PALMITOYLATED CYSTEINE-341 MODULATES PHOSPHORYLATION OF THE BETA(2)-ADRENERGIC RECEPTOR BY THE CAMP-DEPENDENT PROTEIN-KINASE

We previously showed that substitution of a glycine residue for the pa lmitoylated cysteine 341 of the human beta(2)-adrenergic receptor (Gly (341)beta(2)AR), increases the basal level of the receptor phosphoryla tion and reduces its ability to functionally interact with G(s). In th e present study, we show that additional mutation of serines 345 and 3 46 (Ala(345,346)Gly(341)beta(2)AR) restored normal phosphorylation and receptor-G(s) coupling, thus suggesting that the increased phosphoryl ation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. Thi s is supported by the observation that chemical depalmitoylation of pu rified beta(2)AR did not affect the ability of the receptor to stimula te adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser(345,346) in a wild type receptor background (Ala(345,346)beta(2)A R) significantly decreased the rate of agonist-promoted desensitizatio n of the receptor-stimulated adenylyl cyclase activity, supporting a r ole for this phosphorylation site in regulating the functional couplin g of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immedi ately downstream of the palmitoylated cysteine 341, the hypothesis tha t the accessibility of this site may be regulated by the receptor palm itoylation state mas further assessed in vitro. In membrane phosphoryl ation assays, Gly(341)beta(2)AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palm itoylation 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 beta(2)AR and by doing so modulates the responsiv eness of the receptor.