BETA₁-ADRENERGIC RECEPTOR REGULATION REVISITED; THE ROLE OF THE EXTRACELLULAR N-TERMINUS

Abstract

The actions and regulation of cardiomyocyte β₁ARs differ in several respects from the properties described for the prototypical β₂AR subtype; a mechanism to explain the unique properties of the β₁AR subtype has never been obvious. This viewpoint summarizes recent studies that identify a novel signaling paradigm for the β₁AR, implicating the N-terminus as a molecular determinant of β₁AR responsiveness.

SCIENTIFIC METHOD

It is a capital mistake to theorize before one has data. Insensibly one begins to twist the facts to suit theories, instead of theories to suit the facts.

SIR ARTHUR CONAN DOYLE

It is also a good rule not to put too much confidence in experimental results until they have been confirmed by theory.

SIR ARTHUR EDDINGTON

First get your facts; then you can distort them at your leisure.

MARK TWAIN

Beta-adrenergic receptors (βARs) are among the most intensively studied members of the G protein-coupled receptor (GPCR) superfamily. These receptors, which transduce signals derived from catecholamine binding to a cellular response, have garnered considerable interest as therapeutic targets because of their key roles in the physiologic control of cardiovascular performance as well as the pathogenesis of cardiac arrhythmias, ventricular remodeling, and the evolution of heart failure (HF). Breakthroughs in receptor biology, culminating most recently with the development of strategies to obtain high-resolution GPCR structures, have lead to a detailed understanding of the βAR’s two core functions - namely ligand binding to a transmembrane ligand-binding pocket and G protein- or β-arrestin-induced cellular activation ¹. These studies also have exposed desensitization mechanisms that provide a critical homeostatic control to avoid excessive/unrelenting GPCR activation. However, it is important to note that much of our current concepts regarding the mechanisms that activate and/or regulate βARs come from studies of the β₂AR subtype, the first hormone-activated GPCR to be cloned and structurally characterized. Based upon early studies that identified essentially equivalent signaling properties for β₁ARs and β₂ARs in certain model cell types as well as evidence that various aspects of the structural architecture, signaling mechanisms, and regulatory machinery are highly conserved across the GPCR superfamily, the literature has generally viewed the β₂AR as a useful surrogate for the β₁AR subtype. However, this assumption ignores literature that identifies discrepancies between the properties of β₁ vs. β₂ARs. My laboratory has struggled for some time with the nagging suspicion that some aspects of the actions and regulation of the cardiomyocyte β₁AR may not adhere to conventional models described for the prototypical β₂AR subtype.

Studies in transgenic models provided some of the earliest evidence for functional divergence between β₁ and β₂ARs showing that high levels of transgenic β₂AR overexpression are well tolerated, whereas even relatively low levels of transgenic β₁AR overexpression result in maladaptive cardiac remodeling and HF ^{2, 3}. Studies in cell-based models also established that chronic catecholamine stimulation leads to cardiomyocyte apoptosis through the activation of β₁ARs, whereas β₂AR stimulation may in fact be cardioprotective ⁴.

Immunoblotting studies performed to validate the β₁AR knockout mouse model identified another distinctive feature of β₁ARs that defied obvious explanation. These Western blots identified molecular heterogeneity for the β₁AR subtype that was not evident in immunoblotting studies of β₂ARs ⁵. Recent studies from my laboratory establish that β₁ARs are susceptible to N-terminal cleavage ⁶, similar to the processing mechanism originally described for the turkey βAR – the avian homologue of the mammalian β₁AR subtype. The observation that β₁ARs are expressed as both a full-length protein and a smaller N-terminally truncated species raises the first obvious question: What controls the generation of the distinct molecular forms of the β₁AR? Do distinct β₁AR species signal differently?

Other studies exposed differences in susceptibility to agonist-induced desensitization/internalization. The classical paradigm holds that agonist-activated βARs are phosphorylated by G protein-coupled receptor kinases (GRKs), leading to the recruitment of β-arrestin, and receptor desensitization/downregulation. Head-to-head comparisons of β₁ versus β₂ARs indicate that agonist-induced phosphorylation, internalization/sequestration, and/or down-regulation is blunted for the β₁AR subtype, compared to the β₂AR ^7–10. Sequence comparisons provide a likely explanation; β₁- and β₂ARs share considerable sequence homology in their transmembrane ligand-binding pockets, but other regions of these receptors are more divergent. In particular, sites on the intracellular surface of the β₂AR that serve as substrates for GRK phosphorylation and/or docking sites for β-arrestin are not conserved in β₁ARs. Therefore, the observation that HF (a condition characterized by elevated catecholamine levels) leads to selective desensitization/downregulation of the β₁AR subtype and is not accompanied by a commensurate loss of β₂ARs seems paradoxical ¹¹; the prediction based on cell-based studies is that chronic catecholamine-induced, GRK/β-arrestin-dependent desensitization would lead to a decrease in β₂ARs, and that the β₁AR subtype would be relatively spared. Hence, question #2: What mechanism underlies the HF-induced decrease in β₁ARs?

We recently made progress toward answering both of these questions through studies of the β₁AR N-terminus, the relatively short/unstructured extracellular portion of the receptor that differs in length, sequence, and post-translational processing from the β₂AR N-terminus. We showed that the β₁AR N-terminus contains two O-linked glycosylation sites at Ser³⁷ and Ser⁴¹ and that O-glycosylation is required for full-length β₁AR expression; β₁ARs accumulate as N-terminally truncated species under conditions that prevent β₁AR O-glycosylation (⁶ as if the mucin-like O-glycans attached to the N-terminus act as a barrier to prevent protease access and cleavage at an adjacent site, Figure 1). An O-glycosylation-regulated N-terminal cleavage mechanism that gives rise to distinct molecular forms of the β₁AR provides the first credible explanation for the molecular heterogeneity displayed by native β₁ARs in various cardiac preparations. It also emphasizes that previous literature using immunblotting or immunohistochemical methods to track β₁AR expression and/or localization using antibodies to a transgene’s N-terminal tag should be interpreted with caution, since these techniques do not capture N-terminally truncated β₁AR species that lack the N-terminal epitope tag.

Figure 1:

β₁AR N-terminal processing (see text).

Does β₁AR molecular heterogeneity matter?

We recently reported that N-terminally truncated β₁ARs remain signaling competent, but N-terminal truncation alters signaling bias to the cAMP/PKA vs. ERK pathways ⁶. The notion that an O-glycosylation-regulated N-terminal cleavage mechanism can regulate signaling by a GPCR is quite novel and is predicted to have pathophysiological significance since O-linked glycan structures are highly regulated during developmental and in response to inflammatory stimuli, metabolic disorders, and abnormalities in cell growth; alterations in glycan structures are detected in cardiac hypertrophy ¹².

How does the N-terminus function as a molecular determinant of β₁AR responsiveness?

In theory, the N-terminus might function as an allosteric regulator of the β₁AR structure itself, a regulator of β₁AR trafficking to subcellular compartments, and/or a regulator of β₁AR coupling to signaling partners and downstream effector responses. These mechanisms are not mutually exclusive and are the focus of ongoing studies.

What is the mechanism underlying the HF-induced decrease in β₁AR density?

In an effort to identify stimuli that might regulate β₁AR cleavage, we stumbled upon evidence that oxidative stress (a stimulus that contributes to the pathogenesis of HF) leads to a selective decrease in cardiomyocyte β₁AR expression that is not associated with a change in the abundance of the β₂AR subtype ¹³.

Carvedilol to the rescue!

Attempts to identify a mechanism for (and strategies to prevent) the ROS-dependent decrease in β₁AR expression lead to two intriguing observations regarding the pharmacologic properties of carvedilol. First, our studies showed that the redox-dependent decrease in β₁AR expression is prevented by carvedilol (but not a panel of other βAR ligands) and that this cannot be attributed to carvedilol’s ancillary anti-oxidant properties ¹³. While the molecular mechanism underlying carvedilol’s ability to protect β₁ARs from redox-inactivation remains uncertain, it is worth noting that carvedilol contains a bulky aromatic amine substitution (not present in most other adrenergic ligands) that makes unique contacts with an extended β₁AR ligand binding pocket that includes the redox-sensitive cysteines in extracellular loop 2 ¹⁴. In theory, carvedilol might protect β₁ARs from redox-inactivation either by shielding these cysteines from redox-inactivation or producing a conformational rearrangement of the extracellular surface so as to bury the redox-sensitive disulfide bonds.

Carvedilol treatment also leads to the accumulation of N-terminally truncated β₁ARs that constitutively activate the AKT pathway and protect against doxorubicin-induced apoptosis (Figure 1 ¹³). This second cardioprotective property for carvedilol (not shared by other βAR ligands) is predicted to have important clinical and therapeutic implications. First, it identifies a mechanism that presumably underlies the protective effects of prophylactic carvedilol against anthracycline-induced cardiotoxicity in the clinic. Second, these findings may be pertinent to the lingering controversy regarding the interpretation of the COMET trial results, which ascribed significant survival advantage to carvedilol over metoprolol in the treatment of HF. The unique pharmacologic action of carvedilol not shared with metoprolol or other βAR blockers identified in our studies is predicted to offer to meaningful cardioprotection.

This viewpoint presents a revised model of β₁AR action and regulation that challenges conventional dogma in that it identifies a novel role for the β₁AR N-terminus as a molecular determinant of catecholamine responsiveness. We recognize (and are open to the fact that) some aspects of our model might not withstand the scrutiny of future research. Nevertheless, these newer studies set the stage for future research that examines factors (and proteases) that control β₁AR N-terminal cleavage and the role of glycosylation-defective/N-terminally truncated β₁ARs in the evolution of catecholamine-induced pathologic cardiac remodeling in vivo. Our hope is that this line of research will reveal novel strategies targeted to processing events localized to the β₁AR N-terminus that might ultimately be used for therapeutic advantage.