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Arginine 199 and leucine 208 have key roles in the control of adenosine A2A receptor signalling function.

ABSTRACT: One successful approach to obtaining high-resolution crystal structures of G-protein coupled receptors is the introduction of thermostabilising mutations within the receptor. This technique allows the generation of receptor constructs stabilised into different conformations suitable for structural studies. Previously, we functionally characterised a number of mutants of the adenosine A2A receptor, thermostabilised either in an agonist or antagonist conformation, using a yeast cell growth assay and demonstrated that there is a correlation between thermostability and loss of constitutive activity. Here we report the functional characterisation of 30 mutants intermediate between the Rag23 (agonist conformation mutant) and the wild-type receptor using the same yeast signalling assay with the aim of gaining greater insight into the role individual amino acids have in receptor function. The data showed that R199 and L208 have important roles in receptor function; substituting either of these residues for alanine abolishes constitutive activity. In addition, the R199A mutation markedly reduces receptor potency while L208A reduces receptor efficacy. A184L and L272A mutations also reduce constitutive activity and potency although to a lesser extent than the R199A and L208A. In contrast, the F79A mutation increases constitutive activity, potency and efficacy of the receptor. These findings shed new light on the role individual residues have on stability of the receptor and also provide some clues as to the regions of the protein responsible for constitutive activity. Furthermore, the available adenosine A2A receptor structures have allowed us to put our findings into a structural context.

SUBMITTER: Bertheleme N

PROVIDER: S-EPMC3940607 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Arginine 199 and leucine 208 have key roles in the control of adenosine A2A receptor signalling function.

Bertheleme Nicolas N Strege Annette A Bunting Sorrel E SE Dowell Simon J SJ Byrne Bernadette B

PloS one 20140303 3

One successful approach to obtaining high-resolution crystal structures of G-protein coupled receptors is the introduction of thermostabilising mutations within the receptor. This technique allows the generation of receptor constructs stabilised into different conformations suitable for structural studies. Previously, we functionally characterised a number of mutants of the adenosine A2A receptor, thermostabilised either in an agonist or antagonist conformation, using a yeast cell growth assay a ...[more]

PMID: 24595172

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Project description:BackgroundConditions of inflammatory tissue distress are associated with high extracellular levels of adenosine, due to increased adenosine triphosphate (ATP) degradation upon cellular stress or the release of extracellular ATP upon cell death, which can be degraded to adenosine by membrane-bound ecto-enzymes like CD39 and CD73. Adenosine is recognised to mediate anti-inflammatory effects via the adenosine A2a receptor (A2aR), as shown in experimental models of arthritis. Here, using pharmacological interventions and genetic inactivation, we investigated the roles of A2aR in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS).MethodsWe used two independent mouse EAE variants, i.e. active immunization in C57BL/6 with myelin oligodendrocyte glycoprotein (MOG)35-55 or transfer-EAE by proteolipid protein (PLP)139-155-stimulated T lymphocytes and EAE in mice treated with A2aR-agonist CGS21680 at different stages of disease course and in mice lacking A2aR (A2aR(-/-)) compared to direct wild-type littermates. In EAE, we analysed myelin-specific proliferation and cytokine synthesis ex vivo, as well as inflammation and demyelination by immunohistochemistry. In vitro, we investigated the effect of A2aR on migration of CD4(+) T cells, macrophages and microglia, as well as the impact of A2aR on phagocytosis of macrophages and microglia. Statistical tests were Mann-Whitney U and Student's t test.ResultsWe found an upregulation of A2aR in the central nervous system (CNS) in EAE, predominantly detected on T cells and macrophages/microglia within the inflamed tissue. Preventive EAE treatment with A2aR-specific agonist inhibited myelin-specific T cell proliferation ex vivo and ameliorated disease, while application of the same agonist after disease onset exacerbated non-remitting EAE progression and resulted in more severe tissue destruction. Accordingly, A2aR-deficient mice showed accelerated and exacerbated disease manifestation with increased frequencies of IFN-γ-, IL-17- and GM-CSF-producing CD4(+) T helper cells and higher numbers of inflammatory lesions in the early stage. However, EAE quickly ameliorated and myelin debris accumulation was lower in A2aR(-/-) mice. In vitro, activation of A2aR inhibited phagocytosis of myelin by macrophages and primary microglia as well as migration of CD4(+) T cells, macrophages and primary microglia.ConclusionsA2aR activation exerts a complex pattern in chronic autoimmune neurodegeneration: while providing anti-inflammatory effects on T cells and thus protection at early stages, A2aR seems to play a detrimental role during later stages of disease and may thus contribute to sustained tissue damage within the inflamed CNS.

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Arginine 199 and leucine 208 have key roles in the control of adenosine A2A receptor signalling function.

Publications

Arginine 199 and leucine 208 have key roles in the control of adenosine A2A receptor signalling function.

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