Abstract 460: β ₁ -Adrenergic Receptor O-glycosylation Regulates Receptor N-terminal Cleavage and Signaling Responses in Cardiomyocytes

β1-adrenergic receptors (β1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Recent efforts to structurally characterize β1ARs have focused on the ligand binding sites in transmembrane helices and effector docking sites in the intracellular loops and C-terminus. The notion that the β1AR extracellular N-terminus (which is a substrate for both N- and O-linked glycosylation and a target for proteolytic cleavage) functions as a structural determinant of β1AR activation has never been considered. This study takes advantage of site-directed mutagenesis and expression studies in Chinese Hamster ovary cells that harbor a reversible glycosylation defect to map β1ARs O-glycosylation sites to Ser37/Ser41 and show that O-glycosylation at these sites prevents β1AR N-terminal cleavage. We then used an adenoviral overexpression approach to show that both full-length fully-glycosylated β1ARs (β1AR-FL) and N-terminally truncated glycosylation-defective β1ARs (β1AR-Δ52) increase cAMP accumulation and activate the ERK-MAPK signaling cascade in cardiomyocytes. However, isoproterenol-dependent cAMP accumulation is markedly enhanced, and ERK activation is markedly attenuated, in cardiomyocytes that express β1AR-Δ52, compared with β1AR-FL cultures. The conclusion that β1ARs are stabilized in a conformation that is biased toward the cAMP pathway as a result of N-terminal truncation gains further support from additional studies showing that agonistic β1AR autoantibodies induce a more rapid and robust increase in membrane PKA activity in cells that express glycosylation-defective β1ARs, compared to cells that express full-length glycosylated β1ARs. These results identify a novel role for O-glycosylation as a structural determinant of β1AR responsiveness in the heart.