Activation of the beta(2)-adrenergic receptor-Galpha(s) complex leads to rapid depalmitoylation and inhibition of repalmitoylation of both the receptor and Galpha(s).

Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein alpha subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory alpha subunit of trimeric G protein (Galpha(s)) covalently attached to the beta(2)-adrenergic receptor (beta(2)AR) as a carboxyl-terminal extension (beta(2)AR-Galpha(s)) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Galpha(s) moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the beta-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the beta(2)AR and Galpha(s) and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of beta(2)AR-Galpha(s), indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.