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

The beta(1)-adrenergic receptor (beta(1)AR) 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 beta(1)-adrenergic antagonists, such as beta(1)-blockers, relatively little is yet known about its regulation. We have shown previously that beta(1)AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAcT2) specifically O-glycosylates beta(1)AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-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 beta(1)AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the beta(1)AR agonist isoproterenol and its potency in a cAMPaccumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, aGPCRas a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of beta(1)AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases.