Neelakantan T. Vasudevan

Regulation of β-adrenergic receptor function: An emphasis on receptor resensitization

G protein-coupled receptors are the largest family of cell surface receptors regulating multiple cellular processes. β-adrenergic receptor (βAR) is a prototypical member of GPCR family and has been one of the most well studied receptors in determining regulation of receptor function. Agonist activation of βAR leads to conformational change resulting in coupling to G protein generating cAMP as secondary messenger. The activated βAR is phosphorylated resulting in binding of β-arrestin that physically interdicts further G protein coupling leading to receptor desensitization. The phosphorylated βAR is internalized and undergoes resensitization by dephosphorylation mediated by protein phosphatase 2A in the early endosomes. Although desensitization and resensitization are two sides of the same coin maintaining the homeostatic functioning of the receptor, significant interest has revolved around understanding mechanisms of receptor desensitization while little is known about resensitization. In our current review we provide an overview on regulation of βAR function with a special emphasis on receptor resensitization and its functional relevance in the context of fine tuning receptor signaling.

miRNA-548c: A specific signature in circulating PBMCs from dilated cardiomyopathy patients

High fidelity genome-wide expression analysis has strengthened the idea that microRNA (miRNA) signatures in peripheral blood mononuclear cells (PBMCs) can be potentially used to predict the pathology when anatomical samples are inaccessible like the heart. PBMCs from 48 non-failing controls and 44 patients with relatively stable chronic heart failure (ejection fraction of ≤ 40%) associated with dilated cardiomyopathy (DCM) were used for miRNA analysis. Genome-wide miRNA-microarray on PBMCs from chronic heart failure patients identified miRNA signature uniquely characterized by the downregulation of miRNA-548 family members. We have also independently validated downregulation of miRNA-548 family members (miRNA-548c & 548i) using real time-PCR in a large cohort of independent patient samples. Independent in silico Ingenuity Pathway Analysis (IPA) of miRNA-548 targets shows unique enrichment of signaling molecules and pathways associated with cardiovascular disease and hypertrophy. Consistent with specificity of miRNA changes with pathology, PBMCs from breast cancer patients showed no alterations in miRNA-548c expression compared to healthy controls. These studies suggest that miRNA-548 family signature in PBMCs can therefore be used to detect early heart failure. Our studies show that cognate networking of predicted miRNA-548 targets in heart failure can be used as a powerful ancillary tool to predict the ongoing pathology.

Gβγ-Independent Recruitment of G-Protein Coupled Receptor Kinase 2 Drives Tumor Necrosis Factor α–Induced Cardiac β-Adrenergic Receptor Dysfunction

Background— Proinflammatory cytokine tumor necrosis factor-&agr; (TNF&agr;) induces &bgr;-adrenergic receptor (&bgr;AR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results— Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNF&agr; showed that TNF&agr; alone is sufficient to mediate &bgr;AR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling &bgr;AR desensitization independent of sympathetic overdrive. TNF&agr;-mediated &bgr;AR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in &bgr;2AR-overexpressing human embryonic kidney 293 cells showed significant &bgr;AR desensitization, GRK2 upregulation, and recruitment to the &bgr;AR complex following TNF&agr;. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated &bgr;AR phosphorylation and GRK2 recruitment on TNF&agr;. Furthermore, TNF&agr;-mediated &bgr;AR phosphorylation was not blocked with &bgr;AR antagonist propranolol. Additionally, TNF&agr; administration in transgenic mice with cardiac overexpression of G&bgr;&ggr;-sequestering peptide &bgr;ARK-ct could not prevent &bgr;AR desensitization or cardiac dysfunction showing that GRK2 recruitment to the &bgr;AR is G&bgr;&ggr; independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNF&agr;-mediated &bgr;AR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNF&agr;-mediated loss in contractility, showing that TNF&agr;-induced &bgr;AR desensitization is GRK2 dependent. Conclusions— TNF&agr;-induced &bgr;AR desensitization is mediated by GRK2 and is independent of G&bgr;&ggr;, uncovering a hitherto unknown cross-talk between TNF&agr; and &bgr;AR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

Gβγ Independent Recruitment of G-Protein Coupled Receptor Kinase 2 Drives TNFα-Induced Cardiac Beta-Adrenergic Receptor Dysfunction

Background— Proinflammatory cytokine tumor necrosis factor-&agr; (TNF&agr;) induces &bgr;-adrenergic receptor (&bgr;AR) desensitization, but mechanisms proximal to the receptor in contributing to cardiac dysfunction are not known. Methods and Results— Two different proinflammatory transgenic mouse models with cardiac overexpression of myotrophin (a prohypertrophic molecule) or TNF&agr; showed that TNF&agr; alone is sufficient to mediate &bgr;AR desensitization as measured by cardiac adenylyl cyclase activity. M-mode echocardiography in these mouse models showed cardiac dysfunction paralleling &bgr;AR desensitization independent of sympathetic overdrive. TNF&agr;-mediated &bgr;AR desensitization that precedes cardiac dysfunction is associated with selective upregulation of G-protein coupled receptor kinase 2 (GRK2) in both mouse models. In vitro studies in &bgr;2AR-overexpressing human embryonic kidney 293 cells showed significant &bgr;AR desensitization, GRK2 upregulation, and recruitment to the &bgr;AR complex following TNF&agr;. Interestingly, inhibition of phosphoinositide 3-kinase abolished GRK2-mediated &bgr;AR phosphorylation and GRK2 recruitment on TNF&agr;. Furthermore, TNF&agr;-mediated &bgr;AR phosphorylation was not blocked with &bgr;AR antagonist propranolol. Additionally, TNF&agr; administration in transgenic mice with cardiac overexpression of G&bgr;&ggr;-sequestering peptide &bgr;ARK-ct could not prevent &bgr;AR desensitization or cardiac dysfunction showing that GRK2 recruitment to the &bgr;AR is G&bgr;&ggr; independent. Small interfering RNA knockdown of GRK2 resulted in the loss of TNF&agr;-mediated &bgr;AR phosphorylation. Consistently, cardiomyocytes from mice with cardiac-specific GRK2 ablation normalized the TNF&agr;-mediated loss in contractility, showing that TNF&agr;-induced &bgr;AR desensitization is GRK2 dependent. Conclusions— TNF&agr;-induced &bgr;AR desensitization is mediated by GRK2 and is independent of G&bgr;&ggr;, uncovering a hitherto unknown cross-talk between TNF&agr; and &bgr;AR function, providing the underpinnings of inflammation-mediated cardiac dysfunction.

G-Protein Coupled Receptor Resensitization - Appreciating the Balancing Act of Receptor Function

G-protein coupled receptors (GPCRs) are seven transmembrane receptors that are pivotal regulators of cellular responses including vision, cardiac contractility, olfaction, and platelet activation. GPCRs have been a major target for drug discovery due to their role in regulating a broad range of physiological and pathological responses. GPCRs mediate these responses through a cyclical process of receptor activation (initiation of downstream signals), desensitization (inactivation that results in diminution of downstream signals), and resensitization (receptor reactivation for next wave of activation). Although these steps may be of equal importance in regulating receptor function, significant advances have been made in understanding activation and desensitization with limited effort towards resensitization. Inadequate importance has been given to resensitization due to the understanding that resensitization is a homeostasis maintaining process and is not acutely regulated. Evidence indicates that resensitization is a critical step in regulating GPCR function and may contribute towards receptor signaling and cellular responses. In light of these observations, it is imperative to discuss resensitization as a dynamic and mechanistic regulator of GPCR function. In this review we discuss components regulating GPCR function like activation, desensitization, and internalization with special emphasis on resensitization. Although we have used β-adrenergic receptor as a proto-type GPCR to discuss mechanisms regulating receptor function, other GPCRs are also described to put forth a view point on the universality of such mechanisms.

Phosphoinositide 3-kinase γ inhibits cardiac GSK-3 independently of Akt

A kinase promotes cardiac hypertrophy through a kinase-independent mechanism. Making a Bigger Heart Pathological cardiac hypertrophy can be fatal because it can cause congestive heart failure and arrhythmias. Glycogen synthase kinase-3 (GSK-3), which inhibits cardiac hypertrophy, is active when dephosphorylated by the protein phosphatase PP2A, the activity of which is stimulated by methylation mediated by the methyltransferase PPMT-1. Mohan et al. found that mice lacking the γ isoform of phosphoinositide 3-kinase (PI3K) had smaller hearts than wild-type mice and showed decreased phosphorylation of GSK-3. In addition, these mice showed increased activity of PP2A and PPMT-1. Biochemical experiments indicated that PI3Kγ inhibited the interaction between PP2A and PPMT-1. Heart size and phosphorylation of GSK-3 were increased, and the association of PP2A with PPMT-1 was decreased in PI3Kγ knockout mice by expression of a catalytically inactive form of PI3Kγ. Thus, PI3Kγ promotes cardiac hypertrophy by attenuating the PP2A–PPMT-1 interaction and the inactivation of GSK-3 in a kinase-independent manner. Activation of cardiac phosphoinositide 3-kinase α (PI3Kα) by growth factors, such as insulin, or activation of PI3Kγ downstream of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors stimulates the activity of the kinase Akt, which phosphorylates and inhibits glycogen synthase kinase-3 (GSK-3). We found that PI3Kγ inhibited GSK-3 independently of the insulin-PI3Kα-Akt axis. Although insulin treatment activated Akt in PI3Kγ knockout mice, phosphorylation of GSK-3 was decreased compared to control mice. GSK-3 is activated when dephosphorylated by the protein phosphatase 2A (PP2A), which is activated when methylated by the PP2A methyltransferase PPMT-1. PI3Kγ knockout mice showed increased activity of PPMT-1 and PP2A and enhanced nuclear export of the GSK-3 substrate NFATc3. GSK-3 inhibits cardiac hypertrophy, and the hearts of PI3Kγ knockout mice were smaller compared to those of wild-type mice. Cardiac overexpression of a catalytically inactive PI3Kγ (PI3Kγinact) transgene in PI3Kγ knockout mice reduced the activities of PPMT-1 and PP2A and increased phosphorylation of GSK-3. Furthermore, PI3Kγ knockout mice expressing the PI3Kγinact transgene had larger hearts than wild-type or PI3Kγ knockout mice. Our studies show that a kinase-independent function of PI3Kγ could directly inhibit GSK-3 function by preventing the PP2A–PPMT-1 interaction and that this inhibition of GSK-3 was independent of Akt.

Pro-inflammatory cytokines mediate GPCR dysfunction.

Pro-inflammatory reaction by the body occurs acutely in response to injury that is considered primarily beneficial. However, sustained pro-inflammatory cytokines observed with chronic pathologies such as metabolic syndrome, cancer, and arthritis are detrimental and in many cases is a major cardio-vascular risk factor. Pro-inflammatory cytokines such as interleulin-1 (IL-1), IL-6, and tumor necrosis factor α (TNFα) have long been implicated in cardiovascular risk and considered to be a major underlying cause for heart failure. The failure of the anti-TNFα therapy for heart failure indicates our elusive understanding on the dichotomous role of pro-inflammatory cytokines on acutely beneficial effects versus long-tersm deleterious effects. Despite these well-described observations, less is known about the mechanistic underpinnings of pro-inflammatory cytokines especially TNFα in pathogenesis of heart failure. Increasing evidence suggests the existence of an active cross-talk between the TNFα receptor signaling and G-protein coupled receptors (GPCRs) like β-adrenergic receptor (βAR). Given that βARs are the key regulators of cardiac function, the review will discuss current state of understanding on the role of pro-inflammatory cytokine TNFα in regulating βAR function.