Biochemical and Functional Analysis of a Promiscuous beta-Chemokine Receptor, D6

Mendonca, Filipa Lopes de (2002) Biochemical and Functional Analysis of a Promiscuous beta-Chemokine Receptor, D6. PhD thesis, University of Glasgow.

Abstract

Chemokines were initially defined as host defence proteins involved in cellular migration. They are now known to play a central role in the temporal and spatial positioning of leukocytes required for the successful induction of inflammation and the establishment of immunity. Moreover, chemokines exert many biological responses on other cell types and have been implicated in haemopoiesis, angiogenesis, oncogenesis and development. In order to carry out their functions, chemokines must bind to and activate seven transmembrane (TM) domain G protein coupled receptors usually expressed on the surface of target cells. The chemokine system is implicated in many diseases such as chronic inflammation, autoimmunity, allergy, AIDS and cancer. This has lead to considerable efforts aimed towards understanding chemokine/receptor interactions with a view to preventing pathological consequences and this interaction. Human D6 (hD6) is an unusual beta chemokine receptor that binds with high affinity to many pro-inflammatory beta chemokines, yet is not able to couple to signalling pathways activated by other related chemokine receptors. Moreover, immunocytochemistry has revealed that hD6 is absent from peripheral blood leukocytes and rather is expressed by endothelial cells in a subset of lymphatic vessels in the skin, lung, gut and secondary lymphoid tissue. The function of this receptor on these cells is currently uncertain, but its properties are provocative of a role in leukocyte migration, lymphangiogenesis and possibly metastasis. In this thesis, chimaeric receptors have been used to understand the atypical biochemistry of hD6. The ultimate aim was to identify domain(s)/residue(s) responsible for the broad ligand binding promiscuity and the high affinity ligand interactions apparent for this receptor and probe the signalling properties of hD6. This work revealed the many problems associated with this approach to biochemical analysis. Chimaeric constructs bearing domains of CC and CXC receptors and large domain swaps between CC chemokine receptors, were shown to be poorly expressed on the surface of transfected cells. Additionally, these studies highlight the importance of the epitope tag, cell lines and the transfection systems used, thus indicating that the design and interpretation of receptor chimaera studies should be carefully considered. Using small extracellular domain swaps between hD6 and hCCR5 it has been shown that: 1) the first extracellular, and most highly conserved, loop of hD6 is required for high affinity binding to chemokine; 2) the second and third extracellular loops appear to weakly influence ligand interaction, although antibody binding studies suggest that this result may be due to the gross structure of the chimaeric receptor being subtly altered; 2) the N-terminus of hD6 can be replaced with that of hCCR5 with little effect on the binding of most chemokines, although the binding site for RANTES/CCL5 appears to have been altered compared to wild type hD6. Signalling studies on mutant or chimaeric receptors have revealed that a single amino acid change in hD6 is sufficient to introduce ligand-induced signalling via pertussis toxin sensitive G-proteins into this receptor. Specifically, a single point mutation (E to A) to convert the DKYLE motif in the second intracellular loop closer to the conserved DRYLA sequence can allow this receptor to induce weak calcium ion fluxes. A reciprocal mutation in hCCR5 blocked signalling through this receptor. Surprisingly, no other chimaeras of hD6 carrying the intracellular domains of hCCRS were able to induce calcium ion fluxes or enhance the response seen with the E to A mutant. However, these mutants were, unlike the wild type, hD6, able to internalise upon ligand binding. Taken together these data suggest that coupling to calcium ion flux and internalisation are independently regulated events. The results in this thesis have highlighted the technical difficulties associated with chimaeric receptor work, and the interpretation of these studies. Nonetheless, this work has identified the first extracellular loop as crucial for interaction of hD6 with ligand, derived signalling active mutants of hD6 by a single amino acid change, and generated new ideas on GPCR signalling and receptor internalisation. This work should act as a platform for the more detailed analysis of the biochemistry of hD6.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Rob Nibbs
• Keywords: Biochemistry
• Date of Award: 2002
• Depositing User: Enlighten Team
• Unique ID: glathesis:2002-75761
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 18:14
• Last Modified: 19 Nov 2019 18:14
• URI: https://theses.gla.ac.uk/id/eprint/75761