Akt acts as a switch for GPCR transactivation of the TGF-ß receptor type 1.

Transforming growth factor (TGF)-ß signalling commences with the engagement of TGF-ß ligand to cell surface TGF-ß receptors (TGFBR) stimulating Smad2 carboxyl-terminal phosphorylation (phospho-Smad2C) and downstream biological responses. In several cell models, G protein-coupled receptors (GPCRs) transactivate the TGF-ß receptors type-1 (TGFBR1) leading to phospho-Smad2C, however, we have recently published that in keratinocytes thrombin did not transactivate the TGFBR1. The bulk of TGFBRs reside in the cytosol and in response to protein kinase B (Akt phosphorylation) can translocate to the cell surface increasing the cell's responsiveness to TGF-ß. In this study, we investigate the role of Akt in GPCR transactivation of the TGFBR1. We demonstrate that angiotensin II and thrombin do not phosphorylate Smad2C in human vascular smooth muscle cells and in keratinocytes respectively. We used Akt agonist, SC79 to sensitise the cells to Akt and observed that Ang II and thrombin phosphorylate Smad2C via Akt/AS160-dependent pathways. We show that SC79 rapidly translocates TGFBRs to the cell surface thus increasing the cell's response to the GPCR agonist. These findings highlight novel mechanistic insight for the role of Akt in GPCR transactivation of the TGFBR1.

Therapeutic Inhibition of Acid-Sensing Ion Channel 1a Recovers Heart Function After Ischemia-Reperfusion Injury.

BACKGROUND: Ischemia-reperfusion injury (IRI) is one of the major risk factors implicated in morbidity and mortality associated with cardiovascular disease. During cardiac ischemia, the buildup of acidic metabolites results in decreased intracellular and extracellular pH, which can reach as low as 6.0 to 6.5. The resulting tissue acidosis exacerbates ischemic injury and significantly affects cardiac function. METHODS: We used genetic and pharmacologic methods to investigate the role of acid-sensing ion channel 1a (ASIC1a) in cardiac IRI at the cellular and whole-organ level. Human induced pluripotent stem cell-derived cardiomyocytes as well as ex vivo and in vivo models of IRI were used to test the efficacy of ASIC1a inhibitors as pre- and postconditioning therapeutic agents. RESULTS: Analysis of human complex trait genetics indicates that variants in the ASIC1 genetic locus are significantly associated with cardiac and cerebrovascular ischemic injuries. Using human induced pluripotent stem cell-derived cardiomyocytes in vitro and murine ex vivo heart models, we demonstrate that genetic ablation of ASIC1a improves cardiomyocyte viability after acute IRI. Therapeutic blockade of ASIC1a using specific and potent pharmacologic inhibitors recapitulates this cardioprotective effect. We used an in vivo model of myocardial infarction and 2 models of ex vivo donor heart procurement and storage as clinical models to show that ASIC1a inhibition improves post-IRI cardiac viability. Use of ASIC1a inhibitors as preconditioning or postconditioning agents provided equivalent cardioprotection to benchmark drugs, including the sodium-hydrogen exchange inhibitor zoniporide. At the cellular and whole organ level, we show that acute exposure to ASIC1a inhibitors has no effect on cardiac ion channels regulating baseline electromechanical coupling and physiologic performance. CONCLUSIONS: Our data provide compelling evidence for a novel pharmacologic strategy involving ASIC1a blockade as a cardioprotective therapy to improve the viability of hearts subjected to IRI.

Stimulation of the four isoforms of receptor tyrosine kinase ErbB4, but not ErbB1, confers cardiomyocyte hypertrophy.

Epidermal growth factor (EGF) receptors (ErbB1-ErbB4) promote cardiac development and growth, although the specific EGF ligands and receptor isoforms involved in growth/repair versus pathology remain undefined. We challenged ventricular cardiomyocytes with EGF-like ligands and observed that selective activation of ErbB4 (the receptor for neuregulin 1 [NRG1]), but not ErbB1 (the receptor for EGF, EGFR), stimulated hypertrophy. This lack of direct ErbB1-mediated hypertrophy occurred despite robust activation of extracellular-regulated kinase 1/2 (ERK) and protein kinase B. Hypertrophic responses to NRG1 were unaffected by the tyrosine kinase inhibitor (AG1478) at concentrations that are selective for ErbB1 over ErbB4. NRG1-induced cardiomyocyte enlargement was suppressed by small interfering RNA (siRNA) knockdown of ErbB4 and ErbB2, whereas ERK phosphorylation was only suppressed by ErbB4 siRNA. Four ErbB4 isoforms exist (JM-a/JM-b and CYT-1/CYT-2), generated by alternative splicing, and their expression declines postnatally and following cardiac hypertrophy. Silencing of all four isoforms in cardiomyocytes, using an ErbB4 siRNA, abrogated NRG1-induced hypertrophic promoter/reporter activity, which was rescued by coexpression of knockdown-resistant versions of the ErbB4 isoforms. Thus, ErbB4 confers cardiomyocyte hypertrophy to NRG1, and all four ErbB4 isoforms possess the capacity to mediate this effect.

Complex interactions between the angiotensin II type 1 receptor, the epidermal growth factor receptor and TRIO-dependent signaling partners.

Transactivation of the epidermal growth factor receptor (EGFR) by the angiotensin II (AngII) type 1 (AT1) receptor is involved in AT1 receptor-dependent growth effects and cardiovascular pathologies, however the mechanisms underpinning this transactivation are yet to be fully elucidated. Recently, a potential intermediate of this process was identified following the discovery that a kinase called TRIO was involved in AngII/AT1 receptor-mediated transactivation of EGFR. To investigate the mechanisms by which TRIO acts as an intermediate in AngII/AT1 receptor-mediated EGFR transactivation we used bioluminescence resonance energy transfer (BRET) assays to investigate proximity between the AT1 receptor, EGFR, TRIO and other proteins of interest. We found that AngII/AT1 receptor activation caused a Gαq-dependent increase in proximity of TRIO with Gγ2 and the AT1-EGFR heteromer, as well as trafficking of TRIO towards the Kras plasma membrane marker and into early, late and recycling endosomes. In contrast, we found that AngII/AT1 receptor activation caused a Gαq-independent increase in proximity of TRIO with Grb2, GRK2 and PKCζ, as well as trafficking of TRIO up to the plasma membrane from the Golgi. Furthermore, we confirmed the proximity between the AT1 receptor and the EGFR using the Receptor-Heteromer Investigation Technology, which showed AngII-induced recruitment of Grb2, GRK2, PKCζ, Gγ2 and TRIO to the EGFR upon AT1 coexpression. In summary, our results provide further evidence for the existence of the AT1-EGFR heteromer and reveal potential mechanisms by which TRIO contributes to the transactivation process.

BRET-based assay to monitor EGFR transactivation by the AT1R reveals Gq/11 protein-independent activation and AT1R-EGFR complexes.

The type 1 angiotensin II (AngII) receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR), which leads to pathological remodeling of heart, blood vessels and kidney. End-point assays are used as surrogates of EGFR activation, however these downstream readouts are not applicable to live cells, in real-time. Herein, we report the use of a bioluminescence resonance energy transfer (BRET)-based assay to assess recruitment of the EGFR adaptor protein, growth factor receptor-bound protein 2 (Grb2), to the EGFR. In a variety of cell lines, both epidermal growth factor (EGF) and AngII stimulated Grb2 recruitment to EGFR. The BRET assay was used to screen a panel of 9 G protein-coupled receptors (GPCRs) and further developed for other EGFR family members (HER2 and HER3); the AT1R was able to transactivate HER2, but not HER3. Mechanistically, AT1R-mediated ERK1/2 activation was dependent on Gq/11 and EGFR tyrosine kinase activity, whereas the recruitment of Grb2 to the EGFR was independent of Gq/11 and only partially dependent on EGFR tyrosine kinase activity. This Gq/11 independence of EGFR transactivation was confirmed using AT1R mutants and in CRISPR cell lines lacking Gq/11. EGFR transactivation was also apparently independent of ß-arrestins. Finally, we used additional BRET-based assays and confocal microscopy to provide evidence that both AngII- and EGF-stimulation promoted AT1R-EGFR heteromerization. In summary, we report an alternative approach to monitoring AT1R-EGFR transactivation in live cells, which provides a more direct and proximal view of this process, including the potential for complexes between the AT1R and EGFR.

Transactivation of the epidermal growth factor receptor in responses to myocardial stress and cardioprotection.

The epidermal growth factor receptor (EGFR) family comprises the ErbB1 (EGFR) and ErbB4 receptors as well as the 'co-receptors' ErbB2 (which does not bind EGF ligands) and ErbB3 (which lack tyrosine kinase activity). This family of receptors is essential for cardiac development, myocardial, renal and vascular function, and cardiac responses to physiological and pathological perturbations. The EGFR appears critical in protecting cardiac cells from injury, while considerable attention has focussed on neuregulin/ErbB4 signalling in potentially ameliorating cardiomyopathy/heart failure. Indeed, the EGFRs provide a signalling nexus, upon which multiple cardioprotective stimuli appear to converge, including ischaemic preconditioning and various G protein-coupled receptors (opioid, muscarinic, adenosine, adrenergic, bradykinin, sphingosine 1-phosphate). These stimuli engage the EGFR axis (in a process referred to as transactivation) in differing ways, involving both G protein-dependent and -independent mechanisms, to promote myocardial cell survival during and following ischaemia/infarction. Elucidating the molecular processes that underpin EGFR transactivation and mediate cardiac protection will advance our understanding of the intrinsic capacity of the heart to withstand pathological insult. It should also reveal new approaches to facilitate cardioprotective therapy to limit damage during and following myocardial ischaemia/infarction, which despite intense investigation remains an unrealised, yet highly desirable, clinical goal. This review focuses on the cardiovascular functions of the EGFR, its role in cardioprotection, and the potential influences of common disease states on this signalling.

Helix 8 of the angiotensin- II type 1A receptor interacts with phosphatidylinositol phosphates and modulates membrane insertion.

The carboxyl-terminus of the type 1 angiotensin II receptor (AT1A) regulates receptor activation/deactivation and the amphipathic Helix 8 within the carboxyl-terminus is a high affinity interaction motif for plasma membrane lipids. We have used dual polarisation interferometry (DPI) to examine the role of phosphatidylinositdes in the specific recognition of Helix 8 in the AT1A receptor. A synthetic peptide corresponding to Leu(305) to Lys(325) (Helix 8 AT1A) discriminated between PIPs and different charges on lipid membranes. Peptide binding to PtdIns(4)P-containing bilayers caused a dramatic change in the birefringence (a measure of membrane order) of the bilayer. Kinetic modelling showed that PtdIns(4)P is held above the bilayer until the mass of bound peptide reaches a threshold, after which the peptides insert further into the bilayer. This suggests that Helix 8 can respond to the presence of PI(4)P by withdrawing from the bilayer, resulting in a functional conformational change in the receptor.

Unravelling the molecular complexity of GPCR-mediated EGFR transactivation using functional genomics approaches.

To influence physiology and pathophysiology, G protein-coupled receptors (GPCRs) have evolved to appropriate additional signalling modalities, such as activation of adjacent membrane receptors. Epidermal growth factor receptors (EGFRs) mediate growth and remodelling actions of GPCRs, although the precise network of gene products and molecular cascades linking GPCRs to EGFRs (termed EGFR transactivation) remains incomplete. In this review, we describe the current view of GPCR-EGFR transactivation, identifying the established models of receptor cross-talk. We consider the limitations in our current knowledge, and propose that recent advances in molecular and cell biology technology, including functional genomics approaches, will allow a renewed focus of efforts to understand the mechanism underlying EGFR transactivation. Using an unbiased approach for identification of the molecules required for GPCR-mediated EGFR transactivation will provide a contemporary and more complete representation from which to extrapolate therapeutic control in diseases from cardiovascular remodelling to cancer.

A functional siRNA screen identifies genes modulating angiotensin II-mediated EGFR transactivation.

The angiotensin type 1 receptor (AT1R) transactivates the epidermal growth factor receptor (EGFR) to mediate cellular growth, however, the molecular mechanisms involved have not yet been resolved. To address this, we performed a functional siRNA screen of the human kinome in human mammary epithelial cells that demonstrate a robust AT1R-EGFR transactivation. We identified a suite of genes encoding proteins that both positively and negatively regulate AT1R-EGFR transactivation. Many candidates are components of EGFR signalling networks, whereas others, including TRIO, BMX and CHKA, have not been previously linked to EGFR transactivation. Individual knockdown of TRIO, BMX or CHKA attenuated tyrosine phosphorylation of the EGFR by angiotensin II stimulation, but this did not occur following direct stimulation of the EGFR with EGF, indicating that these proteins function between the activated AT1R and the EGFR. Further investigation of TRIO and CHKA revealed that their activity is likely to be required for AT1R-EGFR transactivation. CHKA also mediated EGFR transactivation in response to another G protein-coupled receptor (GPCR) ligand, thrombin, indicating a pervasive role for CHKA in GPCR-EGFR crosstalk. Our study reveals the power of unbiased, functional genomic screens to identify new signalling mediators important for tissue remodelling in cardiovascular disease and cancer.

PAQR3 modulates insulin signaling by shunting phosphoinositide 3-kinase p110α to the Golgi apparatus.

Phosphoinositide 3-kinase (PI3K) mediates insulin actions by relaying signals from insulin receptors (IRs) to downstream targets. The p110α catalytic subunit of class IA PI3K is the primary insulin-responsive PI3K implicated in insulin signaling. We demonstrate here a new mode of spatial regulation for the p110α subunit of PI3K by PAQR3 that is exclusively localized in the Golgi apparatus. PAQR3 interacts with p110α, and the intracellular targeting of p110α to the Golgi apparatus is reduced by PAQR3 downregulation and increased by PAQR3 overexpression. Insulin-stimulated PI3K activity and phosphoinositide (3,4,5)-triphosphate production are enhanced by Paqr3 deletion and reduced by PAQR3 overexpression in hepatocytes. Deletion of Paqr3 enhances insulin-stimulated phosphorylation of AKT and glycogen synthase kinase 3ß, but not phosphorylation of IR and IR substrate-1 (IRS-1), in hepatocytes, mouse liver, and skeletal muscle. Insulin-stimulated GLUT4 translocation to the plasma membrane and glucose uptake are enhanced by Paqr3 ablation. Furthermore, PAQR3 interacts with the domain of p110α involved in its binding with p85, the regulatory subunit of PI3K. Overexpression of PAQR3 dose-dependently reduces the interaction of p85α with p110α. Thus, PAQR3 negatively regulates insulin signaling by shunting cytosolic p110α to the Golgi apparatus while competing with p85 subunit in forming a PI3K complex with p110α.

Silencing relaxin-3 in nucleus incertus of adult rodents: a viral vector-based approach to investigate neuropeptide function.

Relaxin-3, the most recently identified member of the relaxin peptide family, is produced by GABAergic projection neurons in the nucleus incertus (NI), in the pontine periventricular gray. Previous studies suggest relaxin-3 is a modulator of stress responses, metabolism, arousal and behavioural activation. Knockout mice and peptide infusions in vivo have significantly contributed to understanding the function of this conserved neuropeptide. Yet, a definitive role remains elusive due to discrepancies between models and a propensity to investigate pharmacological effects over endogenous function. To investigate the endogenous function of relaxin-3, we generated a recombinant adeno-associated viral (rAAV) vector expressing microRNA against relaxin-3 and validated its use to knock down relaxin-3 in adult rats. Bilateral stereotaxic infusion of rAAV1/2 EmGFP miR499 into the NI resulted in significant reductions in relaxin-3 expression as demonstrated by ablation of relaxin-3-like immunoreactivity at 3, 6 and 9 weeks and by qRT-PCR at 12 weeks. Neuronal health was unaffected as transduced neurons in all groups retained expression of NeuN and stained for Nissl bodies. Importantly, qRT-PCR confirmed that relaxin-3 receptor expression levels were not altered to compensate for reduced relaxin-3. Behavioural experiments confirmed no detrimental effects on general health or well-being and therefore several behavioural modalities previously associated with relaxin-3 function were investigated. The validation of this viral vector-based model provides a valuable alternative to existing in vivo approaches and promotes a shift towards more physiologically relevant investigations of endogenous neuropeptide function.

Angiotensin 1A receptors transfected into caudal ventrolateral medulla inhibit baroreflex gain and stress responses.

AIMS: The caudal ventrolateral medulla (CVLM) is important for autonomic regulation and is rich in angiotensin II type 1A receptors (AT(1A)R). To determine their function, we examined whether the expression of AT(1A)R in the CVLM of mice lacking AT(1A)R (AT(1A)(-/-)) alters baroreflex sensitivity and cardiovascular responses to stress. METHODS AND RESULTS: Bilateral microinjections into the CVLM of AT(1A)(-/-) mice of lentivirus with the phox-2 selective promoter (PRSx8) were made to express either AT(1A)R (Lv-PRSx8-AT(1A)) or green fluorescent protein (Lv-PRSx8-GFP) as a control. Radiotelemetry was used to record mean arterial pressure (MAP), heart rate (HR), and locomotor activity. Following injection of Lv-PRSx8-GFP, robust neuronal expression of GFP was observed with ∼60% of the GFP-positive cells also expressing the catecholamine-synthetic enzyme tyrosine hydroxylase. After 5 weeks, there were no differences in MAP or HR between groups, but the Lv-PRSx8-AT(1A)- injected mice showed reduced baroreflex sensitivity (-25%, P = 0.003) and attenuated pressor responses to cage-switch and restraint stress compared with the Lv-PRSx8-GFP-injected mice. Reduced MAP mid-frequency power during cage-switch stress reflected attenuated sympathetic activation (Pgroup × stress = 0.04). Fos-immunohistochemistry indicated greater activation of forebrain and hypothalamic neurons in the Lv-PRSx8-AT(1A) mice compared with the control. CONCLUSION: The expression of AT(1A)R in CVLM neurons, including A1 neurons, while having little influence on the basal blood pressure or HR, may play a tonic role in inhibiting cardiac vagal baroreflex sensitivity. However, they strongly facilitate the forebrain response to aversive stress, yet reduce the pressor response presumably through greater sympatho-inhibition. These findings outline novel and specific roles for angiotensin II in the CVLM in autonomic regulation.

PAQR10 and PAQR11 mediate Ras signaling in the Golgi apparatus.

Ras plays a pivotal role in many cellular activities, and its subcellular compartmentalization provides spatial and temporal selectivity. Here we report a mode of spatial regulation of Ras signaling in the Golgi apparatus by two highly homologous proteins PAQR10 and PAQR11 of the progestin and AdipoQ receptors family. PAQR10 and PAQR11 are exclusively localized in the Golgi apparatus. Overexpression of PAQR10/PAQR11 stimulates basal and EGF-induced ERK phosphorylation and increases the expression of ERK target genes in a dose-dependent manner. Overexpression of PAQR10/PAQR11 markedly elevates Golgi localization of HRas, NRas and KRas4A, but not KRas4B. PAQR10 and PAQR11 can also interact with HRas, NRas and KRas4A, but not KRas4B. The increased Ras protein at the Golgi apparatus by overexpression of PAQR10/PAQR11 is in an active state. Consistently, knockdown of PAQR10 and PAQR11 reduces EGF-stimulated ERK phosphorylation and Ras activation at the Golgi apparatus. Intriguingly, PAQR10 and PAQR11 are able to interact with RasGRP1, a guanine nucleotide exchange protein of Ras, and increase Golgi localization of RasGRP1. The C1 domain of RasGRP1 is both necessary and sufficient for the interaction of RasGRP1 with PAQR10/PAQR11. The simulation of ERK phosphorylation by overexpressed PAQR10/PAQR11 is abrogated by downregulation of RasGRP1. Furthermore, differentiation of PC12 cells is significantly enhanced by overexpression of PAQR10/PAQR11. Collectively, this study uncovers a new paradigm of spatial regulation of Ras signaling in the Golgi apparatus by PAQR10 and PAQR11.

Relative affinity of angiotensin peptides and novel ligands at AT1 and AT2 receptors.

AT1R (angiotensin type 1 receptor) and AT2R (angiotensin type 2 receptor) are well known to be involved in the complex cardiovascular actions of AngII (angiotensin II). However, shorter peptide fragments of AngII are thought to have biological activity in their own right and elicit effects that oppose those mediated by AngII. In the present study, we have used HEK (human embryonic kidney)-293 cells stably transfected with either AT1R or AT2R to perform a systematic analysis of binding affinities of all the major angiotensin peptides. Additionally, we tested the novel AT2R agonist Compound 21, as well as the MasR (Mas receptor) agonist and antagonist AVE0991 and A-779 respectively, for their ability to bind to AT1R or AT2R. Candesartan, CGP42214 and PD123319 were used as reference compounds. Binding studies using 125I-[Sar1Ile8]AngII on the AT1R-transfected HEK-293 cells revealed only AngII, AngIII [angiotensin III; angiotensin-(2-8)] and candesartan to have high affinity for AT1R. In the AT2R-transfected HEK-293 cells, competition for 125I-[Sar1Ile8]AngII binding was observed for all ligands except candesartan, AVE0991 and A-779, the latter two compounds having negligible affinity at either AT1R or AT2R. The rank order of affinity of ligands at AT2R was CGP42112>AngII≥AngIII>Compound 21≥PD123319≫AngIV [angiotensin IV; angiotensin-(3-8)]>Ang-(1-7) [angiotensin-(1-7)]. Of note, although AngIV and Ang-(1-7) exhibited only modest affinity at AT2R compared with AngII, these two angiotensin peptides, together with AngIII, had substantial AT2R selectivity over AT1R. Collectively, our results suggest that shorter angiotensin peptides can act as endogenous ligands at AT2R.

Determination of the exact molecular requirements for type 1 angiotensin receptor epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy.

Major interest surrounds how angiotensin II triggers cardiac hypertrophy via epidermal growth factor receptor transactivation. G protein-mediated transduction, angiotensin type 1 receptor phosphorylation at tyrosine 319, and ß-arrestin-dependent scaffolding have been suggested, yet the mechanism remains controversial. We examined these pathways in the most reductionist model of cardiomyocyte growth, neonatal ventricular cardiomyocytes. Analysis with [(32)P]-labeled cardiomyocytes, wild-type and [Y319A] angiotensin type 1 receptor immunoprecipitation and phosphorimaging, phosphopeptide analysis, and antiphosphotyrosine blotting provided no evidence for tyrosine phosphorylation at Y319 or indeed of the receptor, and mutation of Y319 (to A/F) did not prevent either epidermal growth factor receptor transactivation in COS-7 cells or cardiomyocyte hypertrophy. Instead, we demonstrate that transactivation and cardiomyocyte hypertrophy are completely abrogated by loss of G-protein coupling, whereas a constitutively active angiotensin type 1 receptor mutant was sufficient to trigger transactivation and growth in the absence of ligand. These results were supported by the failure of the ß-arrestin-biased ligand SII angiotensin II to transactivate epidermal growth factor receptor or promote hypertrophy, whereas a ß-arrestin-uncoupled receptor retained these properties. We also found angiotensin II-mediated cardiomyocyte hypertrophy to be attenuated by a disintegrin and metalloprotease inhibition. Thus, G-protein coupling, and not Y319 phosphorylation or ß-arrestin scaffolding, is required for epidermal growth factor receptor transactivation and cardiomyocyte hypertrophy via the angiotensin type 1 receptor.

The renin-angiotensin system and cancer: old dog, new tricks.

For cancers to develop, sustain and spread, the appropriation of key homeostatic physiological systems that influence cell growth, migration and death, as well as inflammation and the expansion of vascular networks are required. There is accumulating molecular and in vivo evidence to indicate that the expression and actions of the renin-angiotensin system (RAS) influence malignancy and also predict that RAS inhibitors, which are currently used to treat hypertension and cardiovascular disease, might augment cancer therapies. To appreciate this potential hegemony of the RAS in cancer, an expanded comprehension of the cellular actions of this system is needed, as well as a greater focus on translational and in vivo research.

Glucocorticoids suppress growth in neonatal cardiomyocytes co-expressing AT(2) and AT(1) angiotensin receptors.

BACKGROUND: Perinatal glucocorticoid treatment is associated with hypertrophic cardiomyopathy, but the cellular mechanism is controversial. An underlying interaction between glucocorticoids and the renin-angiotensin system may be important, but whether glucocorticoids modulate angiotensin II (AngII)-dependent cardiomyocyte growth responses in the neonate has not been investigated. OBJECTIVES: The major aim of this investigation was to determine whether glucocorticoids modulate the neonatal cardiomyocyte growth response to AngII. In particular we sought evidence to determine whether angiotensin II type 2 (AT(2)) receptor co-expression with angiotensin II type 1 (AT(1)) receptor is of specific importance in this modulatory function. METHODS: In this study, we used AT(1) and AT(2) receptor-expressing adenoviruses (Ad-AT(1) and Ad-AT(2)) in a well-defined in vitro neonatal cardiomyocyte culture model to assess whether glucocorticoids affect cardiomyocyte growth responses (i.e. total protein content). RESULTS: Following addition of AngII (0.1 micromol/l) to neonatal cardiomyocytes infected with Ad-AT(1) alone, a significant growth response was measured (133.2 +/- 4.8%). Expression of Ad-AT(2) alone induced a approximately 20% increase in total cellular protein content, which was unaffected by addition of AngII. Neither corticosterone (1 micromol/l) nor dexamethasone (1 micromol/l) had any significant effect on the AT(1)- or AT(2)-mediated growth responses. In contrast, the growth response to AngII was augmented following co-expression of AT(2) and AT(1) receptors (149.2 +/- 4.2%), which was reduced by approximately 20% in the presence of either corticosterone or dexamethasone (p < 0.05). CONCLUSIONS: The present study provides novel evidence that glucocorticoids suppress neonatal cardiomyocyte growth responsiveness when AT(2 )and AT(1) receptor subtypes are co-expressed.

Beta-arrestin 2 is required for complement C1q expression in macrophages and constrains factor-independent survival.

The beta-arrestins (ARRB1 and ARRB2) regulate G-protein coupled receptor (GPCR) dependent- and independent-signaling pathways and are ubiquitously expressed. Here we show that ARRB2 mRNA and protein expression is enriched in macrophages, and that it regulates complement C1q expression and cell survival. Basal and Toll-like receptor (TLR) inducible expression of mRNAs encoding the complement subcomponents C1qa, C1qb and C1qc was greatly reduced in bone marrow-derived macrophages (BMM) from ARRB2-deficient, but not ARRB1-deficient mice, while factor-independent survival of ARRB2(-/-) BMM was enhanced compared to wildtype BMM. TatARRB2(23), a cell-permeable peptide that contains the MAPK JNK-binding motif from within the ARRB2 C-domain, impaired ARRB2 interaction with JNK3, down-regulated C1q expression and permitted factor-independent survival in BMM, thus suggesting that this peptide antagonises ARRB2 function in macrophages. In addition, TatARRB2(23) transiently activated the phosphorylation of JNK and ERK, but not p38 in BMM. These data imply that ARRB2 acts to limit JNK/ERK activation and survival in macrophages, but is required for basal and TLR-inducible complement C1q expression. Given that loss of C1q function is strongly associated with the development of systemic lupus erythematosus, ARRB2 may act to limit the development of autoimmune disease.

Immunoprecipitation and phosphorylation of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) are integral membrane proteins with seven transmembrane-spanning alpha-helices. Following ligand activation, many GPCRs are rapidly phosphorylated on serine/threonine residues in their cytoplasmic domains, principally the carboxyl-terminus. GPCR phosphorylation recruits arrestin proteins to the activated receptor leading to receptor internalization and desensitization. Arrestins also act as scaffolds to recruit other regulatory and signaling molecules to the receptor. The low level of expression of GPCRs in tissues, the difficulty in developing antibodies that can specifically detect and harvest receptor protein, and the hydrophobic and heterogeneric nature of GPCRs makes examination of their structure, function, and biology an interesting challenge. Receptor phosphorylation is typically performed in cells transfected with wild and mutated receptors usually bearing an epitope tag and equilibrated with [(32)Pi] to radiolabel cellular ATP pools. Following ligand stimulation, receptor protein is extracted using a detergent lysis buffer and immunoprecipitated with antibodies raised against the epitope tag; following separation on SDS-polyacrylamide gel electrophoresis, phosphorylated receptors are quantified using phosphorimaging.

Angiotensin II type 2 receptor antagonizes angiotensin II type 1 receptor-mediated cardiomyocyte autophagy.

Autophagy has emerged as an important process in the pathogenesis of cardiovascular diseases, but the proximal triggers for autophagy are unknown. Angiotensin II plays a central role in the pathogenesis of cardiac hypertrophy and heart failure. In this study, we used angiotensin II type 1 (AT(1)) and type 2 (AT(2)) receptor-expressing adenoviruses in cultured neonatal cardiomyocytes to provide the first demonstration that neonatal cardiomyocyte autophagic activity is differentially modulated by AT(1) and AT(2) receptor subtypes. Angiotensin II stimulation (48 hours) of neonatal cardiomyocytes expressing the AT(1) receptor alone (Ad-AT(1); 10 multiplicities of infection) induced a significant increase in the number of HcRed-LC3 autophagosomes per cell (17.3+/-1.6 versus 33.3+/-4.1 autophagosomes per cell; P<0.05). Coexpression of a high ratio of AT(2):AT(1) (Ad-AT(2):Ad-AT(1) multiplicity of infection ratio: 20:5) receptors completely abrogated the AT(1)-mediated increase in autophagy (9.3+/-1.4 versus 33.3+/-4.1 autophagosomes per cell; P<0.05). Treatment with the AT(2) receptor antagonist PD123319 did not reverse the AT(2)-mediated antiautophagic effect. AT(1)- and AT(2)-mediated autophagic responses were also assessed in cardiomyocytes from a genetic model that exhibits neonatal myocardial growth suppression. In these neonate myocyte cultures, AT(1) receptor activation induced a marked increase in the number of myocytes containing cytoplasmic vacuoles compared with the control (22.7+/-4.1% versus 1.1+/-0.6%; P<0.001) and was characterized by a nonapoptotic autophagic phenotype. The incidence of cardiomyocyte autophagic vacuolization in this myocyte population decreased dramatically to only 0.4+/-0.2% in myocytes infected with a high ratio of Ad-AT(2):Ad-AT(1). This study provides the first description of reciprocal regulation of cardiomyocyte autophagic induction by the AT(1) and AT(2) receptor subtypes.