Role of the cyclic AMP-dependent protein kinase in homologous resensitization of the β1-adrenergic receptor

Abstract

A fundamental question in biology is how the various motifs in G protein-coupled receptors participate in the divergent functions orchestrated by these molecules. Here we describe a fundamental role for a serine residue at position 312 in the third intracellular loop of the human β 1-adrenergic receptor (β1-AR) in endocytic recycling of the agonist-internalized receptor. In receptor recycling experiments that were monitored by confocal microscopy, the agonist-internalized wild-type (WT) β1-AR recycled with a t0.5 of 14 ± 3 min. Mutagenesis of Ser312 to alanine (Ser312 → Asp β1-AR) or to the phosphoserine mimic aspartic acid (Ser 312 → Asp β1-AR) resulted in β 1-AR constructs that were pharmacologically indistinguishable from the WT β1-AR. The internalized Ser312 → Asp β1-AR recycled efficiently with a t0.5 of 11 ± 3 min, whereas the internalized Ser312 → Ala β1-AR was not recycled or functionally resensitized through the endosomal pathway. Because this serine is a putative residue for phosphorylation by the cyclic AMP-dependent protein kinase (PKA), we examined the role of this kinase in recycling of the internalized β1-AR. Inhibition of PKA biochemically or genetically using a dominant negative PKA construct blocked the recycling of the internalized WT β1-AR. Phosphorylation studies revealed that the β1-AR is partially phosphorylated by PKA and that phosphorylation of the β1-AR by the catalytic subunit of PKA occurs exclusively at Ser312. Our results identify a new signaling paradigm in which homologous activation of a kinase provides a reversible modification that shifts the itinerary of the internalized receptor toward recycling and resensitization. Therefore, PKA-mediated phosphorylation of G protein-coupled receptors might result in motif-dependent desensitization or resensitization.