Palmitoylated cysteine 341 modulates phosphorylation of the β2- adrenergic receptor by the cAMP-dependent protein kinase

Abstract

We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human β2-adrenergic receptor (Gly341β2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with G(s). In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341β2AR) restored normal phosphorylation and receptor-G(s) coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified β2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346β2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341β2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoyletion restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the β2AR and by doing so modulates the responsiveness of the receptor.