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Insulin induces IRS2-dependent and GRK2-mediated ?2AR internalization to attenuate ?AR signaling in cardiomyocytes.

ABSTRACT: The counter-regulatory effects of insulin and catecholamines on carbohydrate and lipid metabolism are well studied, whereas the details of insulin regulation of ? adrenergic receptor (?AR) signaling pathway in heart remain unknown. Here, we characterize a novel signaling pathway of insulin receptor (IR) to G protein-coupled receptor kinase 2 (GRK2) in the heart. Insulin stimulates recruitment of GRK2 to ?2AR, which induces ?2AR phosphorylation at the GRK sites of serine 355/356 and subsequently ?2AR internalization. Insulin thereby suppresses ?AR-induced cAMP-PKA activities and contractile response in neonatal and adult mouse cardiomyocytes. Deletion of insulin receptor substrate 2 (IRS2) disrupts the complex of IR and GRK2, which attenuates insulin-mediated ?2AR phosphorylation at the GRK sites and ?2AR internalization, and the counter-regulation effects of insulin on ?AR signaling. These data indicate the requirements of IRS2 and GRK2 for insulin to stimulate counter-regulation of ?AR via ?2AR phosphorylation and internalization in cardiomyocytes.

SUBMITTER: Fu Q

PROVIDER: S-EPMC4333100 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Insulin induces IRS2-dependent and GRK2-mediated β2AR internalization to attenuate βAR signaling in cardiomyocytes.

Fu Qin Q Xu Bing B Parikh Dippal D Cervantes David D Xiang Yang K YK

Cellular signalling 20141125 3

The counter-regulatory effects of insulin and catecholamines on carbohydrate and lipid metabolism are well studied, whereas the details of insulin regulation of β adrenergic receptor (βAR) signaling pathway in heart remain unknown. Here, we characterize a novel signaling pathway of insulin receptor (IR) to G protein-coupled receptor kinase 2 (GRK2) in the heart. Insulin stimulates recruitment of GRK2 to β2AR, which induces β2AR phosphorylation at the GRK sites of serine 355/356 and subsequently ...[more]

PMID: 25460042

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Project description:G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs during heart failure, leads to the overexpression of GRKs and maladaptive downregulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, which is based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and 10 other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the μ-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Furthermore, we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, which explains why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirming that GRK2 predominantly drives internalization of MOR in the cell lines we tested and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases (GRKs) are attractive targets for developing therapeutics for heart failure. We have synthesized a new GRK2 subfamily-selective inhibitor, CCG258747, which has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was used to test the ability of GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicate that CCG258747 blocked the internalization of the μ-opioid receptor most efficaciously because it has the ability to cross cell membranes.

Dataset Information

Insulin induces IRS2-dependent and GRK2-mediated ?2AR internalization to attenuate ?AR signaling in cardiomyocytes.

Publications

Insulin induces IRS2-dependent and GRK2-mediated β2AR internalization to attenuate βAR signaling in cardiomyocytes.

Similar Datasets

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