-activity relationship between the bivalent ligand and the heterodimer was studied with radio-ligand binding assays, functional assays, HIV-1 fusion assays, cell fusion assays, and in silico molecular dynamics. The subsequent bivalent ligand was proven to be a potent inhibitor in both an artificial cell fusion assay mimicking HIV invasion and a native HIV-1 invasion assay using live virus. In all, two novel sets of compounds were synthesized that targeted either CCR5 or the CCR5-MOR heterodimer. For the CCR5 antagonists, as leads for prostate cancer therapeutics, further work needs to be done to ascertain and develop their structure-activity-relationship. This library of novel compounds was shown as promising leads as CCR5 and anti-prostate cancer agents. The bivalent ligand targeting the CCR5-MOR heterodimer proved to be a potent and tissue-specific inhibitor for neuroAIDS where the known treatment, maraviroc, is less efficacious and fails to inhibit virus entry in the presence of morphine. Both projects illustrate the roles that CCR5 plays in these two unique diseases.
3
Davis, Christopher Nathan. "Mammalian and viral chemokines provide insight into the mechanism of chemokine receptor activation." [Gainesville, Fla.] : University of Florida, 2004. http://purl.fcla.edu/fcla/etd/UFE0006481.
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Kiss, Debra Lois. "Regulation of the Chemokine Receptors CXCR4, CXCR7 , and the Androgen Receptor in Prostate Cancer." Thesis, Griffith University, 2013. http://hdl.handle.net/10072/367690.
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The chemokine receptor CXCR4 contributes to tumour cell migration and invasion during the progression of prostate cancer. In particular, this pathway is central to the metastasis of prostate cancer to the bone marrow. Limited therapeutic options exist for prostate cancer patients who have progressed to advanced metastatic disease, and pharmacological interference of the chemokine network may serve to control tumour cell dissemination and the establishment of metastasis. A more detailed knowledge of the mechanisms regulating chemokine receptors is required, in order to further characterise and explore the capacity and effectiveness of targeting these pathways for therapeutic intervention in prostate cancer.
Here, the regulation of CXCR4 protein expression and function was investigated in relation to androgens and the extracellular matrix. Accumulating evidence of CXCR4 regulation by androgens and the androgen receptor have indicated that androgens not only promote the growth and development of prostate cancer, but may actively contribute to the metastatic progression of prostate through modulation of the chemokine network. In the current study, the endogenous protein expression and functionality of the androgen receptor were firstly characterised in the androgen-insensitive prostate cancer cell lines DU145 and PC3, using the androgen-sensitive LNCaP cells as a basis for comparison. Investigations were performed using two-dimensional culture in conjunction with the more physiologically relevant three-dimensional in vitro culture model.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Eskitis Institute for Cell and Molecular Therapies
Science, Environment, Engineering and Technology
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5
Khan, Abid. "CXCR4 chemokine receptor antagonists : new metallodrugs." Thesis, University of Hull, 2009. http://hydra.hull.ac.uk/resources/hull:10418.
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Chemokine receptors are a target of growing interest for new therapeutic drugs, as their role in multiple disease states has been demonstrated. The CXCR4/ CXCL12 pairing has been implicated in HIV and cancer, as well as chronic inflammatory diseases, including asthma and rheumatoid arthritis. HIV uses CXCR4 or CCR5 receptors in the key binding step of the infection process, leading to the idea that drugs could be developed to block this interaction. Cancer metastasis has also been linked to cellular communication via the chemokine pathways and hence, receptor antagonists could potentially inhibit this important pathway of disease progression. Small synthetic CXCR4 antagonists exist including AMD3100 (Mozobil®/Plerixafor), which has been identified as a potent CXCR4 antagonist exhibiting anti-HIV, anti-inflammatory and anti-tumour activity. Configurationally restricted analogues of AMD3100 complexed to metal ions have improved binding characteristics compared to AMD3100 and its metal complexes. Herein we report the binding of a new class of cyclen, cyclam and tris-cyclam based complexes in vitro. Compounds competed effectively in anti-CXCR4 competition assays with the tricyclam linear complex displaying improved binding characteristics. The difference in activity of the compounds is discussed in relation to the different possible binding interactions that are occurring. Furthermore, a monocyclam derivative conjugated to biotin competed effectively in competition with a CXCR4 mAb, however could not directly be detected via a fluorescent conjugated streptavidin molecule. Our most potent compound to date, copper(II) cross-bridged bicyclam was found to have a significant higher relative residence time in CXCR4 compared to AMD3100 and copper(II) AMD3100 in vitro. Moreover, copper(II) cross-bridged bicyclam was able to totally block CXCL12 induced and partially block serum induced, invasion of CXCR4 positive cancer cells with a higher potency than AMD3100 and copper(II) AMD3100. This shows the potential of using such a drug in the clinic. Using CXCR4 mutants, it has been shown that CXCR4 defective degradation and recycling increases invasion in breast cancer cells. Moreover the development of a multicellular tumour spheroid (MTS) is reported that could be used as a preclinical model in the evaluation of the anti-cancer activity of CXCR4 antagonists.
6
Wavre, Silene Tuija. "Endocytic regulation of chemokine receptor expression." Thesis, University College London (University of London), 2007. http://discovery.ucl.ac.uk/1446114/.
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The activity of stimulated cell surface signalling receptors is frequently regulated by endocytosis, which provides a mechanism to internalise and either degrade or reprogram the protein. Clathrin-mediated endocytosis (CME) is one of the principal mechanisms responsible for these events. Although clathrin and many of its associated proteins are relatively well characterised, many aspects of how CME operates are yet to be established. In particular, the early steps of internalisation, the formation of clathrin-coated pits (CCPs) and the mechanism by which receptors are recruited into CCPs remain controversial. This thesis investigates the early events of CME occurring at the plasma membrane using as a model various cell lines expressing CC Chemokine receptor 5 (CCR5), a G-protein coupled receptor (GPCR). Upon agonist binding, CCR5 undergoes rapid phosphorylation by a GPCR kinase (GRK) and protein kinase C (PKC) on four C-terminal serines, p-arrestin is subsequently recruited to the receptor, abrogates CCR5 interaction with the G- protein and links the receptor to the endocytic machinery by binding to AP-2 and clathrin. Using confocal immunofluorescence microscopy and electron microscopy, I show that, on agonist binding, CCR5 relocates in the plasma membrane and clusters into flat clathrin lattices. CCPs were observed to invaginate at the edge of these lattices and proteins from the endocytic machinery were identified by immunolabelling within these domains. As C-terminal serine phosphorylation has been found to be important for efficient agonist-induced internalisation of CCR5, the requirement for specific serines for plasma membrane relocation, association with clathrin lattices and endocytosis were analysed. A mutant lacking all serines failed to be recruited into flat clathrin lattices. Further analysis showed that specific GRK phosphorylation of CCR5 was sufficient for this recruitment, suggesting a distinct role for different kinases in receptor desensitisation and internalisation. This study brings new insights into the mechanism of recruitment of activated GPCRs into CCPs and reveals the importance of flat clathrin lattices in the formation of endocytic clathrin-coated vesicles and CME. Part of the results presented in this thesis have been published (Signoret et al., 2005) (see appendix).
7
Fouillet, Antoine. "Cytokines regulation of chemokine and chemokine receptor in relation to multiple sclerosis." Thesis, Sheffield Hallam University, 2008. http://shura.shu.ac.uk/19675/.
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Expression of chemokines CXCL10 and CCL2 is elevated within inflammatory lesions in the central nervous system (CNS) of multiple sclerosis (MS) patients, particularly in astrocytes. These chemokines play a critical role in the recruitment of inflammatory cells into the CNS during inflammation. However, the cerebrospinal fluid of MS patients also shows high levels of CXCL10 at the time of relapse but by contrast CCL2 is decreased. In the present study, the mechanisms controlling the synthesis and release of these two chemokines in MS were assessed in vitro using primary human brain astrocytes isolated from MS and non-MS individuals. Pro-inflammatory cytokines (interleukin-1beta , tumour necrosis factor and interferon-gamma) increased the expression of both CCL2 and CXCL10 by astrocytes at the mRNA and protein level, as determined by real time PCR and enzyme linked immunosorbent assays (ELISA), respectively. CCL2 binding to astrocytes was then determined to evaluate any autocrine action on astrocytes in a single astrocyte preparation. CCL2 bound constitutively and following cytokine treatment. CCL2-binding was not the result of the interaction with its receptor since astrocytes did not express CCR2 on this astrocyte culture. CCR2-independent binding of CCL2 was confirmed by the absence of intracellular signalling, evidenced by the lack of calcium influx as well as of Erk and Akt phosphorylation, in CCL2-treated astrocytes. Even though astrocytes expressed CXCR3, similar negative results on calcium influx and downstream signalling pathways were observed for CXCL10. D6 chemokine decoy receptor expression was then assessed in vitro and in situ to further investigate the mechanism(s) of chemokine binding to astrocytes. Cultured astrocytes constitutively expressed the D6 decoy receptor at the mRNA and protein level, but levels were unchanged following cytokine treatment. D6 was expressed in situ in MS normal appearing white matter and in control brain tissue, at both the mRNA and protein level. D6 expression was detected on neurons and microglia but not astrocytes using imunohistochemical methods. Incubation of frozen brain sections with biotinylated CCL2 resulted in partial co-localisation with D6 staining. Altogether, these results suggest a role for astrocytes in regulating inflammation through synthesis and secretion of CCL2 and CXCL10. Subsequently, CCL2 binding to astrocytes, either by binding to D6 decoy receptor or by alternative mechanisms, may establish a chemokine gradient in the CNS, and direct the migration of leukocytes.
8
Eltayeb, Sana. "Chemokine receptor expression and function in experimental autoimmune neuroimflammation /." Stockholm, 2007. http://diss.kib.ki.se/2007/978-91-7357-197-5/.
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Duchesnes, Cecile Emmanuelle. "Molecular characterisation of the chemokine receptor CCR3." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.407171.
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Martinelli, Roberta. "Molecular characterisation of the chemokine receptor CCR2." Thesis, Imperial College London, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.398841.
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Rahimpour, Rahbar. "Regulation of chemokine and chemokine receptor expression and function during the inflammatory response." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape17/PQDD_0028/NQ31108.pdf.
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Moyano, Clara. "Elucidating the signalling mechanisms of the CC chemokine receptor 5 upon chemokine stimulation." Thesis, University of East Anglia, 2011. https://ueaeprints.uea.ac.uk/33333/.
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13
Schweneker, Marc. "Identification and characterization of two novel proteins interacting with the chemokine- and HIV-1 co-receptor CCR5." [S.l.] : [s.n.], 2004. http://www.diss.fu-berlin.de/2004/22/index.html.
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Gaspar, Maria Manuela Carvalho. "Expression of chemokine and chemokine receptor networks during rejection and induction of transplantation tolerance." Thesis, University of Oxford, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413973.
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McRobbie, Graeme Walter John. "Configurationally restricted bis-tetraazamacrocyclic complexes : chemokine receptor antagonists." Thesis, University of Hull, 2009. http://hydra.hull.ac.uk/resources/hull:5855.
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The chemokine receptor CXCR4 is a trans-membrane protein which has been implicated in many physiological and pathological processes including cancer, rheumatoid arthritis and most significantly HIV replication. CXCR4 plays a vital role in embryonic development but is not essential at the post-development stage; therefore, it has been identified as a potential therapeutic target. Bis-macrocyclic drugs (e.g. AMDS 100) bind to aspartate residues on the CXCR4 surface and inhibit HIV replication by blocking the interaction of gp!20/gp41 with the protein. The incorporation of transition metals (e.g. zinc(II) and copper(II)) into the macrocyclic cavity increases anti-viral potency. The addition of a bridging ethylene unit to the macrocyclic framework locks the complex into a single configuration, potentially optimising the interaction with the receptor. A series of configurationally restricted macrocyclic compounds have been prepared utilising bis-aminal chemistry. Characterisation by X-ray crystallography and X-ray absorption spectroscopy has confirmed that the complexes possessing an ethylene bridge between adjacent nitrogen atoms are fixed in the trans-II configuration and that complexes containing an ethylene bridge between non-adjacent nitrogen atoms adopt the cis-V configuration. In addition, solution EXAFS has been used as a model to probe the binding of the complexes to aspartate residues on the receptor surface. The zinc(II) trans-II and copper(II) cis-V complexes reported here are more potent against HIV replication than AMDS 100 (IC₅₀ values against IIIB ; 0.00208 µM, 0.00491 µM and 0.018 µM respectively), confirming the importance of coordination interactions for potent binding to CXCR4 and also validating the strategy of configurationally fixing the macrocyclic unit for optimising receptor binding. It is believed that both thermodynamic and kinetic properties are important for effective binding to CXCR4.
16
Comerford, Iain. "Functional analysis of the chemokine receptor, CCX-CKR." Thesis, University of Glasgow, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.421114.
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Ahmed, Mohaned S. A. "New C-C chemokine receptor type 7 antagonists." Thesis, University of Bradford, 2016. http://hdl.handle.net/10454/14623.
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Chemokines are chemotactic cytokines which play an important role in the migration of immune cells to distant tissues or compartments within tissues. These proteins have also been demonstrated to play a major role in cancer metastasis. The C-C chemokine receptor type 7 (CCR7) is a member of the chemokine receptor family. CCR7 along with its ligands CCL19 and CCL21 plays an important role in innate immune response by trafficking of lymphocytes. In cancer, tumour cells expressing CCR7 migrate to lymphoid organs and thus disseminate to other organs. Neutralizing the interactions between CCL21/CCR7 would therefore be expected to inhibit the progression and metastasis of many different types of cancer to regional lymph nodes or distant organs. Our objective was to identify a potent small molecule antagonist of CCR7 as a prelude to the investigation of the role of this axis in cancer metastasis. In this study, we provided a brief description of chemokines and their role in health and disease with an emphasis on the CCR7/CCL19/CCL21 axis, as well as identification of a CCR7 antagonist “hit”. The potency of the CCR7 antagonist “hit” was optimised by synthesizing different CCR7 antagonist analogues. The “hit” optimization process has led to discover the most active compound amongst a series of different analogues which have the ability to bind and block CCR7 receptor. The efficacy of the most active compound and other analogues were evaluated in vitro using a calcium flux assay which is based on detecting fluorescent light emitted upon release of calcium ions. To identify a suitable cell line, which expresses CCR7 and capably respond to it, amongst a panel of cell lines for in vitro assessment of potency of synthesised compounds, we used Western blot assay and later by flow cytometry assay. The activity and selectivity of the most effective compound against CCR7 receptor was evaluated in vitro by other functional assays such as “configured agarose spot assay” and scratch assay. We first configured the existing under agarose assay to fulfil our requirements and then used it to assess activity and selectivity of compounds. The configured agarose spot assay also describes the application of the agarose spot for evaluation of cells chemotactic response to multiple chemokines under identical experiment conditions.
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Wong, Sue Way-Jing. "Chemokine receptor expression in B-cell lymphoproliferative disorders." Thesis, The University of Sydney, 2006. https://hdl.handle.net/2123/28099.
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Chemokine receptors are expressed by many cells, including lymphoid cells, and function to mediate cell trafficking and localisation. Normal B cells have been reported to express CXCR3, CXCR4, CXCRS, CCR6 and CCR7. However, changes in chemokine receptor expression during B-cell development and in B-cell lymphoproliferative disorders (BCLPDs) are incompletely understood, but could provide important information about normal B-cell development as well as behaviour of neoplastic B cells. The objective of this project was to perform a systematic study of chemokine receptor expression on B cells from normal subjects and from patients with a range of BCLPDs. Expression of the above chemokine receptors, and CCR5, was analysed by flow cytometry on lymphocytes from normal controls (n=20), and samples of follicular centre cell lymphoma (FCCL, n=16), precursor B-acute lymphoblastic leukaemia (Prec—B-ALL, n=16), chronic lymphocytic leukaemia (CLL, n=21), small cell lymphocytic lymphoma (SLL, n=9), hairy cell leukaemia (HCL, n=10) and other miscellaneous disorders (n=9). Normal B cells were typically positive for CXCR4, CXCR5 and CCR6, negative for CCR5, and variable for CCR7. Prec-B-ALL cells expressed CXCR4 but were negative for the other receptors. B-CLL cells lost expression of CCR6 but showed strong expression of CCR7. In contrast, the majority of SLL cells failed to express CCR7, but were otherwise similar to CLL cells. HCL cells showed absence of CXCR5 and CCR7, but interestingly, all but one case expressed CCR5, whilst CDZS-negative ‘variant’ HCL cells did not express CCRS. FCCL cells downregulated CXCR4 and CXCR5 expression and lost expression of CCR6 and CCR7. CXCR3 expression was highly variable on normal B cells and on cells from BCLPDs, possibly due to its lability during processing. Distinct changes in chemokine receptor expression accompany B—cell development, whilst some BCLPDs show characteristic alterations that may be useful for phenotyping and understanding biological behaviour.
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Kershaw, Tom. "On the intracellular trafficking of CC chemokine receptor 5." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444245/.
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CC chemokine receptor 5 (CCR5) is a G protein-coupled receptor (GPCR) involved in the recruitment of a subset of leukocytes to sites of inflammation. In addition, CCR5 functions as a co-receptor for several strains of the human immunodeficiency virus (HIV). The finding that agonist-induced internalisation of CCR5 can protect susceptible cells from infection in vitro has stimulated research demonstrating that activated receptors are phosphorylated on C-tail serine residues and bind p-arrestins, which couple them to the clathrin-mediated endocytic machinery. After internalisation, receptors are recycled to the cell surface via a perinuclear recycling compartment, identified as the recycling endosome. Here, I extend these findings, demonstrating that CCR5 also traffics through the trans- Golgi network (TGN), and thus, the perinuclear recycling compartment can be considered to comprise both recycling endosome and TGN elements. Moreover, I show that Rabll regulates trafficking through this compartment and that dynamin inhibition blocks the recycling of CCR5. In combination with other electron microscopy studies, my data support the notion that clathrin and dynamin are involved in the recycling of CCR5 from the recycling compartment. Through morphological and biochemical analysis, I also show that p-arrestins maintain an interaction with CCR5 into the recycling compartment. Kinetic analysis of receptor recycling suggests that p-arrestin2 acts a negative regulator of CCR5 recycling but my data do not discount the possibility that p-arrestins couple CCR5 to clathrin to effect recycling. In keeping with the sustained p-arrestin interaction, CCR5 molecules remain phosphorylated as they traffic to the recycling compartment, but contrary to previous reports showing that receptor C-tail phosphorylation is required for high-affinity p-arrestin binding, I show that a phosphorylation-deficient CCR5 mutant undergoes p-arrestin-dependent agonist-induced internalisation. In addition to its relevance for HIV biology and inflammation, this study contributes to an understanding of GPCR trafficking in general.
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Viney, Jonathan Mark. "Characterisation of antagonist binding sites on chemokine receptor CCR4." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/11128.
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CCR4 is a chemokine receptor notably expressed on T helper 2 and regulatory T cells. CCR4 binds the chemokines CCL17 and CCL22. These are involved in T cell homeostasis and inflammatory diseases including asthma and atopic dermatitis, making CCR4 a potential therapeutic target. Previous studies suggested that CCL22 is dominant over CCL17 with respect to ligand-induced internalisation and desensitisation of CCR4. The biology of CCR4 was investigated in this project using point mutational studies. A C-terminal lysine within the conserved helix VIII region was determined to be dispensable for CCL22-induced chemotaxis but required for CCL17-induced chemotaxis, suggesting that the two chemokines stabilised distinct receptor conformations. The highly conserved GluVII:06 of helix VII was shown to be critical for chemokine binding and receptor function. Seven small molecule allosteric antagonists of CCR4, supplied by GlaxoSmithKline, were hypothesised to bind either a classical intrahelical site (site 1) within the receptor or a novel intracellular site (site 2). 22 amino acids were predicted to be involved in the binding of the antagonists. Antagonist binding was indirectly investigated by inhibiting either function or chemokine binding of the receptor mutants. Mutation of leucine 118 in transmembrane helix III significantly reduced CCR4 sensitivity to site 1 antagonism in chemotaxis and chemokine-binding assays. Mutants of phenylalanine 305 and leucine 307 at the end of transmembrane helix VII also showed a reduction in antagonist 2 sensitivity. Direct investigation of the effects of mutation on antagonist binding was performed using tritium-labelled antagonists. Mutation of GluVII:06 prevented site 1 antagonist binding to CCR4. Further investigation of site 1 antagonist binding was hindered by high non-specific binding of the compounds. A low-affinity site for the tritium-labelled site 2 antagonist was identified on untransfected cells, possibly within endogenously expressed chemokine receptors. This antagonist therefore may have potential as a broad-spectrum chemokine receptor inhibitor.
21
Yu, Tian. "Role of atypical chemokine receptor-2 in ocular inflammation." Thesis, University of Aberdeen, 2015. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=229021.
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The atypical chemokine receptor-2 (ACKR2) is a chemokine decoy receptor that recognises pro-inflammatory CC chemokines. Many studies showed up-regulated inflammation and delayed resolution of inflammatory responses in ACKR2-/- mice. Furthermore, in the absence of ACKR2, lymphatic endothelial cells (LEC) fail to regulate the expression of pro-inflammatory CC chemokines leading to the excessive peri-lymphatic accumulation of leukocytes. As a result, the migration of antigen presenting cells (APC) through lymphatic vessels may be impaired due to lymphatic congestion. In addition, ACKR2 was shown to regulate lymphatic vessel density in the embryonic skin by regulating the proximity of pro-lymphangiogenic macrophages to LEC. Therefore, to address the role of ACKR2 and its significance in 1) APC migration and 2) inflammation-associated lymphangiogenesis, three models of ocular inflammation were used in this work, experimental autoimmune uveoretinitis (EAU), corneal graft rejection and herpes simplex keratitis (HSK). With regard to APC migration, in both EAU and HSK models, this process was fine-tuned to the level of disease in that migration was significantly compromised in ACKR2-/- mice during severe inflammation, but not under mild inflammatory conditions. Furthermore, while the severity of EAU was associated with the migration of APC, this was not so in HSK. In order to study lymphangiogenesis, the transparent avascular cornea provides a good substrate and corneal lymphangiogenesis was studied using both corneal graft model and HSK model. I found that lymphatic vessel density was increased in ACKR2-/- mice compared to wild type mice in corneal graft induced lymphangiogenesis (macrophage mediated), but not altered during early stages of HSK associated lymphangiogenesis (non-macrophage mediated). These findings confirmed that ACKR2 indirectly regulates the process of lymphangiogenesis in a macrophage dependent manner. Although the severity of HSK correlated with the level of lymphangiogenesis, this does not seem to correlate with viral load but rather associated with inflammatory infiltrations in the cornea.
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Dalton, Richard S. J. "Chemokine and chemokine receptor gene expression in human PBMCs in the early renal post-transplant period." Thesis, University of Southampton, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.418008.
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Subileau, Eva-Anne. "Chemokine and chemokine receptor expression by human brain endothelium : an 'in vitro' and 'in situ' study." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.434967.
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Petersen, Desiree C. "The role of chemokine and chemokine receptor genes in genetic susceptibility to HIV infection in South Africa." Thesis, Stellenbosch : Stellenbosch University, 2002. http://hdl.handle.net/10019.1/53158.
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Zhu, John Z. "Sulfotyrosines impart ligand specificity in a chemokine receptor model system." [Bloomington, Ind.] : Indiana University, 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3380144.
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Thesis (Ph.D.)--Indiana University, Dept. of Biochemistry, 2009.Title from PDF t.p. (viewed on Jul 20, 2010). Source: Dissertation Abstracts International, Volume: 70-12, Section: B, page: 7377. Advisers: Martin J. Stone; Carl E. Bauer.
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Ueda, Satoshi. "Synthetic studies on chemokine receptor CXCR4 and its specific antagonists." 京都大学 (Kyoto University), 2007. http://hdl.handle.net/2433/137117.
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Masters, Jennefer. "Myxoma virus induced activation of CC-chemokine receptor 5, CCR5." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0019/MQ53479.pdf.
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Zeelenberg, Ingrid Saskia. "Chemokine receptor signals: role in migration, invasion and cancer metastasis." [S.l. : Amsterdam : s.n.] ; Universiteit van Amsterdam [Host], 2004. http://dare.uva.nl/document/72874.
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Banfield, Graham Kaye. "Chemokine receptor 4 expression on T lymphocytes in allergic rhinitis." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.428323.
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Hurson, Catherine Eileen. "Expression and function of the atypical chemokine receptor CCX-CKR." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2718/.
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The ability to clear infections and repair injury is dependent on the coordinated migration of immune cells, or leukocytes. These cells can directly destroy invading pathogens and also produce a variety of bioactive factors that promote pathogen clearance. Interactions between immune cells occur both at the site of inflammation and in specialised lymphoid organs throughout the body. The efficiency and specificity of these interactions relies on the production of a family of molecules called chemotactic cytokines, or chemokines, that drive leukocyte migration. Cells express specific profiles of chemokine receptors to ensure they are directed to the appropriate location to exert their immunological function. The field of chemokine biology, already complex, has been further complicated by the discovery of a subfamily of receptors, the atypical chemokine receptors. These molecules lack the ability to couple to signal transduction pathways used by the other chemokine receptors, and are proposed to act as chemokine scavengers or transport molecules. The atypical chemokine receptor CCX-CKR was discovered more than a decade ago but its function in vivo remains unclear. At the beginning of my project, information about this molecule was very limited. The murine receptor binds the CC chemokines CCL19, CCL21 and CCL25, which have well-characterised and critical roles in the development and homeostasis of the immune system as well as in the immune response to infection. Thus, identification of this new receptor, which unlike classical receptors does not induce cell migration in response to ligand binding, presented some exciting possibilities as to how these processes might be regulated in vivo. Reports describing the pattern of expression of CCX-CKR have thus far provided only limited and sometimes contradictory information. Additionally, while in vitro studies from our lab have provided some important clues as to the potential role of the receptor, published in vivo studies were, at the time of commencing this work, limited to one report describing an unvalidated EGFP reporter knock-in transgenic mouse and a conflicting online resource detailing data generated using a LacZ reporter mouse. To understand the true function of this molecule, it is critical to know where it is expressed in vivo and to explore its function on these cells. In this project I set out with the aim of identifying murine tissues and cells expressing CCX-CKR, as well as examining its potential as an in vivo scavenger of chemokine. Related to this, I hoped to uncover any impact of deletion of CCX-CKR on lymphoid tissue cellularity and/or function, both in resting and inflamed conditions. In chapter 3, I present data that identify lymphoid tissues and “barrier” tissues as sites of robustly detectable CCX-CKR mRNA expression. I describe how I have established a novel fluorescent chemokine tetramer-based protocol for the detection of CCL19 receptors, with emphasis on the application of this protocol to identify CCX-CKR activity on specific cell subsets. Using this method, I present evidence that some CD11b+ CD11c+ myeloid subsets in the inguinal lymph node exhibit CCX-CKR dependent internalisation of chemokine. I also describe attempts to fractionate tissues to identify cell populations responsible for the detected whole-tissue expression of CCX-CKR mRNA. The results described in chapter 4 provide support for the hypothesis that CCX-CKR regulates levels of its ligands in vivo, with alterations in chemokine levels in serum and inguinal lymph nodes in the absence of CCX-CKR. I also present evidence demonstrating that deletion of the receptor can influence mRNA levels of the related receptor CCR7. Following on from this, chapter 5 details my analysis of the impact of CCX-CKR on the cellularity of various lymphoid compartments. I present evidence that CCX-CKR influences lymphocyte populations in the peritoneal cavity, with both innate-like and conventional lymphocytes significantly overrepresented in this compartment. The cellularity of the inguinal lymph node, but not the spleen, is subtly altered by deletion of the receptor. Splenic leukocyte cellularity is not affected, either in number or in localisation. In chapter 6, I turn my attention to the possible role of CCX-CKR during the inflammatory response by examining various experimental parameters during a short-term model of induced cutaneous inflammation. This study shows that CCX-CKR deletion alters the cellularity of the myeloid compartment in the draining lymph node and again highlights myeloid subsets as displaying CCX-CKR dependent chemokine internalisation. Finally, I present preliminary data suggesting a protective effect of CCX-CKR deletion during a long-term model of inflammation-driven tumorigenesis. Taken together, my data provide tentative support for the theory that CCX-CKR acts as a chemokine scavenger in vivo. They further indicate that CCX-CKR is involved in regulating cellularity of various lymphoid compartments both at rest and during induced inflammation. In chapter 7 I discuss in detail the implications of my findings in the context of work published since my project began, and highlight growing evidence to suggest a role for CCX-CKR in regulating immune function.
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Petit, Sarah Joanna. "Molecular Characterisation of the Chemokine CXCL16 and its Receptor CXCR6." Thesis, Imperial College London, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495432.
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The chemokine CXCL16 is selectively expressed by antigen presenting cells and unli ke most chemokines, is attached to the cell surface by means of a mucin stalk. This allows CXCL16 to function as an adhesion molecule, binding leukocytes such as T-cells which display the cognate receptor, CXCR6. Cleavage of CXCL16 by metalloproteinases can also release a soluble form of CXCLI6 which can induce the migration of CXCR6 expressing cells. In addition, membrane-bound CXCL16 can also ftinction as a scavenger receptor for a variety of ligands, including oxidised low density lipoprotein. Collectively, these functions support a role for both CXCL16 and CXCR6 in the process of atherosclerosis. The CXCL16:CXCR6 relationship appears to be monogamous and the nature of this interaction was investigated by a programme of mutagenesis used to examine CXCR6 function in vitro, assessed by adhesion, chemotaxis and ligand binding assays. CXCL16 function was also examined, in particular, the effects of a single nucleotide polymorphism associated with disease. In a third arm of the project, recombinant CXCL16 was produced in E. Coli using two different systems and subjected to assays of biological function. In contrast to other chemokine receptors, mutation of the CXCR6 N-terminus and inhibition of post-translational modifications were without effect on function. Likewise, N-terminal extension of CXCL16 by 24 amino-acids resulted in a protein with decent potency and efficacy in chemotaxis and not as anticipated, a CXCR6 antagonist. Cells expressing the CXCR6 mutants Asp-176-Asn and Glu274- Gln were unable to interact with soluble CXCLI6, suggesting that these residues are critical for ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the Glu-274-Gln mutant was able ·to promote robust adhesion to membrane-anchored CXCL16, suggesting that the soluble and membrane-bound forms of CXCL16 possess distinct conformations. Glycosylation of CXCL16 was found to be critical for cell-cell adhesion function, and a Ala-181-Val polymorphic variant of CXCL16 was found to have no capacity for mediating cell-cell adhesion, suggesting a potential relevance of this mutation in disease. Collectively, the data suggests that the CXCL16:CXCR6 interaction is distinct from other chemokine-chemokine interactions described in the literature, and that current models for chemokine receptor activation, in which the chemokine N-terminus ligand protrudes into an intrahelical pocket, do not apply to the CXCR6:CXCLI6 interaction. This may have implications for successful antagonism of the receptor by small molecules.
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Finley, Matthew James. "Molecular Basis for Kappa-Opioid Regulation of Chemokine Receptor Function." Diss., Temple University Libraries, 2009. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/62878.
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Molecular Biology and GeneticsPh.D.
Opioid receptor-mediated regulation of chemokine receptors is vital for the host immune response, development, and neurological function. Previous studies have demonstrated that the kappa opioid receptor (KOR) activation results in decreased infectivity of human immunodeficiency virus 1 (HIV-1) in human peripheral blood mononuclear cells (PBMCs). We have found this effect is due to down-regulation of the major HIV-1 co-receptors, CCR5 and CXCR4. Using molecular techniques, CCR5 and CXCR4 mRNA levels drop dramatically following KOR activation. To dissect the mechanism involved, we used transcription factor binding arrays and compared control cell extracts to KOR activated cell extracts. We determined that the interferon regulatory factors (IRFs) and signal transducers and activators of transcription (STATs) could be involved in the KOR-mediated repression of CCR5 and CXCR4 transcription and protein expression. Using chemical inhibitors and small interfering RNA (siRNA) molecules, we determined that JAK2, STAT3, and IRF2 are critical members of this signal transduction pathway. The understanding of these particular mechanisms should prove to be beneficial for the development of potential pharmacological agents targeted at HIV-1 binding and infection since virus infection requires expression of the co-receptors CXCR4 and CCR5. Understanding the molecular basis for KOR-induced inhibition of co-receptor expression may provide a basis for the development of KOR agonist-based therapeutics to treat individuals infected with HIV.
Temple University--Theses
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Dworzak, Jenny. "A role for neuronal chemokine receptor Cx3cr1 in Alzheimer's disease." Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708171.
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Hosogi, Hisahiro. "Chemokine receptor CXCR3 promotes colon cancer metastasis to lymph nodes." Kyoto University, 2008. http://hdl.handle.net/2433/135806.
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Bennett, Laura Danielle. "Trafficking regulation of the chemokine receptor CCR2B compared to CCR5." Thesis, University of York, 2012. http://etheses.whiterose.ac.uk/3169/.
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The closely-related CC chemokine receptors 2B and 5 are seven-transmembrane domain receptors coupled to heterotrimeric G proteins. The two receptors bind inflammatory chemokines and play important complementary roles in the recruitment of specific leukocyte sub-populations to sites of infection. To enable fine-tuning of cellular responses to chemokines, CCR2B and CCR5, like other GPCRs, can be desensitised in response to agonist stimulation or cross-talk with other receptors. This involves down-modulation of cell surface active receptor through two essential transportation events, endocytosis and recycling. The CCR5 endocytic and recycling pathways are well established and several mechanisms involved have been clearly defined. Conversely, less is known about the route followed by CCR2B upon stimulation. This study investigated the regulation, trafficking and fate of CCR2B in the context of THP-1 cells endogenously expressing the receptor and HEK293 transfectants. Comparison with CCR5 highlighted marked differences in the behavious of the two receptors. However, my initial findings indicate that certain aspects of the regulation of CCR5 as well as CCR2B may be cell type-dependent. Flow cytometry, immunofluorescence and biochemical analyses showed that unlike CCR5, internalised CCR2B can be both degraded and recycled following agonist stimulation. In HEK293, CCR2B follows an EGF receptor-like pathway, transiting through early endosomes containing EEA1, transferrin and Rab4, reaching CD63 and Lamp1 positive late endosomes/lysosomes before being degraded. Importantly, I showed that CCR2B cell surface molecules are N- and O-glycosylated, and only this glycosylated form of the receptor is targeted for agonist-induced degradation. This thesis also presents findings from proteomics approaches developed in an attempt to identify interacting proteins implicated in the trafficking of each receptor. This study brings new insights to the endocytic regulation of agonist-treated CC chemokine receptors, revealing receptor- and cell type-specific behaviours, which add complexity to a relatively conserved process.
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Ebert, Lisa Michelle. "The regulation of chemokine receptor expression upon T lymphocyte activation." Title page, contents and abstract only, 2002. http://web4.library.adelaide.edu.au/theses/09PH/09phe165.pdf.
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Reinecke, Bethany A. "Development of Bivalent Ligands Targeting the Putative Mu Opioid Receptor and Chemokine Receptor CXCR4 Heterodimer." VCU Scholars Compass, 2019. https://scholarscompass.vcu.edu/etd/5754.
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Human immunodeficiency virus (HIV) and opioid abuse have been described as synergistic epidemics. Pharmacologically, it has been found that opioids have the capacity to enhance HIV infection and replication. Research has shown that activation of the mu-opioid receptor (MOR) elevates the expression of the HIV-1 entry co-receptor CXCR4 on T-lymphocytes in the peripheral nervous system, thus allowing for enhanced viral entry and invasion. Although the exact mechanism for opioid modulation of CXCR4 expression and subsequent exacerbation of HIV is unknown, several hypotheses exist. One hypothesis is that MOR and CXCR4 are functionally interacting through the formation of a heterodimer. This hypothesis is supported by studies substantiating the ability for MOR and CXCR4 to form heterodimers with other GPCRs, and the finding that MOR and CXCR4 were co-expressed in several central and peripheral regions including immune cells. To test this hypothesis, a series of bivalent ligands containing both a mu opioid receptor (MOR) antagonist and a CXCR4 antagonist pharmacophore was designed and synthesized to understand the pharmacological role of the putative CXCR4-MOR heterodimer in opioid exacerbated HIV progression.
These bivalent ligands were evaluated for their binding and functional activities in radioligand binding, antibody binding, [35S]GTPγS, and calcium mobilization assays. In these assays, the bivalent ligands were shown to maintain binding and functional activities in both MOR and CXCR4 monoclonal cell lines. In addition, these bivalent ligands were evaluated for their ability to block HIV entry in a reverse transcriptase assay, and for their ability to inhibit morphine exacerbated HIV invasion in an LTR-luciferase assay. In these assays, the bivalent ligands were shown to inhibit HIV entry in a dose dependent manner. However, due to experimental limitations in our morphine exacerbated reporter system, the ability for the bivalent ligands to inhibit viral entry upon morphine co-exposure was not fully validated. Finally, molecular modeling approaches were utilized to visualize the putative binding modes of the bivalent ligands in a constructed MOR-CXCR4 heterodimer model.
Overall, these studies have provided a solid basis for the utility of bivalent ligands in studying MOR-CXCR4 interactions and their involvement in opioid potentiated HIV progression. Further studies are ongoing to optimize the bivalent ligands construct and explore new analyses to evaluate their ability to block opioid modulation of the virus.
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Leach, Katie. "Pharmacological analysis of the CC chemokine receptors, CCR4 and CCR5 signalling properties and receptor-drug interactions." Thesis, University of Reading, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.427859.
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King, Vicky. "Assessment of the therapeutic potential of the atypical chemokine receptor, D6." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/2165/.
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Infiltration of inflammatory cells into the tissue during the inflammatory response is beneficial to the host. Chemokines and their receptors are instrumental in this process by influencing the migration and behaviour of leukocytes in the tissue. However, prolonged inflammation is associated with many diseases. In recent years, a family of atypical receptors have emerged which do not seem to signal. One of these receptors, D6, is able to internalise and degrade 12 pro-inflammatory CC chemokines and has a role in the resolution of the inflammatory response. Here, using a murine transgenic approach, the potential therapeutic role of D6 in suppressing cutaneous inflammation in vivo has been investigated, using a well-characterised model of skin inflammation. In addition, expression of D6 in a range of inflammatory disorders has also been characterised. Transgenic mice were generated (K14D6), using an epidermis-specific transgene, in which expression of the D6 transgene was driven by the human keratin 14 promoter in epidermal keratinocytes. K14D6 mice were validated and we have shown that D6 is expressed in K14D6 but not in wild-type epidermal keratinocytes. The K14D6 transgene was shown to be functional as only K14D6 keratinocytes were able to bind CCL2 and progressively deplete extracellular CCL3. K14D6 mice can dampen down cutaneous inflammation in response to a topical application of TPA. In addition, K14D6 mice displayed reduced infiltration of epidermal T cells and mast cells compared to wild-type mice. Using a microarray approach, we examined the transcriptional consequences of non-ligated D6 and after ligand binding in primary murine keratinocytes from K14D6 and wild-type mice. Although limited conclusions could be made from the microarray data, our results suggest the possibility that non-ligated D6 in murine keratinocytes may have a negative impact on the transcription of some genes, such as chemokines. In a previous study, D6 null mice displayed a human psoriasis-like pathology after chemical induced skin inflammation, suggesting a possible involvement of D6-dysfunction as a contributing factor in the pathogenesis of psoriasis. We have investigated the possible correlation between D6 expression levels and cutaneous disease development. Analysis of skin biopsies revealed that D6 mRNA levels were 8-fold higher in uninvolved psoriatic skin compared to matching psoriatic lesional skin, atopic dermatitis and control skin. In PBMCs, there was no significant difference in D6 mRNA expression in psoriasis patients compared to control. A preliminary study examining surface D6 expression on leukocytes from control and rheumatoid arthritis patients revealed enhanced D6 expression on B cells and myeloid DCs. In this study, we have shown for the first time that increased expression of D6 in vivo can limit cutaneous inflammation, therefore providing a rationale for exploring the therapeutic potential of D6 in human inflammatory diseases. In addition, we provide evidence that D6 expression is dysregulated in inflammatory disorders further suggesting an involvement of this receptor in the pathogenesis of these diseases.
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Bordon, Yvonne. "The role of the D6 chemokine receptor in immunity and inflammation." Thesis, University of Glasgow, 2007. http://theses.gla.ac.uk/6552/.
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D6 is a novel chemokine receptor, homologous to other members of the CC-chemokine receptor family, which recognises a number of inflammatory CC-chemokines with high affinity. The aims of this thesis were to further our understanding of the biology of D6, chiefly through characterisation of immune responses in D6-deficient animals. Firstly, as described in Chapter 3, I analysed the cellular composition of lymphoid tissues of D6 KO mice. These studies revealed higher proportions of CD11c+ and F4/80+ cells in the D6 KO spleen compared with WT controls, suggesting that increased accumulation of myeloid lineage cells was occurring at this site. In Chapter 4, I examined the role of D6 in myeloid cell responses, by comparing monocyte recruitment to the inflamed peritoneum and dendritic cell development from bone-marrow (BM) cultures. I found that while the accumulation of inflammatory monocytes/macrophages appeared quantitatively similar in WT and D6 KO animals, D6 KO cells expressed greater levels of CD11c, suggesting preferential accumulation of DC-like cells in the inflamed peritoneum. D6 may influence the development and function of myeloid lineage cells. As D6 is expressed at high levels in the small and large intestine, I next investigated both tolerogenic and inflammatory intestinal responses in D6 KO animals. As detailed in Chapter 5 of this thesis, the induction of oral tolerance in response to a high dose feeding protocol was normal in D6 KO mice. However, D6 KO mice showed increased resistance to experimental colitis. As described in Chapter 6, various D6 KO populations displayed differential chemokine receptor profiles compared with their WT counterparts. The results suggest a role for D6 in the normal development of leukocytes populations, with absence of this atypical receptor leading to the dysregulated expression of other chemokine receptors. Taken together, my data suggest that the biological functions of D6 may be more complicated than previously appreciated. Indeed, I found no evidence for a decoy role of D6 in vivo, but D6-deficient animals were characterised by altered leukocyte development, aberrant chemokine receptor expression and increased resistance to experimental colitis induction.
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Schäuble, Karin [Verfasser]. "Structural and Functional Dissection of the Chemokine Receptor CCR7 / Karin Schäuble." Konstanz : Bibliothek der Universität Konstanz, 2011. http://d-nb.info/1081016698/34.
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Lopes, de Mendonca Filipa. "Biochemical and functional analysis of a promiscuous #beta#-chemokine receptor, D6." Thesis, University of Glasgow, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.394766.
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Fujiyoshi, Keiko. "The effect of biomaterials on the regulation of chemokine receptor expression." Thesis, University of Liverpool, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.420292.
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Jacques, Richard. "Pharmacological insights into C-C motif chemokine receptor 5 mediated chemotaxis." Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/42415/.
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Aim: Despite being well validated as a therapeutic target, no chemokine receptor antagonists to be used as therapeutic agents in inflammatory or metastatic disease have made it to market. This is in part due to receptor redundancy, but also a lack of understanding with regard to cytoplasmic signal transduction linking activated chemokine receptors to chemotaxis. Resolving signal transduction pathways in model chemokine receptor systems may allow intracellular drug targets to be identified, bypassing the difficulties associated with extracellular chemokine receptor blockade. Methodology: Experimentation was undertaken in THP-1 monocytes expressing the chemokine receptors CCR5 and CCR1 and in stably CCR5-transfected HeLa and CHO cell lines. Small molecule inhibition and protein overexpression was used before chemotaxis and calcium release assays to measure cellular responses. Immunocytology was used to determine the effect of protein blockade on receptor internalisation, protein localisation and the formation of cellular structures associated with migration. Experiments were also performed in activated primary tissue for comparative analysis and validation of results in normal human tissue. Results: A systematic blockade of signalling proteins by small molecule means revealed that Gβγ, ERK1/2, p38 and PI3K are not required for CCL3 stimulated monocyte migration. GRK2 and PKC inhibition along with internalisation blockade showed antagonistic effects on the ability of cells to migrate, suggesting arrestin dependent signalling was involved in chemotaxis. Inhibition of dynamin, Grb2 and non-receptor tyrosine kinases were equally effective at blocking migration in THP-1 cells but less effective at blocking CXCL11 stimulated migration in activated PBLs. Conclusions: This study has shown that CCL3 stimulated chemotaxis through CCR5 does not occur through typical G-protein mediated signalling, but maybe therapeutically accessible by inhibition of dynamin and Grb2. Additionally the differences in dynamin inhibitor efficacy suggest that the production of migration specific dynamin inhibitors may be possible. Overall the research in this thesis has identified novel targets for therapeutic intervention in diseases where dysregulation of chemokine receptor mediated migration are causative.
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Kerr, Jason Stuart. "Heterotrimeric G-protein interactions and activation in chemokine receptor 5 signalling." Thesis, University of East Anglia, 2010. https://ueaeprints.uea.ac.uk/10571/.
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Activation of the G-protein coupled receptor (GPCR) chemokine receptor 5 (CCR5) has been shown to result in activation of the Gαi family of heterotrimeric G-proteins. However, little is currently known about the temporal characteristics of G-protein heterotrimer activation by CCR5. Furthermore, this simplistic model does not account for the range of changes CCR5 activation brings about. Recent evidence suggests that GPCRs may be able to signal through numerous permutations of heterotrimer. In order to assess G-protein activation and interaction with CCR5 functional stably transfected cell lines were created expressing Gαi2 fused to ECFP and Gβ1 fused to EYFP. The interaction of these constructs was confirmed by measuring fluorescent resonance energy transfer (FRET). Stably transfected cells exhibited a FRET ratio of 2.63% (±0.345%, n=3) over that of control cells. Following CCR5 stimulation with CCL3, Gαi2β1γ activation could be monitored in real time, in whole cell populations, on a fluorescent plate reader by monitoring FRET emissions. This assay system represents a novel approach to measuring G-protein activation which can be used as a foundation to build more powerful FRET based assays. Measurement of G-protein interactions was further invested by BRET based studies. Transfection of dominant negative and constitutively active G-proteins alongside siRNA knockdown of G-proteins revealed that CCR5 is capable of signalling through other members of the Gαi family, with strikingly similar efficacy and potency to CCL3 stimulation. Dual knockdown of Gαi subunits and Gαq resulted in much attenuated calcium release following CCL3 stimulation, providing evidence that CCR5 may functionally couple to several types of G-proteins. Findings also support the theory that GPCRs can participate in domain swapping in order to rescue function. Treatment with gallein resulted in higher resting cytosolic cAMP but did not prevent CCR5 mediated inhibition of cAMP production. Gallein treatment also resulted in significant increases in calcium release following CCR5 activation, highlighting Gβγ as a potential target for modulating CCR5 signalling events. Data herein emphasize the complexity of GPCR signalling, and also provide a foundation for exploring GPCR signalling using fluorescence resonance energy transfer methods in the future.
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Teoh, Pek Joo. "The role of the atypical chemokine receptor D6 in the placenta." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5098/.
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D6 is an atypical chemokine receptor related to CCR1-5 that binds to many inflammatory CC chemokines. Experiments using transfected cell lines have shown that upon binding to a chemokine ligand D6 does not trigger cellular signalling pathways, but rather acts to scavenge the bound ligand. It achieves this by constitutively travelling to and from the cell surface via early and recycling endosomes, internalising chemokines bound when it is at the cell surface. Over time, D6 removes a large amount of ligands from the extracellular compartment. In vivo, this scavenging activity is thought to regulate the level of CC chemokines, and thus controls inflammation locally and systemically. Lack of D6 has been shown to result in elevated amounts of bioavailable chemokines, and is associated with over exuberant inflammatory responses. In human, D6 mRNA and protein is highly expressed in trophoblast-derived gestational tissues. The expression of D6 mRNA in the placenta is by far the highest, compared to other solid tissues being studied. The importance of D6 in protecting the offspring has been demonstrated in animals. In pigs, a defect in D6 expression was discovered in placental attachment sites in endometrium from arresting fetuses. In mice, lack of D6 results in an increase in fetal loss after challenge with lipopolysaccharide (LPS) or antiphospolipid autoantibodies (aPL), and an increase in the number of abnormal pups when mouse embryos are transferred into fully allogeneic pseudo-pregnant female recipients. In view of these results suggesting a critical role for D6 in placental mediated complications, the expression and molecular function of D6 in primary human trophoblast cells were studied, as to date in vitro human studies have utilised the choriocarcinoma cell line BeWo or immortalised cell lines engineered to over-express exogenous D6. Secondly the impact of D6 deficiency on placental structure, chemokine expression and leukocyte abundance in mice was examined. Chapter 3 presents the results of experiments on primary human trophoblasts. Protocols for routine primary trophoblast isolation, purification and culture from fresh term placentas were optimised in our laboratory. D6 mRNA was detected in these primary cells. Using Western blotting, immunofluorescence and flow cytometry, D6 was shown to be present predominantly in the intracellular vesicles of the cells. Competition chemokine uptake assays, analysed by flow cytometry, showed that CCL2 was internalised by trophoblasts using D6. Competitive chemokine scavenging assays, analysed by quantitative Western blot, confirmed that D6 was functioning as a chemokine scavenger on primary human trophoblasts and that it progressively removed substantial quantities of chemokine from medium bathing the cells. This is the first set of experiments that confirms D6 is present, and functioning as a chemokine scavenger in primary human cells. Chapter 4 contains the results from the mouse experiments. Even in an unchallenged environment it was shown that, on the DBA-1 genetic background, D6 deficiency in the mother and pups leads to higher rates of stillbirth and neonatal deaths, resulting in a reduction in the number of pups weaned per litter than their WT counterparts. By gestational age E14, pup weight was significantly smaller in the D6 KO mice. Using stereological techniques, the placenta of the D6 KO mice at this gestation was found to have a smaller labyrinthine zone. The volume of the labyrinthine zone was positively correlated with pup/placenta ratio. These phenotypes could be due to a maternal or fetal effect of D6 deficiency. To ascertain the answer to this question, the experiment at E14 was extended by breeding DBA-1 females heterozygous for the deleted D6 allele (D6 HET) with D6 deficient (D6 KO) males. In this model the phenotypes of D6 KO pups and placentas could be compared with their D6 HET siblings that developed in a mother expressing some D6 (i.e. D6 HET). Although there were no differences in pup weight, placental weight and pup/placenta ratio between these two groups, stereology revealed a decrease in labyrinthine zone volume fraction in the D6 KO placentas in comparison to their D6 HET siblings. The observed fetal compromise and placental defect at E14 was not apparent at the later gestational age of E18. Luminex multiplex protein assay showed an elevated level of circulating chemokine CCL2 in the serum of D6 KO pregnant mice in comparison to their WT counterpart, so loss of chemokine regulation could be responsible for the defects observed in D6 deficient placentas. In summary, D6 deficiency results in an increase in perinatal death, a fundamental defect in placental formation (reduced labyrinthine zone) and dysregulation of circulating chemokine levels. Chapter 5 discusses the mechanisms of D6 in regulating placental formation and reproductive outcome and the novel insights that this work provided into placental D6 function. It also describes the design of future experiments to reveal the precise role of D6 in chemokine regulation and cell signalling in reproductive immunology, and discusses how D6 might contribute to pregnancy outcome in humans.
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Briukhovetska, Daria [Verfasser]. "The roles of C5a receptor 1 and chemokine receptor 5 in Toxoplasma gondii infection / Daria Briukhovetska." Lübeck : Zentrale Hochschulbibliothek Lübeck, 2017. http://d-nb.info/1142648001/34.
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Vacchini, A. "ANALYSIS OF BIASED SIGNALING IN THE CHEMOKINE SYSTEM." Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/365864.
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Chemokines constitute a family of almost 50 small secreted cytokines, recognized by about 20 different 7TM spanning G protein coupled receptors (GPCRs), that activating pertussis toxin sensitive G proteins induce cell migration. These receptor are abundantly expressed by leukocytes and, controlling cell migration, they dictate leukocyte positioning during homeostatic patrolling within peripheral tissues, their maintenance in bone marrow during maturation and in addition mediate their recruitment to inflamed tissues. Upon inflammation in fact a number of chemokines are produced or activated by inflammatory mediators and diffuse within the tissue, generating a chemical gradient along which leukocytes migrate to reach the center of inflammation to contain and remove the insulting factor. This system needs an extremely tight control, since its dysregulation has been demonstrated to be at the basis of different inflammatory diseases, auto-immunity and has also been linked to cancer development. In particular, in this thesis we focused our attention on two regulatory system: post-translational modifications of chemokines, mediated by enzymes specifically released upon inflammation also by immune cells, and on the activity of atypical chemokine receptors, a subfamily of chemokine receptors that despite high structural homology and similar binding properties compared to conventional chemokine receptors, are unable to drive chemotaxis but act instead as key regulators of the chemokine system activity. In detail we looked at the ability of these regulatory mechanisms to modulate chemokine signaling properties generating a biased signaling, an emerging feature of GPCR pharmacology that describes the ability of a given receptor to elicit different or even opposite functional activities depending on the ability of different agonists to stabilize different receptor’s active structural conformation, resulting in different phenotypes mediated by the same receptor. In the chemokine system biased signaling has already been described to occur on different receptors upon binding of their different ligands, therefore during our investigation on chemokine regulatory system signaling we maintained our focus on the ability of these systems to bias chemokine signaling properties in order to better understand how this regulation occurs.
To this point we assessed the ability of differently post-translationally modified chemokines to elicit signaling activities on different receptors by measuring in HEK293 cells their potential in inhibiting adenylyl cyclase, a proximal downstream signal of Gα inhibitory proteins activation, and in inducing β-arrestin recruitment to the receptors in energy transfer-based assays. We also compared signaling properties of an atypical chemokine receptor to the ones elicited by a conventional receptor analyzing the phosphoproteome modifications occurring constitutively and after stimulation with the same agonist.
Our results indicate that regulation of CXCL5 and CXCL8 chemokine activity by post-translational modifications is more prone to regulate chemokine activity modifying chemokine potency, rather than generating a bias in their signaling properties. Truncation of chemokine NH2-terminus increases both CXCL5 and CXCL8 activity, while citrullination of the most NH2-terminal arginine results in opposite effects on the two agonists since on CXCL8 increases chemokine potency, while it reduces CXCL5 activity. We investigated the properties of ACKR2 in recruiting β-arrestins, demonstrating that this receptor is able to associate both β-arrestin 1 and 2 in basal conditions while upon agonist stimulation preferentially increases its association with β-arrestin 1, resulting in a completely different agonist-induced outcome of proteome phosphorylation, compared to CCR5, in terms of kinetics, protein phosphorylation modifications, biological function of the regulated proteins and signal mediators activated. Taken together, these results indicate that chemokine system regulation is based not only on chemokine post-translational modifications that modulate chemokine potency, but also on the activity of structurally biased atypical chemokine receptors, as in the case of ACKR2 that interacts with different effectors and kinetics to generate distinct functional outcomes, compared to the conventional chemokine receptor CCR5.
In this thesis it has also been attempted to translate the investigation of chemokine signaling to a clinical intervention for inflammatory diseases. We assessed the modulation of CXCR1 signaling activity exerted by Reparixin, a leukocyte migration inhibitor that blocks cell recruitment to inflamed tissues, that binds to its target receptors in an allosteric binding site, without inhibiting chemokine binding to the receptor. In our assays performed on HEK293 cells we could not detect any inhibition of the signaling pathways assayed, possibly indicating that HEK293 cells are not the best model to assay the activity of this molecule, with the need to assess in this cellular model drug inhibitory activity on read-outs already evaluated in literature on other cell types.
In conclusion, we can say that observations described in this thesis allow to better understand chemokine system regulation, that occurs by biased signaling activity in the case of atypical chemokine receptors that by selected interaction with signaling mediators induce opposite biological outcomes compared to conventional chemokine receptors, while post-translational modifications regulate chemokines activity modulating their potency, rather than biasing their signaling properties.
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Coke, Christopher James. "Cannabinoid Receptor 2 and C-X-C Chemokine Receptor 4 Interact to Abrogate CXCL12-Mediated Cellular Response." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 2017. http://digitalcommons.auctr.edu/cauetds/76.
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The expression of C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with increased metastatic potential of cancer cells. CXCR4 increases tumor malignancy by encouraging tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis provides a good strategy to inhibit the metastatic spread of tumor cells and slow cancer progression. Various studies suggest that cannabis may have anti-proliferative as well as anti-metastatic properties, though a biochemical mechanism describing how this occurs has yet to be discovered. Our lab has confirmed that agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) can form heterodimers that play a role in decreasing cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231 and the prostate cancer cell line PC-3, with CXCL12 and AM1241, a synthetic ligand for CB2, desensitizes the intrinsic cellular response to migrate toward areas of high CXCL12 concentration. Furthermore, through co-immunoprecipitation and
proximity ligation assays (PLA), we have determined that there is increased interaction between the two receptors with co-stimulation of respective agonists, providing evidence for the therapeutic notion that treating tumors that endogenously secrete CXCL12 with exogenous ligands for the cannabinoid can induce dimerization. Moreover, when CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Finally, to determine whether simultaneously–treated, dimerized receptors inhibited activity of respective receptors, calcium mobilization assays to determine G-protein coupled receptor activation were employed. Results showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treated with their individual ligands. Phosphorylation of ERK and AKT were abrogated in response to simultaneous stimulation indicating loss in downstream signaling. Therefore, we believe that the interaction of CB2 with CXCR4 may play a role in inhibiting the cells response to CXCL12, leading to a loss in metastatic potential of cells expressing these receptors.
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Masuda, Ryo. "Development and Application of Imaging Probes for CXC Chemokine Receptor 4 (CXCR4)." 京都大学 (Kyoto University), 2012. http://hdl.handle.net/2433/157894.
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