Oligomerisation of chemokine receptors CXCR1 and CXCR2

Wilson, Shirley Risk (2005) Oligomerisation of chemokine receptors CXCR1 and CXCR2. PhD thesis, University of Glasgow.

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Abstract

Chemokine receptors CXCRl and CXCR2 are expressed on several immune cell types including granulocytes and monocytes. The two receptors share 78% sequence identity and primarily function to mediate chemotaxis. Homo- and hetero-oligomerisation of CXCRl and CXCR2 was investigated in this study. CXCRl and CXCR2 were modified with either N- or C-terminal epitope tags in order to facilitate the detection of receptor oligomers. The modified receptors were predominantly cell surface expressed and underwent prominent internalisation following exposure to Interleukin-8 (IL-8). The modifications were found to have no effect on affinity for IL-8 determined by competition radio ligand binding assays. The modified receptors retained the ability to couple to G proteins assessed by agonist-induced reduction in forskolin- stimulated cAMP levels. Co-immunoprecipitation experiments demonstrated the existence of constitutive CXCRl and CXCR2 homo-oligomers and CXCR1/CXCR2 hetero-oligomers following expression in HEK293T cells. Single cell FRET imaging experiments also confirmed constitutive CXCRl and CXCR2 homo- and hetero-oligomerisation. A lower level of energy transfer was observed between both CXCRl and CXCR2 and the less homologous alphaiA-adrenoceptor. Saturation BRET experiments indicated that CXCR1/CXCR2 homo- and hetero-oligomers formed with equal propensity. This approach also indicated that the interaction between CXCRl and the alphaiA-adrenoceptor detected in FRET studies was non-specific as a linear relationship between donor and acceptor was shown to exist. Tr-FRET experiments indicated that constitutive CXCRl and CXCR2 homo- and hetero-oligomers exist pre-formed at the cell surface. The effect of agonist on oligomerisation was investigated. Experiments using BRET and Tr-FRET did not demonstrate any modulation of energy transfer following exposure to IL-8. A novel endoplasmic reticulum (ER) trapping strategy was developed in order to investigate the location and selectivity of oligomer formation. CXCRl was modified by the addition of an ER retention sequence to the C terminal tail that prevented expression of the receptor at the cell surface. Upon co-expression with wild type CXCRl and CXCR2, CXCRl-ER reduced their cell surface expression. This suggested that oligomerisation between the wild type receptors and CXCRl-ER was initiated during protein synthesis and maturation and prior to cell surface delivery. Cell surface expression of the alphaiA- adrenoceptor was unaffected by co-expression of CXCRl-ER indicating a lack of interaction. Despite cell surface constitutive CXCRl/CXCR2 hetero-oligomers being indicated by Tr-FRET, no co-internalisation of CXCRl and CXCR2 was observed in response to GRO-a, a ligand specific for CXCR2. Expression of opioid receptors on immune cells has been demonstrated and opioid agonists have been demonstrated to modulate chemotaxis. CXCR2 is also expressed in the brain. Co-immunoprecipitation experiments indicated oligomerisation between CXCR2 and DOP, MOP and KOP receptors. Single cell FRET also indicated oligomerisation between CXCR2 and the opioid receptors. This highlights the potential existence of chemokine and opioid hetero-oligomers in physiologically relevant settings that could be of great potential interest in drug development for the treatment of inflammatory conditions.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Graeme Milligan
Keywords: Immunology
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-71198
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71198

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