Maradumane L. Mohan

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.

Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

β2-Adrenergic receptors (β2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a β2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for β2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes β2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following βAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling.

G protein-coupled receptors directly bind filamin A with high affinity and promote filamin phosphorylation

Although interaction of a few G protein-coupled receptors (GPCRs) with Filamin A, a key actin cross-linking and biomechanical signal transducer protein, has been observed, a comprehensive structure–function analysis of this interaction is lacking. Through a systematic sequence-based analysis, we found that a conserved filamin binding motif is present in the cytoplasmic domains of >20% of the 824 GPCRs encoded in the human genome. Direct high-affinity interaction of filamin binding motif peptides of select GPCRs with the Ig domain of Filamin A was confirmed by nuclear magnetic resonance spectroscopy and isothermal titration calorimetric experiments. Engagement of the filamin binding motif with the Filamin A Ig domain induced the phosphorylation of filamin by protein kinase A in vitro. In transfected cells, agonist activation as well as constitutive activation of representative GPCRs dramatically elicited recruitment and phosphorylation of cellular Filamin A, a phenomenon long known to be crucial for regulating the structure and dynamics of the cytoskeleton. Our data suggest a molecular mechanism for direct GPCR–cytoskeleton coupling via filamin. Until now, GPCR signaling to the cytoskeleton was predominantly thought to be indirect, through canonical G protein-mediated signaling cascades involving GTPases, adenylyl cyclases, phospholipases, ion channels, and protein kinases. We propose that the GPCR-induced filamin phosphorylation pathway is a conserved, novel biochemical signaling paradigm.

A mechanism of global shape-dependent recognition and phosphorylation of filamin by protein kinase A

Background: The mechanism of filamin Ser2152 phosphorylation by PKA is unclear. Results: Autoinhibitory filamin is resistant to phosphorylation despite exposed Ser2152, but ligand binding alters the filamin conformation, triggering PKA recognition. Conclusion: Filamin Ser2152 phosphorylation is conformation-dependent on ligand binding. Significance: The overall conformation of substrate, not just the exposed phosphorylation site, regulates the kinase substrate recognition in signaling. Protein phosphorylation mediates essentially all aspects of cellular life. In humans, this is achieved by ∼500 kinases, each recognizing a specific consensus motif (CM) in the substrates. The majority of CMs are surface-exposed and are thought to be accessible to kinases for phosphorylation. Here we investigated the archetypical protein kinase A (PKA)-mediated phosphorylation of filamin, a major cytoskeletal protein that can adopt an autoinhibited conformation. Surprisingly, autoinhibited filamin is refractory to phosphorylation by PKA on a known Ser2152 site despite its CM being exposed and the corresponding isolated peptide being readily phosphorylated. Structural analysis revealed that although the CM fits into the PKA active site its surrounding regions sterically clash with the kinase. However, upon ligand binding, filamin undergoes a conformational adjustment, allowing rapid phosphorylation on Ser2152. These data uncover a novel ligand-induced conformational switch to trigger filamin phosphorylation. They further suggest a substrate shape-dependent filtering mechanism that channels specific exposed CM/kinase recognition in diverse signaling responses.

G Protein-Coupled Receptor Resensitization Paradigms

Cellular responses to extracellular milieu/environment are driven by cell surface receptors that transmit the signal into the cells resulting in a synchronized and measured response. The ability to provide such exquisite responses to changes in external environment is mediated by the tight and yet, deliberate regulation of cell surface receptor function. In this regard, the seven transmembrane G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors that regulate responses like cardiac contractility, vision, and olfaction including platelet activation. GPCRs regulate these plethora of events through GPCR-activation, -desensitization, and -resensitization. External stimuli (ligands or agonists) activate GPCR initiating downstream signals. The activated GPCR undergoes inactivation or desensitization by phosphorylation and binding of β-arrestin resulting in diminution of downstream signals. The desensitized GPCRs are internalized into endosomes, wherein they undergo dephosphorylation or resensitization by protein phosphatase to be recycled back to the cell membrane as naïve GPCR ready for the next wave of stimuli. Despite the knowledge that activation, desensitization, and resensitization shoulder an equal role in maintaining GPCR function, major advances have been made in understanding activation and desensitization compared to resensitization. However, increasing evidence shows that resensitization is exquisitely regulated process, thereby contributing to the dynamic regulation of GPCR function. In recognition of these observations, in this chapter we discuss the key advances on the mechanistic underpinning that drive and regulate GPCR function with a focus on resensitization.