Reengineering the collision coupling and diffusion mode of the A2A-adenosine receptor: palmitoylation in helix 8 relieves confinement.
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.
Institute of Pharmacology, Center of Physiology and Pharmacology, Medical University of Vienna, Währinger Strasse 13A, 1090 Vienna, Austria.
Simon
S
Keuerleber
Patrick
P
Thurner
Christian W
CW
Gruber
Jürgen
J
Zezula
Michael
M
Freissmuth
D019943:Amino Acid Substitution; D002460:Cell Line; D006801:Humans; D054878:Lipoylation / Q000502:physiology*; D021962:Membrane Microdomains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D045663:Quantum Dots; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D043705:Receptor, Adenosine A2A