GRK2-Mediated Crosstalk Between ß-Adrenergic and Angiotensin II Receptors Enhances Adrenocortical Aldosterone Production In Vitro and In Vivo.

Aldosterone is produced by adrenocortical zona glomerulosa (AZG) cells in response to angiotensin II (AngII) acting through its type I receptors (AT1Rs). AT1R is a G protein-coupled receptor (GPCR) that induces aldosterone via both G proteins and the adapter protein ßarrestin1, which binds the receptor following its phosphorylation by GPCR-kinases (GRKs) to initiate G protein-independent signaling. ß-adrenergic receptors (ARs) also induce aldosterone production in AZG cells. Herein, we investigated whether GRK2 or GRK5, the two major adrenal GRKs, is involved in the catecholaminergic regulation of AngII-dependent aldosterone production. In human AZG (H295R) cells in vitro, the ßAR agonist isoproterenol significantly augmented both AngII-dependent aldosterone secretion and synthesis, as measured by the steroidogenic acute regulatory (StAR) protein and CYP11B2 (aldosterone synthase) mRNA inductions. Importantly, GRK2, but not GRK5, was indispensable for the ßAR-mediated enhancement of aldosterone in response to AngII. Specifically, GRK2 inhibition with Cmpd101 abolished isoproterenol's effects on AngII-induced aldosterone synthesis/secretion, whereas the GRK5 knockout via CRISPR/Cas9 had no effect. It is worth noting that these findings were confirmed in vivo, since rats overexpressing GRK2, but not GRK5, in their adrenals had elevated circulating aldosterone levels compared to the control animals. However, treatment with the ß-blocker propranolol prevented hyperaldosteronism in the adrenal GRK2-overexpressing rats. In conclusion, GRK2 mediates a ßAR-AT1R signaling crosstalk in the adrenal cortex leading to elevated aldosterone production. This suggests that adrenal GRK2 may be a molecular link connecting the sympathetic nervous and renin-angiotensin systems at the level of the adrenal cortex and that its inhibition might be therapeutically advantageous in hyperaldosteronism-related conditions.

Not all arrestins are created equal: Therapeutic implications of the functional diversity of the ß-arrestins in the heart.

The two ubiquitous, outside the retina, G protein-coupled receptor (GPCR) adapter proteins, ß-arrestin-1 and -2 (also known as arrestin-2 and -3, respectively), have three major functions in cells: GPCR desensitization, i.e., receptor decoupling from G-proteins; GPCR internalization via clathrin-coated pits; and signal transduction independently of or in parallel to G-proteins. Both ß-arrestins are expressed in the heart and regulate a large number of cardiac GPCRs. The latter constitute the single most commonly targeted receptor class by Food and Drug Administration-approved cardiovascular drugs, with about one-third of all currently used in the clinic medications affecting GPCR function. Since ß-arrestin-1 and -2 play important roles in signaling and function of several GPCRs, in particular of adrenergic receptors and angiotensin II type 1 receptors, in cardiac myocytes, they have been a major focus of cardiac biology research in recent years. Perhaps the most significant realization coming out of their studies is that these two GPCR adapter proteins, initially thought of as functionally interchangeable, actually exert diametrically opposite effects in the mammalian myocardium. Specifically, the most abundant of the two ß-arrestin-1 exerts overall detrimental effects on the heart, such as negative inotropy and promotion of adverse remodeling post-myocardial infarction (MI). In contrast, ß-arrestin-2 is overall beneficial for the myocardium, as it has anti-apoptotic and anti-inflammatory effects that result in attenuation of post-MI adverse remodeling, while promoting cardiac contractile function. Thus, design of novel cardiac GPCR ligands that preferentially activate ß-arrestin-2 over ß-arrestin-1 has the potential of generating novel cardiovascular therapeutics for heart failure and other heart diseases.

Deletion of Osteopontin Enhances ß2-Adrenergic Receptor-Dependent Anti-Fibrotic Signaling in Cardiomyocytes.

Cardiac ß2-adrenergic receptors (ARs) are known to inhibit collagen production and fibrosis in cardiac fibroblasts and myocytes. The ß2AR is a Gs protein-coupled receptor (GPCR) and, upon its activation, stimulates the generation of cyclic 3',5'-adenosine monophosphate (cAMP). cAMP has two effectors: protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac). Epac1 has been shown to inhibit cardiac fibroblast activation and fibrosis. Osteopontin (OPN) is a ubiquitous pro-inflammatory cytokine, which also mediates fibrosis in several tissues, including the heart. OPN underlies several cardiovascular pathologies, including atherosclerosis and cardiac adverse remodeling. We found that the cardiotoxic hormone aldosterone transcriptionally upregulates OPN in H9c2 rat cardiac myoblasts-an effect prevented by endogenous ß2AR activation. Additionally, CRISPR-mediated OPN deletion enhanced cAMP generation in response to both ß1AR and ß2AR activation in H9c2 cardiomyocytes, leading to the upregulation of Epac1 protein levels. These effects rendered ß2AR stimulation capable of completely abrogating transforming growth factor (TGF)-ß-dependent fibrosis in OPN-lacking H9c2 cardiomyocytes. Finally, OPN interacted constitutively with Gαs subunits in H9c2 cardiac cells. Thus, we uncovered a direct inhibitory role of OPN in cardiac ß2AR anti-fibrotic signaling via cAMP/Epac1. OPN blockade could be of value in the treatment and/or prevention of cardiac fibrosis.

Co-IP assays for measuring GPCR-arrestin interactions.

ßarrestin1 and -2 (also known as arrestin2 and -3, respectively) are G protein-coupled receptor (GPCR) adapter proteins, performing three major functions in the cell: functional desensitization, i.e., G protein uncoupling from the receptor, GPCR internalization via clathrin-coated pits, and formation of signalosomes. The ßarrestins elicit a large part of the G protein-independent signaling emanating from GPCRs. Several methodologies have been developed over the past 15 years or so to quantify the GPCR-arrestin interaction/binding, especially since the latter's roles in signal transduction were discovered. One of the simplest and most traditional of these methodologies is the assay of co-immunoprecipitation (co-IP), followed by western blotting. This assay is also one of the most reliable ones, since it does not require any chemical modification of either component in the complex (i.e., neither of the receptor nor of the arrestin). Therefore, it is the only assay that can detect and semi-quantify interactions between native GPCRs and native arrestins. The caveat of this assay is of course that its reliability depends on the quality (specificity and sensitivity) of the utilized antibodies. Here, we describe a simple protocol for performing this co-IP assay to get a measurement of the steady-state levels of agonist-elicited GPCR-arrestin interaction in cells.

Novel Insights into the Crosstalk between Mineralocorticoid Receptor and G Protein-Coupled Receptors in Heart Adverse Remodeling and Disease.

The mineralocorticoid hormone aldosterone regulates sodium and potassium homeostasis but also adversely modulates the maladaptive process of cardiac adverse remodeling post-myocardial infarction. Through activation of its mineralocorticoid receptor (MR), a classic steroid hormone receptor/transcription factor, aldosterone promotes inflammation and fibrosis of the heart, the vasculature, and the kidneys. This is why MR antagonists reduce morbidity and mortality of heart disease patients and are part of the mainstay pharmacotherapy of advanced human heart failure. A plethora of animal studies using cell type⁻specific targeting of the MR gene have established the importance of MR signaling and function in cardiac myocytes, vascular endothelial and smooth muscle cells, renal cells, and macrophages. In terms of its signaling properties, the MR is distinct from nuclear receptors in that it has, in reality, two physiological hormonal agonists: not only aldosterone but also cortisol. In fact, in several tissues, including in the myocardium, cortisol is the primary hormone activating the MR. There is a considerable amount of evidence indicating that the effects of the MR in each tissue expressing it depend on tissue- and ligand-specific engagement of molecular co-regulators that either activate or suppress its transcriptional activity. Identification of these co-regulators for every ligand that interacts with the MR in the heart (and in other tissues) is of utmost importance therapeutically, since it can not only help elucidate fully the pathophysiological ramifications of the cardiac MR's actions, but also help design and develop novel better MR antagonist drugs for heart disease therapy. Among the various proteins the MR interacts with are molecules involved in cardiac G protein-coupled receptor (GPCR) signaling. This results in a significant amount of crosstalk between GPCRs and the MR, which can affect the latter's activity dramatically in the heart and in other cardiovascular tissues. This review summarizes the current experimental evidence for this GPCR-MR crosstalk in the heart and discusses its pathophysiological implications for cardiac adverse remodeling as well as for heart disease therapy. Novel findings revealing non-conventional roles of GPCR signaling molecules, specifically of GPCR-kinase (GRK)-5, in cardiac MR regulation are also highlighted.

Biased Agonism/Antagonism of Cardiovascular GPCRs for Heart Failure Therapy.

G protein-coupled receptors (GPCRs) are among the most important drug targets currently used in clinic, including drugs for cardiovascular indications. We now know that, in addition to activating heterotrimeric G protein-dependent signaling pathways, GPCRs can also activate G protein-independent signaling, mainly via the ßarrestins. The major role of ßarrestin1 and -2, also known as arrestin2 or -3, respectively, is to desensitize GPCRs, i.e., uncoupled them from G proteins, and to subsequently internalize the receptor. As the ßarrestin-bound GPCR recycles inside the cell, it serves as a signalosome transducing signals in the cytoplasm. Since both G proteins and ßarrestins can transduce signals from the same receptor independently of each other, any given GPCR agonist might selectively activate either pathway, which would make it a biased agonist for that receptor. Although this selectivity is always relative (never absolute), in cases where the G protein- and ßarrestin-dependent signals emanating from the same GPCR result in different cellular effects, pharmacological exploitation of GPCR-biased agonism might have therapeutic potential. In this chapter, we summarize the GPCR signaling pathways and their biased agonism/antagonism examples discovered so far that can be exploited for heart failure treatment. We also highlight important issues that need to be clarified along the journey of these ligands from bench to the clinic.