N-glycosylation of α1D-adrenergic receptor N-terminal domain is required for correct trafficking, function, and biogenesis

Abstract

G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by posttranslational modifications, including N-glycosylation of asparagine residues. α 1D-adrenergic receptors (α 1D-ARs)-key regulators of central and autonomic nervous system function-contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α 1D-AR between transmembrane domain 2 and 3. Labelfree dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α 1D-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α 1D-AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α 1D-AR n-terminal glycosylation is required for complete translation of nascent, functional receptor. G protein-coupled receptors (GPCRs) are essential membrane proteins that regulate the vast majority of physiological functions in the human body. As a result, GPCRs have been estimated to be targeted by approximately one third of all currently approved medications 1. Adrenergic receptors (ARs) are a clinically relevant subfamily of GPCRs. Activated by the endogenous sympathetic neurotransmitters epinephrine and norepinephrine, adrenergic GPCRs consist of three major subtypes: α 1 , α 2 , and β. The α 1 sub-family-containing α 1A , α 1B , and α 1D subtypes 2-are targets for medications that regulate blood pressure 3,4 , bladder 5,6 , prostate 7,8 , and central nervous system function 9-11. Thus, understanding the molecular and cellular mechanisms regulating α 1-AR function will help spur the development of new medications associated with aberrant α 1-AR signaling, such as hypertension, PTSD, schizophrenia, and benign prostatic hypertrophy 12-15. Among the three α 1 subtypes, the α 1D-AR remains poorly understood due to technical challenges. Relative to the closely related α 1A and α 1B-AR subtypes, α 1D-AR displays limited functional responses and minimal plasma membrane expression when expressed in heterologous cell culture 16-18. Although pharmacologically detectable in intact isolated aortae in organ-tissue bath assays 19 , α 1D-AR functional expression rapidly disappears in primary vascular smooth muscle cell cultures within 24-48 hours 20. Also, immortalized cell lines that endogenously express α 1D-ARs have yet to be discovered. Combined, these experimental clues indicate the molecular and cellular ...

Development of a Novel SNAP-Epitope Tag/Near-Infrared Imaging Assay to Quantify G Protein-Coupled Receptor Degradation in Human Cells

“…We and others have utilized SNAP-tag technology to monitor GPCR protein expression and localization in vitro. 16–25 Here, we investigated the utility of incorporating SNAP-tag technology into traditional CHX-chase assays to permit calculation of GPCR protein half-lives when expressed in cultured human cells. Our experimental workflow involved fusing the SNAP-tag to the N-terminal domain of select GPCRs, transiently transfecting SNAP-GPCR cDNA constructs into HEK293 cells, and then treating cells with 50 µg/mL CHX for 0–24 h. Cells were then lysed and treated with the irreversible SNAP substrate BG-782, subjected to PAGE, and analyzed with LICOR Odyssey NIR quantitative analysis.…”

“…Thus, the primary goal of this study was to develop a simple, reproducible approach that can be used to quantify degradation half-lives of GPCRs (and possibly other membrane proteins) in high throughput relative to traditional immunoprecipitation/immunoblotting. Previous studies in which the SNAP-tag was leveraged to identify and characterize the α 1D -AR:Scribble:dystrophin-associated protein complex, 34 determined the importance of type I PDZ ligands for GPCR functional expression, 15 or facilitated the serendipitous discovery that the α 1D -AR N-terminal domain is endogenously cleaved 19 guided the decision to incorporate the SNAP-tag into CHX-chase assays. Herein, we demonstrated that the SNAP-epitope tag is remarkably stable and does not induce degradation of β-actin, and thus is not likely enhancing GPCR degradation rates.…”

Post-Translational Modifications of G Protein–Coupled Receptors Control Cellular Signaling Dynamics in Space and Time

“…A more recent report indicates that a mutant purinergic P2Y 2 receptor deficient in glycosylation undergoes ER-associated proteasomal degradation pathway, possibly due to retention in ER lipid rafts and failure of traffic to the Golgi (Nakagawa et al, 2017). A similar observation was made for a 1D -adrenoreceptor, where a glycosylation-deficient mutant displays impaired plasma membrane expression likely because of degradation via ERAD (Janezic et al, 2020). Collectively, substantial evidence suggests that N-glycosylation of GPCRs during maturation in the biosynthetic pathway is essential to achieve optimal cell surface expression.…”

N-Glycans on the extracellular domain of the Notch1 receptor control Jagged-1 induced Notch signalling and myogenic differentiation of S100β resident vascular stem cells

“…Future studies using additional mutations on both the essential asparagine residue and on other proximal non-essential residues could reveal the impact of the amino acid alteration on protein expression, function, and stability. Many studies which involve the mutation of N-glycosylation sites on a glycoprotein also use simultaneous multi-site mutation to investigate whether the combined impact of losing two or more N-glycans differs from the effect observed following loss of a single N-glycan ( 52 , 60 , 61 ). This is a strategy that could reveal crucial information about the synergistic roles of N-glycans on the Notch1 ECD.…”