Functional analysis of the chemokine receptor, CCX-CKR

Comerford, Iain (2005) Functional analysis of the chemokine receptor, CCX-CKR. PhD thesis, University of Glasgow.

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Chemokines comprise a large family of low molecular weight, mainly secreted proteins, well known for their role in directing leukocyte traffic, a vital component of immune and inflammatory responses. However, chemokines also regulate a broad range of other biological functions, including cell survival, growth, and differentiation. All these responses are mediated through cognate interactions with chemokine receptors, seven transmembrane (T/M) spanning G-protein coupled receptors. Importantly, the role of chemokines and their receptors in an enormous range of pathologies is apparent from a large number of studies in the last two decades, and these molecules have become popular targets for the pharmaceutical industry. Despite the explosion in our understanding of chemokine networks, much less is known about regulatory mechanisms which must be in place to control and facilitate the biological effects of chemokines in vivo. Recent work has suggested the existence of a subset of atypical chemokine receptors that sequester their ligands to help remove or transport chemokines. This focus of this thesis regards the function of one of these molecules, CCX-CKR. This acts as a receptor for CCL19, CCL21 and CCL25, chemokines involved in constitutive and inflammatory trafficking of key leukocytic subsets in lymphoid tissue and small intestine. However, CCX-CKR does not appear to directly mediate these migratory events and, despite displaying high affinity binding, this receptor does not activate downstream signaling pathways typically involved in chemokine receptor function. Instead, roles for CCX-CKR in regulating chemokine levels or localisation have been proposed. The studies described in this thesis have examined the biochemistry of CCX-CKR in vitro with a view to determining what this receptor is capable of These studies form a molecular framework for the interpretation of phenotypes emerging from CCX-CKR null mice generated as part of this study. The results from in vitro assays demonstrate that, in heterologous transfectants, human CCX-CKR mediates the internalisation and intracellular degradation of the chemokine CCL19. Importantly, unlike CCR7, CCX-CKR is primed, not desensitised, by previous exposure to ligand, allowing continuous CCL19 sequestration. This endows CCX-CKR with an impressive ability to sequester and destroy its ligands in continuous culture and therefore modify adjacent CCR7-driven responses. Mechanistically, CCL19 uptake by CCX-CKR is dependent on dynamin but, unlike CCR7, not reliant on beta-arrestins. Genetic inhibition of both clathrin mediated endocytosis and early endosome function had no apparent effect upon CCL19 uptake by CCX-CKR, but inhibited uptake by CCR7. In contrast, over-expression of caveolin-1 dramatically inhibited CCX-CKR mediated CCL19 uptake while leaving CCR7 function unaffected, implicating caveolae in CCX-CKR endocytosis. The significance of these alternative pathways of receptor internalisation is discussed. In parallel, the in vivo significance of CCX-CKR function was examined by generating CCX-CKR knock-out mice. These mice did not display any gross phenotypic abnormalities, yet detailed analysis revealed a significant reduction in the cellularity of inguinal lymph nodes compared with wild-type mice. In addition, the phenotype of splenic macrophages appeared altered in CCX-CKR null mice. Moreover, the topical application of irritant or hapten, to these mice, did not cause the subsequent inguinal lymph node hypertrophy observed in wild-type couterparts. However, CCX-CKR null mice were capable of mounting relatively normal immune responses although proliferative responses to antigen appeared to persist longer in the lymph nodes of null animals. Taken together, the results from this study suggest that CCX-CKR contributes to the organisation and regulation of the immune system during homeostasis and immune response. This may be achieved through the control of chemokine availability such that the responses through the receptors CCR7 and CCR9 are modified. Importantly, the work described in this thesis represents the first attempt to dissect the function of CCX-CKR and provides the foundation for future studies aimed at more precisely defining the function of this atypical chemokine receptor.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Rob Nibbs
Keywords: Immunology
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-74198
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Sep 2019 15:33
Last Modified: 23 Sep 2019 15:33

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