Insulin induces IRS2-dependent and GRK2-mediated β2AR internalization to attenuate βAR signaling in cardiomyocytes
Introduction
G-protein-coupled receptors and tyrosine-kinase receptors represent two prominent modalities in cell signaling. Cross regulation between members of both receptor super families has been reported, including the counter-regulatory effects of insulin on β-adrenergic action [1]. β2-Adrenergic receptor (β2AR) displays acute homologous desensitization in response to βAR agonists as well as counter-regulation by insulin [2]. Insulin stimulates a rapid tyrosine phosphorylation and sequestration of the β2AR [3]. This counter-regulatory effect of insulin on βAR signaling is observed in either DDT1MF-2 smooth muscle cells or Chinese hamster ovary cells (CHO) [4]. Insulin-stimulated internalization of β2AR is dependent upon insulin receptor (IR) kinase-catalyzed phosphorylation of tyrosyl residue at position 350 of the β2AR [4], which creates a docking site for SH2 domains of a variety of proteins, including Grb2 and dynamin. The integrity of Y350 and its phosphorylation in response to insulin are essential for the inhibitory regulation of β2AR functions and β2AR sequestration [5]. These studies largely focus on insulin action in skeletal muscle, liver and adipose tissues, including phosphorylation of β2AR in HEK293 cells and adipocytes [1], [2], [6], [7]. As a result, insulin induces an acute reduction in the ligand binding capacity of βΑR in rat adipocytes [8]; and stimulation of fat cells with insulin promotes a marked attenuation of βAR-mediated activation of AC [8], [9].
By comparison, little is known on how insulin influences βAR trafficking as well as the counter-regulation of βAR signaling in heart tissues. Current literatures report conflict views on cross-regulation between these two distinct classes of receptors in heart tissues [10], [11]. Insulin enhances myocardial contractility response to β-adrenergic action in isolated rat cardiac papillary muscle [10]. However, insulin also suppresses β-adrenergic-induced cardiac dysfunction and cell injury in myocardial ischemia and reperfusion [11].
We have recently showed that phosphorylation of β2AR by GRK is required for rapid receptor internalization and desensitization in cardiomyocytes [12]. Disruption of the GRK sites of β2AR prolongs isoproterenol-induced myocyte contraction response [12]. A recent study reported that insulin induced membrane translocation of GRK2 in cultured adult rat ventricular cardiomyocytes [13]. In the current work, we probed the role of GRK2 in trafficking of β2AR after insulin stimulation in cardiomyocytes. The results revealed a physical interaction between GRK2 and insulin receptor in heart. Moreover, insulin treatment increased interaction between GRK2 and β2AR, revealing a GRK2-linked pathway between insulin receptor and β-adrenergic signaling. Our data show that a GRK2-mediated β2AR phosphorylation and internalization are necessary for the counter-regulation of insulin on β-adrenergic signaling in cardiomyocytes.
Section snippets
Cell culture
Animal protocols were approved by the IACUC of the University of California at Davis according to NIH regulation. Neonatal cardiomyocytes were isolated from 1- to 2-day-old wild type, β1AR knockout (KO), and β2AR-KO mouse pups. Adult mouse cardiomyocytes were isolated as described previously [14]. H9c2 cardiac myoblasts and Mouse Embryonic Fibroblasts (MEFs) from wild type mice and insulin receptor substrate 2 (IRS2) KO mice (a gift from Dr. Morris White, Harvard University) were cultured in
Insulin induces β2AR internalization via GRK phosphorylation of β2AR in cardiac myoblasts
We have previously shown that GRK2 is necessary for βAR agonist-induced GRK2 phosphorylation of β2ARs at serine 355/356. Interestingly, we observed that IR formed a complex with GRK2 in mouse hearts and MEFs (Fig. 1A and 1B). Insulin stimulation increased GRK2-IR interaction (Fig. 1B). Consistently, when H9c2 cardiac myoblasts were exposed to insulin, GRK2 displayed a membrane translocation (Fig. 1C). Next, we directly addressed whether insulin-induced activation of IR leads to GRK2
Discussion
Receptor phosphorylation and internalization are prominent features of agonist-induced desensitization of GRCRs. β2ARs are substrates of several protein kinases [20]. Catecholamines activate both protein kinase A and protein kinase C, as well as GRK2 that phosphorylate serine and threonine residues of β2AR. In addition, β2ARs are phosphorylated on tyrosyl residues by insulin stimulation [1], [4]. Activation of both pathways blunts or abolishes β2AR action and promotes sequestration of β2ARs.
Conclusion
This study reveals the counter-regulation of insulin on βAR signaling in cardiomyocytes, highlighting the role of IRS2 and GRK2 in insulin-induced β2AR phosphorylation and internalization in cardiomyocytes. Thus our study elucidates a distinct mechanism on insulin regulation of βAR signaling in cardiomyocytes in comparison to other tissues, providing a basis to understand cross talk between these two regulatory signaling pathways in the heart.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This study was supported by a National Natural Science Foundation of China grant 81473212 and a Central Authorities of an Institution of Higher Learning of Scientific Research Special Fund of China grant 2014QN031 to QF, a NIH grant RO1 HL082846 and a National Natural Science Foundation of China grant 81428022 to YKX. YKX is an AHA established investigator.
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