Altered O‐glycosylation of β1‐adrenergic receptor N‐terminal single‐nucleotide variants modulates receptor processing and functional activity

N‐terminal nonsynonymous single‐nucleotide polymorphisms (SNPs) of G protein‐coupled receptors (GPCRs) are common and often affect receptor post‐translational modifications. Their functional implications are, however, largely unknown. We have previously shown that the human β 1 ‐adrenergic receptor (β 1 AR) is O‐glycosylated in the N‐terminal extracellular domain by polypeptide GalNAc transferase‐2 that co‐regulates receptor proteolytic cleavage. Here, we demonstrate that the common S49G and the rare A29T and R31Q SNPs alter these modifications, leading to distinct effects on receptor processing. This was achieved by in vitro O‐glycosylation assays, analysis of native receptor N‐terminal O‐glycopeptides, and expression of receptor variants in cell lines and neonatal rat ventricular cardiomyocytes deficient in O‐glycosylation. The SNPs eliminated (S49G) or introduced (A29T) regulatory O‐glycosites that enhanced or inhibited cleavage at the adjacent sites (P 52 ↓L 53 and R 31 ↓L 32 ), respectively, or abolished the major site at R 31 ↓L 32 (R31Q). The inhibition of proteolysis of the T29 and Q31 variants correlated with increased full‐length receptor levels at the cell surface. Furthermore, the S49 variant showed increased isoproterenol‐mediated signaling in an enhanced bystander bioluminescence energy transfer β‐arrestin2 recruitment assay in a coordinated manner with the common C‐terminal R389G polymorphism. As Gly at position 49 is ancestral in placental mammals, the results suggest that its exchange to Ser has created a β 1 AR gain‐of‐function phenotype in humans. This study provides evidence for regulatory mechanisms by which GPCR SNPs outside canonical domains that govern ligand binding and activation can alter receptor processing and function. Further studies on other GPCR SNPs with clinical importance as drug targets are thus warranted.