Norepinephrine- and Epinephrine-induced Distinct β2-Adrenoceptor Signaling Is Dictated by GRK2 Phosphorylation in Cardiomyocytes

Abstract

Agonist-dependent activation of G protein-coupled receptors induces diversified receptor cellular and signaling properties. Norepinephrine (NE) and epinephrine (Epi) are two endogenous ligands that activate adrenoceptor (AR) signals in a variety of physiological stress responses in animals. Here we use cardiomyocyte contraction rate response to analyze the endogenous ␤ 2 AR signaling induced by Epi or NE in cardiac tissue. The Epi-activated ␤ 2 AR induced a rapid contraction rate increase that peaked at 4 min after stimulation. In contrast, the NE-activated ␤ 2 AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas both drugs activated ␤ 2 AR coupling to G s proteins, only Epi-activated receptors were capable of coupling to G i proteins. Subsequent studies showed that the Epi-activated ␤ 2 AR underwent a rapid phosphorylation by G protein-coupled receptor kinase 2 (GRK2) and subsequent dephosphorylation on serine residues 355 and 356, which was critical for sufficient receptor recycling and G i coupling. In contrast, the NE-activated ␤ 2 ARs underwent slow GRK2 phosphorylation, receptor internalization and recycling, and failed to couple to G i . Moreover, inhibiting ␤ 2 AR phosphorylation by ␤ARK C terminus or dephosphorylation by okadaic acid prevented sufficient recycling and G i coupling. Together, our data revealed that distinct temporal phosphorylation of ␤ 2 AR on serine 355 and 356 by GRK2 plays a critical role for dictating receptor cellular events and signaling properties induced by Epi or NE in cardiomyocytes. This study not only helps us understand the endogenous agonist-dependent ␤ 2 AR signaling in animal heart but also offers an example of how G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings.GPCRs 3 comprise the largest known family of cell-surface receptors and are fundamentally involved in mammalian physiology (1, 2). This receptor superfamily represents the largest single target for modern drug therapy. A growing body of evidence indicates that divergent efficacies of an activated GPCR are both agonist-and tissue-dependent (3-5), which presents a great challenge on clinical application when a specific receptor is targeted with drugs. Many of the drug-dependent effects have been attributed to the distinct receptor conformational changes induced by different ligands, leading to different subsequent cellular events and signaling properties (5, 6). Thus, there is great interest in elucidating the mechanisms underlying the drug-dependent cellular events in physiologically relevant contexts.Interestingly, ␤ARs, a family of prototypical GPCRs, can be activated by two endogenous ligands NE and Epi. The receptors play critical roles in the regulation of cardiovascular (7) and pulmonary function (8), as well as other physiological processes. NE is primarily released from sympathetic nerve terminal on the innervated tissues, whereas Epi is primarily released from adrenal gland to the circulating plasma. The distinct ...

Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates β2 Adrenoceptor Coupling to Gi Proteins in Cardiomyocytes

“…3A). However, stimulation with 10 M Iso induced a time-dependent clustering of ␤ 2 ARs in the cells, similar to those reported previously (8), which is a characteristic of receptor internalization (Fig. 3A).…”

“…At both low and high concentrations of agonist, the activated ␤ 2 ARs undergo the PKA-mediated phosphorylation. In comparison, only high concentrations of agonist induce the GRK-mediated phosphorylation of the ␤ 2 ARs for subsequent internalization, which is also necessary for sufficient receptor coupling to G i proteins (8,9). Our studies link together various components of the ␤ 2 AR, including receptor phosphorylation, receptor trafficking, and differential receptor/G protein coupling in cardiac cells, which may allow the activation of the ␤ 2 AR signaling pathway to function as either a stimulatory or protective mechanism for cardiac cells under different levels of stress.…”

“…The ␤ 2 AR phosphorylation by PKA mediates the switch of coupling from G s to G i (6,7), presumably operating in a feedback after receptor activation. Our previous studies have revealed that ␤ 2 AR/G i coupling is also dependent on receptor internalization and recycling (8,9). Meanwhile, Wang et al have shown that direct inhibition of GRK2 prevents ␤ 2 AR/G i coupling in mouse cardiac myocytes (8), supporting a role of the ␤ 2 AR phosphorylation by GRK in receptor/G i coupling.…”

Differential Signaling of the Endogenous Agonists at the β2-Adrenergic Receptor

“…The different release mechanisms and functional roles of the two endogenous agonists raise the possibility that they might also display distinct mechanisms of receptor activation and signaling. Such different activities between epinephrine and norepinephrine have recently been reported for cardiomyocyte ␤ 2 AR, where norepinephrine was observed to induce slower GRK2-mediated phosphorylation, receptor internalization and recycling, and no coupling to G i , when compared with epinephrine (18).…”

Pathophysiology of Takotsubo Syndrome

“…We combined this observation of apical-basal βAR and basal-apical neural gradients with a second interesting pharmacological observation. Epinephrine at low and medium doses is a positive inotrope, but at the highest doses it becomes a negative inotrope via the β2AR, by activating a switch from the stimulatory Gs to the cardioinhibitory Gi pathway in a process known as stimulus trafficking (Figure 3), 21,22 which could explain the negative inotropy observed in the apical myocardium after exposure to very high levels of circulating epinephrine. We developed a rat TTS model and demonstrated that an intravenous bolus of high-dose epinephrine triggered acute apical hypokinesia, with preserved basal contractility, recapitulating the clinical features of TTS.…”