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