Redox and proteolytic regulation of cardiomyocyte β1-adrenergic receptors – a novel paradigm for the regulation of catecholamine responsiveness in the heart

Conventional models view β1-adrenergic receptors (β1ARs) as full-length proteins that activate signaling pathways that influence contractile function and ventricular remodeling - and are susceptible to agonist-dependent desensitization. This perspective summarizes recent studies from my laboratory showing that post-translational processing of the β1-adrenergic receptor N-terminus results in the accumulation of both full-length and N-terminally truncated forms of the β1AR that differ in their signaling properties. We also implicate oxidative stress and β1AR cleavage by elastase as two novel mechanisms that would (in the setting of cardiac injury or inflammation) lead to altered or decreased β1AR responsiveness.

1 Introduction b-Adrenergic receptors (bARs) are among the most intensively studied members of the G protein-coupled receptor superfamily primarily because they control physiologic mechanisms that impact on the pathogenesis of cardiovascular disease; they are clinically important targets for drug discovery.Conventional models hold that bARs function to rapidly adjust cardiac output by recruiting a Gs-adenylyl cyclase pathway that leads to the accumulation of cAMP, activation of protein kinase A, and phosphorylation of membrane and sarcomeric proteins involved in excitation-contraction coupling (Figure 1).bAR-driven inotropic and chronotropic responses provide hemodynamic support in the setting of acute heart failure.However, with chronic heart failure, agonist-occupied bARs are stabilized in an active conformation that is phosphorylated by G protein-coupled receptor kinase (GRK); GRK-phosphorylated receptors then recruit b-arrestin, an adapter protein that acts to both sterically interdict bAR-G protein interactions and scaffold binding partners that trigger a second wave of signaling to Gs-independent growth regulatory responses such as extracellular-signal regulated kinase (ERK) and AKT (1,2).While these pathways generally have been implicated in cardioprotection, studies in cardiomyocytes also link chronic bAR activation the activation of proapoptotic pathways and a spectrum of changes (including cardiomyocyte hypertrophy and apoptosis, interstitial fibrosis, and contractile dysfunction) that contribute to the pathogenesis of heart failure (3,4).
It is worth noting that conventional models describing the molecular basis for bAR signaling responses derive from literature heavily biased towards an analysis of the b 2 AR.While the literature has generally tended to assume that the signaling properties of b 1 ARs and b 2 ARs are similar (and that b 2 ARs can serve as meaningful surrogates for the b 1 AR subtype), this ignores a substantial body of evidence showing that b 1 ARs are relatively resistant to agonist-induced GRK-dependent phosphorylation, they engage b-arrestin only weakly (5), and they show little-to-no agonist-induced internalization (6, 7).These differences should not be surprising, since the 54% overall homology between b 1 AR and b 2 AR subtypes is largely confined to their transmembrane, ligand-binding pockets; their N-and C-termini are quite distinct (8).This perspective summarizes our recent studies focusing on the b 1 AR N-terminus, the relatively short/unstructured extracellular portion of the receptor that differs in length, sequence, and posttranslational processing from the b 2 AR N-terminus.Our studies characterize an O-glycan regulated N-terminal cleavage mechanism that is specific for the b 1 AR (does not apply to the b 2 AR) that results in the generation of N-terminally truncated b 1 ARs with signaling properties that differ from that described for full-length b 1 ARs (9, 10).Our results force a reexamination of current concepts regarding the molecular basis for b 1 AR-dependent signaling responses, since conventional models have been derived from literature predicated on the assumption that b 1 ARs signal exclusively as full-length receptors.This perspective focuses on the novel notion b 1 AR cleavage results in the generation of distinct b 1 AR species that differ in their signaling properties.2A).In way of background, N-glycosylation results from the en bloc transfer of a preformed complex glycan structure to an asparagine residue in a N-x-S/T consensus motif, with the glycan then further trimmed/modified to yield higher-order glycan structures.In contrast, O-glycosylation is a non-template driven post-translational modification that is initiated by the enzymatic transfer of a single a-GalNAc to an acceptor serine or threonine residue (a reaction catalyzed by a member of the multi-gene family of polypeptide GalNAc-transferases that differ in their tissue distribution and substrate specificities), followed by the step-wise enzymatic transfer of additional sugars (galactose, GlcNac, fucose, etc.) to yield a spectrum of complex higher-order linear and/or branched glycan structures (11).This mechanism allows for a high level of structural diversity/microheterogeneity, even at single sites within a given protein.Evidence that clusters of O-glycans can serve as barriers to prevent protein cleavage by proteases (11) provided the rationale to examine whether O-glycosylation play a role in the maturational processing of b 1 ARs.
We used a range of biochemical and mutagenesis approaches [described in detail in previous publications (9,10)] to map the It is worth noting that the O-glycan-regulated N-terminal cleavage mechanism identified in our studies provides the first credible explanation for the molecular heterogeneity observed for native b 1 AR in various cardiac preparations.Our findings also emphasize that immunblotting or immunohistochemistry studies that rely on antibodies to N-terminal epitope tags to track b 1 AR expression and/or localization should be interpreted with caution, since these techniques do not capture N-terminally truncated forms of the b 1 AR.

b 1 AR N-terminal cleavage
We used mutagenesis and pharmacologic strategies [described in (9,10)] to map the major O-glycan-regulated b 1 AR N-terminal cleavage site to R 31 ↓L 32 and show that cleavage at this site is attributable to the cellular actions of a disintegrin and metalloproteinase 17 [ADAM17; Figure 2A (9,10)].We also identified a secondary ADAM17-dependent N-terminal cleavage at S 41 ↓L 42 that is specifically inhibited by the O-glycan modification at S 41 (10).
The observation that the b 1 AR N-terminus can serve as a target for proteolytic cleavage by ADAM17 raised the obvious question of whether full-length b 1 ARs on the cell surface are cleaved by other proteases.Since the R 31 ↓L 32 cleavage site conforms to a consensus trypsin cleavage motif, we examined whether b 1 ARs are cleaved by trypsin.We showed that trypsin cleaves full-length b 1 ARs and that cleavage is specifically at the N-terminal R 31 ↓L 32 cleavage site (Figure 2A (12),).This mechanism should interest laboratories that interrogate b 1 AR signaling in isolated adult cardiomyocytescells typically extracted from intact ventricular using trypsin digests (13).The in vivo significance of cardiomyocyte b 1 AR cleavage by trypsin (a digestive enzyme found in the gastrointestinal tract) is dubious.
Therefore, we turned our attention to other, more pathophysiologically relevant proteases.Preliminary studies performed in CHO-Pro5 cells failed to identify significant levels (A) The b 1 AR N-terminal sequence showing the single N-glycosylation site at N 15 , O-glycosylation sites at T 28 , S 37 , and S 41 , and the major ADAM17 cleavage site at R 31 ↓L 32 as well as a second ADAM17 cleavage site at S 41 ↓L 42 (that is exposed only when S 41 O-glycosylation is prevented).(B) b 1 AR N-terminal cleavage by neutrophil elastase occurs only when the O-linked glycan at T 28 is not capped by a terminal sialic acid; O-linked glycans at S 37 and S 41 do not impact on elastase-dependent b 1 AR N-terminal cleavage.Studies in neonatal rat ventricular cardiomyocytes expose an additional intramolecular cleavage site for elastase (that is not dependent on N-terminal glycan desialylation) that disrupts catecholamine responsiveness.
of b 1 AR cleavage by various Since terminal charged acid residues have been implicated as regulators of glycoprotein cleavage (14-16) and O-glycan attachments on the b 1 AR Nterminus are heavily sialylated (9), we repeated the protease screens in cells treated with neuraminidase (an enzyme that removes terminal sialic acid residues).These studies exposed an action of elastase to cleave full-length (but not N-terminally truncated) b 1 ARs under conditions that disrupt glycoprotein sialylation [Figure 2B (17)].We then used a mutagenesis strategy to identify the sialylated O-glycan attachment that prevents elastase cleavage.Preliminary studies effectively ruled out roles for previously identified N-or O-glycosylation sites at N 15 , S 37 , or S 41 .Therefore, we considered a possible role for T 28 , a residue previously reported to be O-glycosylated in the context of reductionist in vitro assays (18).These studies showed that a sialylated O-glycan at T 28 plays only a minor role in the maturational processing of b 1 ARs to full-length receptors, but it fully protects b 1 ARs from elastase-dependent cleavage (17).This novel mechanism for b 1 AR regulation is predicted to have pathophysiologic importance, given that neuraminidase is released along with elastase by activated neutrophils at sites of inflammation or injury (Figure 2).
We then used a mutagenesis strategy in an attempt to map the elastase cleavage site.Our studies excluded possible roles for previously identified MMP/ADAM17-sensitive sites at R 31 ↓L 32 or S 41 ↓L 42 indicating that elastase cleavage must be at another site on the b 1 AR N-terminus (17).In this regard, it is intriguing to note that T 28 is strategically positioned adjacent to an elastase consensus cleavage motif (i.e., elastase typically cleaves scissile bonds Cterminal to small residues such as Ala, Gly, or Val; Figure 2A).
An elastase-dependent cleavage mechanism restricted to the b 1 AR N-terminus would generate a uniform population of signaling-competent N-terminally truncated receptorsa molecular form of the b 1 AR that constitutively couples to the cardioprotective AKT pathway.Hence, this type of proteolytic cleavage mechanism is predicted to afford survival advantage in the setting of heart failure.

b 1 AR cleavage at an intramolecular site
The actions of elastase also were examined in the more physiologically relevant cardiomyocyte context.Here, elastase treatment (even under conditions that do not disrupt protein Oglycosylation or sialylation) leads to a decrease in the abundance of the b 1 AR in association with the accumulation of ~40 kD Nterminal and ~25 kD C-terminal fragments, consistent with an intramolecular cleavage in extracellular loop 2 (Figure 2B (17),).The additional observation that elastase treatment results in a pronounced defect in isoproterenol-dependent (but not basal or forskolin-dependent) cAMP accumulation (17) supports the conclusion that the b 1 AR fragments that accumulate in elastasetreated cardiomyocytes are signaling-incompetent.
Our studies linking elastase treatment to an intramolecular cleavage that disrupts b 1 AR responsiveness were performed in neonatal cardiomyocyte cultures.It is important to note that differences in b 1 AR glycosylation or trafficking patterns between neonatal and adult cardiomyocytes or in cardiomyocytes that have been induced to hypertrophy could in theory lead to differences in b 1 AR protease-sensitivity. Hence, the functional consequences of b 1 AR cleavage would depend upon whether cleavage is restricted to the N-terminus (resulting in a uniform population of cardioprotective b 1 ARs) or whether cleavage is at an intramolecular site that disrupts catecholamine responsiveness.Mechanisms that fine-tune b 1 AR protease-sensitivity are the subject of ongoing studies.

b 1 AR regulation by oxidative stress
There is considerable evidence that chronic heart failure (which is associated with elevated catecholamine levels) leads to a loss of cardiac reserve due to decreased expression and signaling by bARs.This loss of catecholamine responsiveness traditionally has been attributed to homologous desensitization and/or bAR downregulation.However, the notion that a single mechanism underlies the heart failure-induced defects in signaling by both the b 1 AR (the predominant bAR subtype and principal driver of catecholaminedriven sympathetic responses in the healthy heart) and the b 2 AR subtype is difficult to reconcile with clinical studies showing that b 1 ARs and b 2 ARs are regulated differently in heart failure; heart failure leads to a selective downregulation of the b 1 AR subtype that is not accompanied by a commensurate loss of b 2 ARs.This formulation also is at odds with cell-based studies showing that b 2 ARs undergo agonist-dependent desensitization/down-regulation, but b 1 ARs are relatively resistant agonist-induced desensitization/internalization (6,19).In this regard, it is worth noting that much like the N-termini, b 1 AR and b 1 AR C-termini and intracellular loops show little sequence homology; sites on the b 2 AR that serve as substrates for GRK phosphorylation and/or docking sites for b-arrestin are not conserved in the b 1 AR subtype.This raises a fundamental question as to the mechanism underlying the defect in b 1 AR responsiveness in heart failure.Our recent studies address this conundrum by showing that oxidative stress (a stimulus that contributes to the pathogenesis of heart failure and various other cardiomyopathic syndromes) decreases b 1 AR expression and isoproterenol responsiveness in cardiomyocytes; oxidative stress does not lead to changes in the expression of the b 2 AR subtype (20).Hence, these studies implicate oxidative stress as a mechanism that selectively decreases b 1 AR (but not b 2 AR) expression that would underly the decreased cardiac catecholamine responsiveness that is a hallmark of heart failure.

Discussion
Studies in model cell types have traditionally ignored possible differences in the biological controls and cellular actions of b 1 ARs vs. b 2 ARs.However, studies in genetic models of receptor overexpression provide compelling evidence that the deleterious effects of chronic sympathetic overdrive that contribute to the pathogenesis of cardiac hypertrophy and heart failure can be attributed to the cardiac actions of the b 1 AR subtype.These studies show that moderate levels of transgenic b 1 AR leads to maladaptive remodeling and heart failure (21,22) whereas even high levels of transgenic b 2 AR overexpression are relatively well tolerated (23-25).Our studies add an additional dimension to the analysis by showing that the b 1 AR subtype accumulates as both full-length and N-terminally truncated forms and that these distinct molecular forms of the b 1 AR display importance differences in their coupling to pro-vs.antiapoptotic signaling pathways.The notion that the b 1 AR Nterminus functions as a novel molecular determinant of b 1 AR signaling responses suggests that therapeutic strategies designed to influence b 1 AR N-terminal cleavage might be exploited for the treatment of heart failure.Our studies also implicate oxidative stress and proteolytic cleavage as two pathophysiologicaly relevant stimuli that act to either disrupt or alter catecholamine-driven b 1 AR growth and/or injury responses in the setting of heart failure, cardiac inflammation, or myocardial infarction-induced cardiac injury.Collectively, the novel signaling paradigms for cardiomyocyte b 1 ARs identified in our studies add a new dimension to our understanding of the evolution of heart failure and other cardiomyopathic disorders.

2
Post-translational processing of the b 1 AR 2.1 b 1 AR O-glycosylation b 1 ARs and b 2 ARs both contain sites for N-glycosylation, but b 1 ARs uniquely serve as targets for O-glycosylation and proteolytic cleavage (Figure

FIGURE 1
FIGURE 1 Schematic showing the classical paradigm for signaling by full-length b 1 ARs (top) and the distinct signaling properties of the N-terminally truncated form of the b 1 AR (bottom).