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J Biol Chem. 2012 Dec 7;287(50):42104-18. doi: 10.1074/jbc.M112.393579. Epub 2012 Oct 15.

Reengineering the collision coupling and diffusion mode of the A2A-adenosine receptor: palmitoylation in helix 8 relieves confinement.

Author information

1
Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Währinger Strasse 13A, 1090 Vienna, Austria.

Abstract

The A(2A)-adenosine receptor undergoes restricted collision coupling with its cognate G protein G(s) and lacks a palmitoylation site at the end of helix 8 in its intracellular C terminus. We explored the hypothesis that there was a causal link between the absence of a palmitoyl moiety and restricted collision coupling by introducing a palmitoylation site. The resulting mutant A(2A)-R309C receptor underwent palmitoylation as verified by both mass spectrometry and metabolic labeling. In contrast to the wild type A(2A) receptor, the concentration-response curve for agonist-induced cAMP accumulation was shifted to the left with increasing expression levels of A(2A)-R309C receptor, an observation consistent with collision coupling. Single particle tracking of quantum dot-labeled receptors confirmed that wild type and mutant A(2A) receptor differed in diffusivity and diffusion mode; 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 (estimated radius 110 nm). These were rarely seen with the palmitoylated A(2A)-R309C receptor, the preferred diffusion mode of which was a random walk in both the basal and the agonist-activated state. 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 A(2A) receptor. The experiments allowed for visualizing local confinement of an agonist-activated G protein-coupled receptor in an area consistent with the dimensions of a lipid raft.

PMID:
23071116
PMCID:
PMC3516756
DOI:
10.1074/jbc.M112.393579
[Indexed for MEDLINE]
Free PMC Article

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