Altered phosphorylation and desensitization patterns of a human beta 2-adrenergic receptor lacking the palmitoylated...

Exposure of beta 2-adrenergic receptors to agonists causes a rapid desensitization of the receptor-stimulated adenylyl cyclase, associated with an increased phosphorylation of the receptor. Agonist-promoted phosphorylation of the beta 2-adrenergic receptor (beta 2AR) by protein kinase A and the beta-adrenergic receptor kinase (beta ARK) is believed to promote a functional uncoupling of the receptor from the guanyl nucleotide regulatory protein Gs. More recently, palmitoylation of Cys341 of the receptor has also been proposed to play an important role in the coupling of the beta 2-adrenergic receptor to Gs. Here we report that substitution of the palmitoylated cysteine by a glycine (Gly341 beta 2 AR) using site directed mutagenesis leads to a receptor being highly phosphorylated and largely uncoupled from Gs. In Chinese hamster fibroblasts (CHW), stably transfected with the human receptor cDNAs, the basal phosphorylation level of Gly341 beta 2AR was found to be approximately 4 times that of the wild type receptor. This elevated phosphorylation level was accompanied by a depressed ability of the receptor to stimulate the adenylyl cyclase and to form a guanyl nucleotide-sensitive high affinity state for agonists. Moreover, exposure of this unpalmitoylated receptor to an agonist did not promote any further phosphorylation or uncoupling. A modest desensitization of the receptor-stimulated adenylyl cyclase response was observed but resulted from the agonist-induced sequestration of the unpalmitoylated receptor and could be blocked by concanavalin A. This contrasts with the agonist-promoted phosphorylation and uncoupling of the wild type receptor.(ABSTRACT TRUNCATED AT 250 WORDS)