G Protein Coupled Receptors - receptors with new tricks up their sleeves

G protein receptors (GPCRs) continue to fascinate and confound. Since 2012 when Brian Kobilka and Robert Lefkowitz received the Nobel Prize in Chemistry for their pioneering work on the beta-adrenergic receptor structure, GPCRs have been the subject of over 9,250 articles appearing on PubMed. Approximately 13% of these deal with drug discovery or development. Overall, about 35% of all Food and Drug Administration approved small molecule drugs target GPCRs.¹ Yet many fundamental features of signaling by GPCR family members, including the dynamics and mechanisms that fine-tune their interactions with heterotrimeric G proteins and other binding partners and the functional consequences of various modes of GPCR activation, have only recently been exposed.^2–4 This issue of the Journal of Cardiovascular Pharmacology contains a special series entitled Cardiovascular Regulation by GPCRs organized by one of us (Susan Steinberg) that includes 7 outstanding invited review articles, each entry updating a different recent development pertaining to a novel aspect of GPCR biology.

Laura M. Wingler and Andrew P. Feld discuss nanobodies as novel tools for both probing and regulating GPCR activation.⁵ Nanobodies are fashioned on the variable regions of camelid heavy chain-only antibodies and are typically designed to “capture” a native GPCR in a specific conformation, either active or inactive. They discuss strategies for developing nanobodies targeted to specific surfaces of the GPCR, including nanobodies designed to act at intracellular transducer-binding or allosteric sites and nanobodies that recognize orthosteric or allosteric sites on the extracellular surface. They discuss how one might potentially use a nanobody approach as a strategy to achieve biased agonism, to activate the receptor in a manner that favors signaling through either G protein- or β-arrestin-dependent signaling pathways. Nanobodies also can be exploited to expose GPCR signals that are confined to a specific subcellular compartment. For example, by using a nanobody linked to a fusion partner that confines its subcellular localization, this approach was used to show that the pool of β₁-adrenergic receptors (β₁ARs) that are localized to the Golgi compartment stimulates a signaling pathway that involves phosphatidylinositol-4-phosphate hydrolysis and culminates in cardiomyocyte hypertrophy. These studies emphasize the power of nanobodies as an approach that could potentially take GPCR blockade to a whole new level of pharmacological sophistication.

The β₁AR, the predominant beta-adrenergic receptor subtype in cardiomyocytes, is responsible for rapid adjustments to cardiac contractility by the sympathetic nervous system. However, chronic persistent activation of the β₁AR drives adverse cardiac remodeling and contributes to the evolution of heart failure. This finding underlies the clinical effectiveness of beta blockers in the management of heart failure with reduced ejection fraction (HFrEF). However, the precise mechanism(s) linking β₁AR activation to pathologic changes remains uncertain, because cardiomyocyte β₁ARs activate an elaborate signaling network that couples to both pro- and anti-apoptotic responses. The article by Susan F. Steinberg describing recent studies in her laboratory that begin to address this dilemma by showing that cardiomyocytes express both full length and N-terminally truncated forms of the β₁AR, that the N-terminally truncated form of the β₁AR is generated as a result of an O-glycan-regulated proteolytic cleavage of the N-terminus by ADAM17, and that N-terminal truncation provides a mechanism to alter the β₁AR’s signaling phenotype.⁶ N-terminally truncated β₁ARs acquire a unique property to constitutively activate a PTX-sensitive Gi-dependent pathway that leads to the activation of AKT, a pathway that confers cardioprotection. Adding a layer of complexity to the story are their findings that oxidative stress, and not homologous desensitization, is the likely mechanism for β₁AR downregulation in heart failure. Of note, this redox-dependent mechanism for β₁AR downregulation is uniquely prevented by carvedilol, and not a panel of other β₁AR inhibitors. The additional observation that carvedilol enhances expression of the N-terminally truncated cardioprotective form of the β₁AR exposes a novel strategy to target the β₁AR for therapeutic advantage in the treatment of heart failure.

Many cardiovascular diseases, such as atherosclerosis, hypertension, and heart failure, are associated with the production of circulating autoantibodies against GPCRs. W.H. Wilson Tang and Sathyamangla V. Naga Prasad discuss the various ways for that to occur.⁷ Their article is focused on β₁AR agonistic autoantibodies, which play a causal role in dilated cardiomyopathy. However, as noted by the authors, autoantibodies may not necessarily exert harmful actions. In fact, under some conditions β₁AR agonistic antibodies of the IgG3 subclass might have beneficial effects in patients with chronic systolic heart failure. Their binding to an external “allosteric” site may bias agonist/antagonist signaling from the orthosteric site to a cardioprotective pathway.

Another means to allosterically modulate the activity of GPCRs are pepducins, which are small lipidated peptides based on the intracellular loops of the receptor. Depending upon their design, they may act as antagonists, partial agonists, or biased agonists. They have been shown to have efficacy in preclinical models and favorable pharmacokinetics in humans. As discussed by Heli Xu and Douglas G. Tilley, pepducins may have the advantage of providing stable, long lasting effects that are reversible.⁸ These authors discuss the development of pepducins for several GPCRs, namely protease-activated receptors, C-X-C motif chemokine receptors, formyl peptide receptors, and β₂ARs. Pepducins targeted to these receptors may have therapeutic utility in the control of platelet aggregation and inflammation, and for the treatment of sepsis, myocardial infarction, atherosclerosis, heart failure, and certain cancers.

While chronic persistent stimulation of the β₁AR contributes to cardiac injury and the development of heart failure, the roles of other cardiac ARs (the β₂, α_1A and α_1B subtypes) remain more ambiguous. In this context, Brian C. Jensen and colleagues present a comprehensive summary of adrenergic receptor subtype regulation of mitochondrial biogenesis, focusing on the dynamics of mitochondrial fusion and fission, the regulation of mitochondrial calcium content, and the regulation of oxidative phosphorylation and ROS productions.⁹ The authors provide a detailed summary of the literature, which includes conflicting findings and highlights the many unresolved issues. As the authors note, future experiments involving genetic and molecular approaches designed to unravel divergent AR signaling responses in cardiac myocytes, with a focus on the mechanisms that might be time- or injury-specific, hold promise for the development of new pharmaceutical strategies.

β₁ and β₂ARs can activate a similar Gs-dependent pathway that leads to the generation of cAMP, and yet these two βAR subtypes play markedly different roles in the regulation of cardiac contraction, the induction of cardiac hypertrophy, and the evolution of adverse cardiac remodeling. Wenhui Wei and Alan V. Smrcka discuss literature that implicates spatial localization as a mechanism to control βAR access to its binding partners and downstream effectors (i.e., as a mechanism to fine-tune catecholamine-dependent signaling responses). For example, they note that β₂ARs are largely confined to the sarcolemma, whereas cardiomyocytes contain pools of signaling competent β₁ARs at various intracellular compartments. The additional observation that cell surface and intracellular β₁ARs couple to distinct functional responses, with cell surface β₁ARs providing a rapid mechanism to enhance contractility and intracellular β₁ARs providing a more chronic/persistent mechanism to regulate gene expression and contribute to pathologic cardiac remodeling, has implications for the choice of βAR inhibitor in the therapy of heart failure; β₁AR inhibitors that are membrane-impermeant would not gain access to intracellular β₁ARs. These studies also provide a rationale for the design of genetically encoded, subcellular-targeted nanobody-based β-blockers as alternative strategies for the development of more efficacious β-blockers.

HFrEF is associated with hyperactivation of both the sympathetic nervous system and the renin angiotensin aldosterone system (RAAS), which further exacerbate the work demands on the heart and contribute to maladaptive cardiac remodeling. Anastasios Lymperopoulos and colleagues describe roles for ARs and angiotensin II (AngII) receptors to control adrenal catecholamine secretion from adrenal chromaffin cells and aldosterone secretion from cells in the zona glomerulosa.¹⁰ They note that adrenal chromaffin α₂-adrenergic receptors have been shown to play a role to inhibit catecholamine release and that this autocrine negative feedback mechanism is impaired in heart failure due to the upregulation of G protein coupled receptor kinase 2 (GRK2) and desensitization/downregulation of α₂ARs. The authors note that this provides a rationale to target adrenal GRK2 for the therapy of heart failure. They also describe studies that focus on the Ang II type 1 (AT₁) GPCR as a major stimulator of aldosterone secretion from the zona glomerulosa. The authors describe observations in the Lymperopoulos laboratory showing that AT₁-mediated aldosterone synthesis and secretion is mediated by both G protein- and β-arrestin-1-dependent signaling pathways, findings that have practical implications for the choice of AT₁ receptor blocker in the therapy of HFrEF.

Each of these 7 contributions provides a timely update on a unique feature of cardiovascular GPCRs. Clearly, literature over the last 5 years describing various novel aspects in the control of signaling by these receptors will influence efforts to design new pharmaceutical approaches. Yet, from reading these reviews, it is also clear that recent progress (at least in part due to the development and application of new experimental strategies to interrogate the properties of these GPCRs) has served to expose previously unappreciated features of these receptor that call for further study. We hope that our review series will be a catalyst to spur further interest and research into these versatile receptors.