Mutagenesis of acidic residues in the β2-adrenergic receptor abolishes the requirement of low pH for receptor dephosphorylation

We have previously shown that acidic pH activates β2-adrenergic receptor (B2AR) dephosphorylation both in vitro as well as in cells, where sequestered B2ARs are localized to endosomes. To further define the role of acidic pH in receptor dephosphorylation, vesicles containing phosphorylated B2AR and bacteriorhodopsin were incubated with PP-2A. Upon illumination, an acidic environment is created inside the vesicles due to the ability of bacteriorhodopsin to act as a light-driven proton pump. While little dephosphorylation of phosphorylated B2AR was observed when the reaction was performed in the dark, illumination resulted in a 10-fold stimulation of the amount of B2AR dephosphorylated. Since only the intravesicular side of the receptor was exposed to the low pH environment, these data confirm a low pH-induced conformational change within the receptor. As acidic residues have been reported to play a role in conformational changes elicited by the low pH found in endosomes, we mutated 3 acidic residues located in the second extracellular loop of the B2AR to Ala. Compared to wild type (WT) B2AR, mutant receptor displayed an increased rate of resensitization, a process which requires receptor dephosphorylation. Further, while WT B2AR was not dephosphorylated upon incubation with phosphatase at neutral pH but only at acidic pH, the mutant B2AR was dephosphorylated at neutral pH. These data suggest that the conformation of the mutant mimics that of the WT receptor in endosomes and underscore the involvement of acidic residues in the pH-induced conformational change in the receptor.

Duke Scholars

Author Robert J. Lefkowitz Medicine, Cardiology