Site-specificO-Glycosylation by PolypeptideN-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.
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 byO-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specificallyO-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. Usingin vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient inO-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 impairedO-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-specificO-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.
From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Christoffer K
CK
Goth
the Medical Research Center Oulu, Research Unit of Biomedicine, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland.
Hanna E
HE
Tuhkanen
the Medical Research Center Oulu, Research Unit of Biomedicine, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland.
Hamayun
H
Khan
the Medical Research Center Oulu, Research Unit of Biomedicine, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland.
Jarkko J
JJ
Lackman
From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Shengjun
S
Wang
From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Yoshiki
Y
Narimatsu
the Department of Clinical Biochemistry, Rigshospitalet, Copenhagen University Hospital, DK-2100 Copenhagen Ø, Denmark and.
Lasse H
LH
Hansen
the Centre for Blood Research, Department of Oral Biological and Medical Sciences, and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Christopher M
CM
Overall
From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
Henrik
H
Clausen
From the Copenhagen Center for Glycomics, Department of Cellular and Molecular Medicine, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark, Schjoldager@sund.ku.dk.
Katrine T
KT
Schjoldager
the Medical Research Center Oulu, Research Unit of Biomedicine, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland, ulla.petaja-repo@oulu.fi.
Ulla E
UE
Petäjä-Repo
D000595:Amino Acid Sequence; D000818:Animals; D055786:Gene Knockout Techniques; D006031:Glycosylation; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D051381:Rats; D018342:Receptors, Adrenergic, beta-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
D017350:N-Acetylgalactosaminyltransferases; D020033:Protein Isoforms; D018342:Receptors, Adrenergic, beta-1; D000097763:Polypeptide N-acetylgalactosaminyltransferase
ADAM; G protein-coupled receptor (GPCR); adrenergic receptor; glycoprotein; glycosyltransferase; matrix metalloproteinase (MMP); protein processing; receptor structure-function