β1-adrenergic receptor N-terminal cleavage by ADAM17; the mechanism for redox-dependent downregulation of cardiomyocyte β1-adrenergic receptors

https://doi.org/10.1016/j.yjmcc.2021.01.012Get rights and content

Highlights

  • The β1AR N-terminus functions as a structural determinant of β1AR responses in cardiomyocytes.

  • The β1AR N-terminus is cleaved at R31↓L32 by ADAM17.

  • β1ARs contain a second O-glycan-regulated ADAM17-dependent N-terminal cleavage site at S41↓L42.

  • β1AR inactivation during oxidative stress is due to ADAM17-dependent cleavage at R31↓L32.

  • N-terminally truncated β1ARs afford protection against doxorubicin-dependent apoptosis.

Abstract

β1-adrenergic receptors (β1ARs) are the principle mediators of catecholamine action in cardiomyocytes. We previously showed that the β1AR extracellular N-terminus is a target for post-translational modifications that impact on signaling responses. Specifically, we showed that the β1AR N-terminus carries O-glycan modifications at Ser37/Ser41, that O-glycosylation prevents β1AR N-terminal cleavage, and that N-terminal truncation influences β1AR signaling to downstream effectors. However, the site(s) and mechanism for β1AR N-terminal cleavage in cells was not identified. This study shows that β1ARs are expressed in cardiomyocytes and other cells types as both full-length and N-terminally truncated species and that the truncated β1AR species is formed as a result of an O-glycan regulated N-terminal cleavage by ADAM17 at R31↓L32. We identify Ser41 as the major O-glycosylation site on the β1AR N-terminus and show that an O-glycan modification at Ser41 prevents ADAM17-dependent cleavage of the β1-AR N-terminus at S41↓L42, a second N-terminal cleavage site adjacent to this O-glycan modification (and it attenuates β1-AR N-terminal cleavage at R31↓L32). We previously reported that oxidative stress leads to a decrease in β1AR expression and catecholamine responsiveness in cardiomyocytes. This study shows that redox-inactivation of cardiomyocyte β1ARs is via a mechanism involving N-terminal truncation at R31↓L32 by ADAM17. In keeping with the previous observation that N-terminally truncated β1ARs constitutively activate an AKT pathway that affords protection against doxorubicin-dependent apoptosis, overexpression of a cleavage resistant β1AR mutant exacerbates doxorubicin-dependent apoptosis. These studies identify the β1AR N-terminus as a structural determinant of β1AR responses that can be targeted for therapeutic advantage.

Introduction

Catecholamines enhance the mechanical performance of the heart by activating cardiac β-adrenergic receptors (βARs). While cardiomyocytes co-express β1AR and β2AR, the β1AR is the predominant subtype (constituting ~75–80% of the total βAR population) and the principle driver of catecholamine-dependent sympathetic responses in the healthy heart [1]. β1ARs provide hemodynamic support in the setting of acute stress by activating a Gs-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway that phosphorylates substrates that enhance excitation/contraction coupling [2]. Although β1ARs also couple to cardioprotective Gs-independent mechanisms such as extracellular signal-regulated kinase (ERK), chronic/persistent β1AR activation leads to a spectrum of changes (including cardiomyocyte hypertrophy/apoptosis, interstitial fibrosis, and contractile dysfunction) that contribute to the pathogenesis of heart failure [3,4].

Like other G protein-coupled receptor (GPCR) superfamily members, βARs are characterized by an extracellular N-terminus, seven membrane-spanning α-helical segments that are joined by three extracellular loops and three intracellular loops, and an intracellular C-terminus. While βARs have served as prototypes in structural studies designed to elucidate the molecular dynamics of GPCR activation, studies to date have focused on specific amino acid side chains in the transmembrane helices that form the ligand binding pocket and effector docking sites on the βAR's intracellular surface [5]. The relatively short/unstructured βAR N-terminus - a region traditionally viewed as having a negligible role in mechanisms that contribute to receptor activation and regulation - has routinely been removed for these structural studies [5]. However, our recent studies challenge the notion that the β1AR N-terminus plays a negligible role in receptor activation/regulation showing that the β1AR extracellular N-terminus is a target for glycan-regulated proteolytic cleavage and that this mechanism influences β1AR signaling responses in cardiomyocytes [6]. We showed that the β1AR is detected in cardiomyocytes as both full-length and N-terminally truncated species, that N-terminal cleavage is controlled by an O-glycan modification on the β1AR N-terminus at Ser37/Ser41 (residues in the vicinity of predicted β1AR N-terminal cleavage sites) and that the β1AR extracellular N-terminus functions as a heretofore-unrecognized structural determinant of β1AR activation [6]. N-terminally truncated β1ARs are signaling competent, but their signaling properties differ from that of the full-length β1AR; they display differences in their signaling bias to cAMP/PKA vs. ERK pathways – and only the N-terminally truncated form of the β1AR constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes [6,7].

Efforts to date to identify the O-glycan modifying enzymes and specific proteases that participate in the maturational processing of the β1AR N-terminus have relied on in vitro approaches. On the basis of in vitro cleavage assays (using short peptides based upon the β1AR N-terminal sequence as substrate and purified matrix metalloproteinase [MMP] or adisintegrin and metalloproteinase [ADAM] enzymes), Goth et al. concluded that the β1AR N-terminus contains two major cleavage sites at R31↓L32 and P52↓L53. They showed that O-glycosylated and unmodified forms of these peptides can serve as substrates for many different MMP and ADAM family enzymes and that O-glycosylation influences the efficiency of peptide cleavage at some sites by some enzymes [8]. It is important to note that this previous study interrogated the role of O-glycosylation by subjecting peptides to in vitro O-glycosylation reactions with GalNAc-T2, a reaction that adds a single GalNAc moiety to one or more sites on the peptide. Results obtained in this type of reductionist assay system may not necessarily recapitulate the proteolytic cleavage events that accompany maturational processing of full-length β1ARs in a more physiologically relevant context (such as in differentiated cells such as a cardiomyocyte). First, this approach does not evaluate the role of more physiologically relevant extended and sialylated O-glycan structures that decorate the β1AR N-terminus in a cellular context (and are predicted to undergo remodeling in the setting of inflammation, metabolic disorders, and/or cardiac hypertrophy [9,10]). Second, in vitro cleavage assays are not suited to evaluate cleavage events on membrane proteins, a situation where substrate and enzyme may localize to distinct membrane subdomains or (when tethered to the same membrane) be conformationally restrained. Third, the previous study did not resolve the importance of individual O-glycosylation sites in the control of β1AR N-terminal cleavage. Finally, the in vitro approach cannot be used to explore the role of glycan-regulated β1AR N-terminal cleavage events in the response to pathophysiologic cardiac injury. This study addresses these issues by mapping the O-glycosylation sites that regulate β1AR N-terminal cleavage in cardiomyocytes, identifying ADAM17 as the protease required for β1AR N-terminal cleavage in cardiomyocytes, and implicating ADAM17-dependent β1AR N-terminal cleavage in an oxidative stress-dependent mechanism that calibrates cardiomyocyte catecholamine responsiveness.

Section snippets

Materials

Antibodies were from the following sources: Rabbit polyclonal anti-β1AR (ab3442, raised against residues 394–408 in human β1AR) was from Abcam (Cambridge, MA). Mouse monoclonal anti-FLAG M2 and anti-β-actin were from Sigma-Aldrich (Saint Louis, MO). Rabbit monoclonal anti-cleaved caspase-3 (Asp175) (5A1E) was from Cell Signaling Technology (Danvers, MA). IRDye 800CW or 680RD goat anti-rabbit or goat anti-mouse IgG (H + L) were from LI-COR Biosciences (Lincoln, NE). GM6001, GI254023X (ADAM-10

Maturational processing of the β1AR N-terminus; proteolytic cleavage at R31↓L32

A previous study concluded that the β1AR N-terminus contains two major cleavage sites at R31↓L32 and P52↓L53 (Fig. 1A) based upon in vitro cleavage assays using short peptides corresponding to the β1AR N-terminal sequence and purified ADAM or MMP enzymes [8]. Since the proteolytic cleavage events that can be identified in this type of reductionist assay system may not necessarily reflect proteolytic cleavage events that regulate the maturational processing of the full-length β1AR in cells, we

Discussion

Cardiomyocyte βARs are among the most intensely studied members of the GPCR superfamily primarily because they control a large number of physiologically important processes that impact on the pathogenesis of cardiovascular disease (and hence constitute clinically important targets for drug discovery). Studies to date implicate cardiomyocyte β1ARs in the activation of cellular responses required for normal physiological control of cardiac contractility as well as responses that contribute to the

Funding source

This work is supported by the National Institutes of Health, National Heart, Blood, and Lung Institute grant HL138468.

Declaration of competing interest

None.

Acknowledgements

The authors thank HaeJung Chung for technical assistance.

References (33)

  • V.O. Rybin et al.

    Differential targeting of β-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling pathway

    J. Biol. Chem.

    (2000)
  • E.P. van der Vorst et al.

    A disintegrin and metalloproteases: molecular scissors in angiogenesis, inflammation and atherosclerosis

    Atherosclerosis

    (2012)
  • M. Satoh et al.

    Expression of tumor necrosis factor-alpha--converting enzyme and tumor necrosis factor-alpha in human myocarditis

    J. Am. Coll. Cardiol.

    (2000)
  • J. Wang et al.

    G-protein-coupled receptors in heart disease

    Circ. Res.

    (2018)
  • S.F. Steinberg

    Beta1-adrenergic receptor regulation revisited

    Circ. Res.

    (2018)
  • C. de Lucia et al.

    New insights in cardiac β-adrenergic signaling during heart failure and aging

    Front. Pharmacol.

    (2018)
  • Cited by (14)

    • Dissecting Beta-Adrenergic Receptors: The Sum of Many Parts

      2023, JACC: Basic to Translational Science
    • Glycosylation-dependent cleavage of the human β<inf>1</inf>-adrenoceptor

      2021, Journal of Molecular and Cellular Cardiology
    View all citing articles on Scopus
    View full text