Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage

Abstract

The β₁-adrenergic receptor (β₁AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β₁AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by <i>O</i>-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically <i>O</i>-glycosylates β₁AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using <i>in vitro O</i>-glycosylation and proteolytic cleavage assays, a cell line deficient in <i>O</i>-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β₁AR. Furthermore, we demonstrate that impaired <i>O</i>-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific <i>O</i>-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β₁AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.