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This article is part of the supplement: 18th Scientific Symposium of the Austrian Pharmacological Society (APHAR)

Open Access Meeting abstract

Restricted collision coupling of the adenosine A2A receptor is due to its agonist-induced confinement in the membrane

Patrick Thurner, Simon Keuerleber, Ingrid Gsandtner, Christoph Gruber, Michael Freissmuth and Jürgen Zezula*

Author Affiliations

Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria

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BMC Pharmacology and Toxicology 2012, 13(Suppl 1):A81  doi:10.1186/2050-6511-13-S1-A81

The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/2050-6511/13/S1/A81


Published:17 September 2012

© 2012 Thurner et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

The A2A adenosine receptor is of interest because of several reasons. (i) It is a frequently blocked pharmacological target, because it is the site of action of caffeine. (ii) It has a long C-terminus that provides a docking site for several proteins, which direct the fate of the receptor from its synthesis to its lysosomal degradation. (iii) The A2A receptor can only promote activation of a limited number of available Gs molecules. This coupling mode was termed restricted collision coupling. (iv) Most G protein-coupled receptors carry one or several cysteine residues in their C-terminus which is subject to palmitoylation to anchor and stabilize the amphipathic helix 8; the A2A receptor lacks this palmitoylation site. We explored the hypothesis that there is a causal link between the absence of a palmitoyl moiety and restricted collision coupling.

Methods

We constructed a mutant A2A receptor, R309C, which underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. Radioligand binding, cAMP accumulation and Western blotting were performed to determine its signaling properties. Using single particle tracking of quantum dot-labeled receptors we compared diffusivity and diffusion mode of wild-type and mutant A2A receptors.

Results

In contrast to the wild-type receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A2A receptor R309C. Single particle tracking demonstrated that agonist activation resulted in a decline in mean square displacement of both receptors, but the drop was substantially more pronounced for the wild-type receptor. In addition, in the agonist-bound state, the wild-type receptor was frequently subject to confinement events; these were rarely seen with the palmitoylated A2A receptor R309C.

Conclusions

Taken together, the observations link restricted collision coupling to diffusion limits imposed by the absence of a palmitoyl moiety in the C-terminus of the A2A receptor.

Acknowledgements

This work was supported by the Austrian Sciences Fund (FWF) and the Medical University of Vienna.