Phosphatidyl serine preconditions the A2AAR for G-protein coupling via a conserved motif on TM6 and TM7.

Abstract

19F NMR of A2AAR in nanodiscs of defined composition indicates that negative charge modulates activation of the receptor. The fully active state is obtained either when bound to G-protein, or without G-protein but in a membrane environment containing negatively charged headgroups. Mutagenesis indicates that a trio of residues on the intracellular ends of TM6 and TM7 are responsible for the lipid-dependent activity. In order to determine the mechanism, a series of molecular dynamics simulations were performed. Comparison of simulations of the inactive and the active, G-protein bound state shows that a glutamic acid on the helix of the G-protein coordinates interactions between these three residues. This suggested a hypothesis in which a negatively charged headgroup can stand-in for the glutamic acid side chain, coordinating the ends of TM6 and TM7 and preconditioning the receptor for G-protein coupling. To test this idea, simulations of the fully active state were performed with and without phosphatidyl serine headgroups, but without the G-protein. Consistent with our hypothesis, the TM6 -- TM7 interactions are similar to the G-protein coupled state in the presence of PS, but similar to the inactive state in the absence of PS. Moreover, the interaction of a PS headgroup with a key residue on TM6 recapitulates the same interaction with the glutamic acid, but only when the receptor is in the active state.