Liza Barki-harrington - Academia.edu (original) (raw)

Papers by Liza Barki-harrington

Research paper thumbnail of Oligomerisation of angiotensin receptors: novel aspects in disease and drug therapy

Journal of Renin-Angiotensin-Aldosterone …, 2004

The concept that 7 transmembrane receptors (7TMRs) exist and function as independent monomers has... more The concept that 7 transmembrane receptors (7TMRs) exist and function as independent monomers has facilitated a therapeutic approach of selective targeting of receptors. However, oligomerisation of 7TMRs is now being recognised as an important biological phenomenon that adds a level of complexity to their signalling. In vitro, many 7TMR heterodimers display altered binding, signalling, and trafficking properties compared to their constituent monomeric units. This review discusses an emerging concept regarding the role of 7TMR heterodimerisation in vivo, and its significance to drug therapy. Studies of angiotensin receptor signalling indicate a pivotal role for heterodimerisation in the pathogenesis of human disorders. Furthermore, the occurrence of angiotensin receptor heterodimers has a profound effect on the responsiveness to treatment with 7TMR blockers. Global assessment of the prevalence of different heterodimers during disease and their responsiveness to drug therapy is likely to optimise patient treatment and reduce side-effects associated with 7TMR blockers.

Research paper thumbnail of Functional and quantitative measures of receptor-coupled G proteins in human mononuclear leukocytes: No change with age

Experimental …, 1996

Aging has been associated with alterations in signal transduction for a number of neurotransmitte... more Aging has been associated with alterations in signal transduction for a number of neurotransmitter receptors in human tissues. Heterotrimeric G proteins play a pivotal role in postreceptor information transduction, by coupling a variety of hormone and neurotransmitter receptors to several ...

Research paper thumbnail of Down-regulation of cyclooxygenase-2 by the carboxyl tail of the angiotensin II type 1 receptor

The Journal of biological chemistry, Jan 7, 2014

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the pr... more The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT1) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT1 also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or β-arrestin-dependent signaling. Instead, AT1 enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequen...

Research paper thumbnail of Underwater trauma causes a long-term specific increase in the expression of cyclooxygenase-2 in the ventral CA1 of the hippocampus

Psychoneuroendocrinology, 2014

The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal ne... more The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal neurons, but its role in emotional trauma is not known. Here we show that a single acute stress caused by a near-drowning experience results in heightened anxiety-like behavior one month after the trauma. Biochemical analyses of dorsal and ventral hippocampal CA1, CA3 and dentate gyrus revealed decreased ubiquitination and elevated levels of COX-2 in the traumatized animals only in the ventral CA1. To reveal the identity of the ubiquitin E3 ligase that targets COX-2, we tested the effect of several representative E3 ligases on COX-2 expression in vitro. We found that while AIP4 and Nedd4 had no effect, Mdm2 lowered COX-2 expression by nearly 50%, an effect that was not observed by its dominant negative form. To test whether this also occurs in the hippocampus, we immunoprecipitated Mdm2 from dorsal and ventral CA1 of traumatized and control animals and probed for the presence of COX-2. Our results showed that the levels of Mdm2 were not affected by the trauma but there was significantly less COX-2 associated with Mdm2 in the ventral but not dorsal CA1 of the traumatized animals. Together these data propose that an increase in COX-2 expression in ventral CA1 following trauma is likely due to its attenuated degradation. Unraveling the pathways and mechanisms that control hippocampal COX-2 degradation is important to boost the development of novel therapeutic approaches designed to treat stress-related pathologies.

Research paper thumbnail of Receptor-Signaling Pathways in Heart Failure

Principles of Molecular Cardiology, 2005

Heart failure, a progressive disorder characterized by deterioration of cardiac function and prem... more Heart failure, a progressive disorder characterized by deterioration of cardiac function and premature myocardial cell death, results from several common heart diseases such as coronary atherosclerosis, hypertension, and valvular diseases (1,2). With almost 550,000 new cases diagnosed each year, heart failure affects an estimated 4.7 million Americans, and costs associated with the disease range from 10 billion to10 billion to 40 billion per year (3). The aggregate 5-year mortality of patients with heart failure is about 50%, while the 1-year mortality of patients with advanced disease may exceed 50% (3). To maintain adequate myocardial contractility in response to injury, the heart uses several mechanisms, including hypertrophy of myocardial cells, changing the energetics of myocardial cell contraction, and upregulating transcription of several genes (4,5).

Research paper thumbnail of β1-Adrenergic receptor downregulates the expression of cyclooxygenase-2

Biochemical and Biophysical Research Communications, 2014

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the generation of prostanoids, and i... more Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the generation of prostanoids, and is thus one of the key players in the inflammatory process. Contrary to the constitutively expressed isoform COX-1, the expression of COX-2 is rapidly and transiently upregulated following pathological stimuli but little is known about pathways that mediate its degradation. Here we show that co-expression of COX-2 together with the β1 adrenergic receptor (β1AR) specifically lowers the expression of COX-2 in a dose-dependent manner. We further find that stimulation of the receptor for prolonged periods of time does not reverse the β1AR-induced decrease in COX-2, suggesting that this effect does not occur via classical β1-mediated signaling pathways. Rather we find that the half-life of COX-2 is significantly decreased in the presence of β1AR and that inhibition of the proteasome reverses the effect of the receptor on COX-2. Together these findings ascribe a new role for β1AR in the downregulation of COX-2.

Research paper thumbnail of Lessons From Overexpressed Mouse Models

The Adrenergic Receptors, 2006

ABSTRACT

Research paper thumbnail of Tyrosine Phosphorylation of the 2B Subunit of the NMDA Receptor Is Necessary for Taste Memory Formation

Journal of Neuroscience, 2009

We aimed to test whether tyrosine phosphorylation of the NMDA receptor (NMDAR) in the insular cor... more We aimed to test whether tyrosine phosphorylation of the NMDA receptor (NMDAR) in the insular cortex is necessary for novel taste learning. We found that in rats, novel taste learning leads to elevated phosphorylation of tyrosine 1472 of the NR2B subunit of the NMDAR and increases the interaction of phosphorylated NR2B with the major postsynaptic scaffold protein PSD-95. Injection of the tyrosine kinase inhibitor genistein directly into the insular cortex of rats before novel taste exposure prevented the increase in NR2B tyrosine phosphorylation and behaviorally attenuated taste-memory formation. Functionally, tyrosine phosphorylation of NR2B after learning was found to determine the synaptic distribution of the NMDAR, since microinjection of genistein to the insular cortex altered the distribution pattern of NMDAR caused by novel taste learning.

Research paper thumbnail of Elevated dopamine receptor-coupled Gs protein measures in mononuclear leukocytes of patients with schizophrenia

Schizophrenia Research, 2001

Heterotrimeric G proteins play a pivotal role in post-receptor information transduction and were ... more Heterotrimeric G proteins play a pivotal role in post-receptor information transduction and were previously implicated in the pathophysiology and treatment of mood disorders. Changes previously detected in G protein levels in post-mortem brain of patients with schizophrenia could reflect effects of antipsychotic medication. The present study aims at quantitatively and functionally evaluating receptor-coupled G proteins in mononuclear leukocytes obtained from 23 untreated patients with schizophrenia and 30 healthy subjects in an attempt to unravel a pattern of G protein measures in schizophrenia distinctive from patterns previously obtained in mood disorders. Dopamineenhanced guanine nucleotide binding capacity to G s protein through D 1 /D 5 receptor in mononuclear leukocytes of untreated patients with schizophrenia was significantly increased in comparison with healthy subjects, and positively correlated with both the total PANSS score and the positive subscale. b-Adrenergic and muscarinic receptor-coupled G protein functions, as well as G s a, G i a and Gb immunoreactivities, were similar to healthy subjects. These findings, distinctive for schizophrenia, unrelated to drug treatment, and differential from previous findings in mania and depression, may potentially help to differentially diagnose, after the first psychotic episode, between the major psychoses: schizophrenia and manic-depressive illness.

Research paper thumbnail of Brain region-specific methylation in the promoter of the murine oxytocin receptor gene is involved in its expression regulation

Psychoneuroendocrinology, 2014

Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsi... more Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsion and breast-feeding, and has been recently implicated in mammalian social behavior. The actions of both central and peripheral oxytocin are mediated through the oxytocin receptor (Oxtr), which is encoded by a single gene. In contrast to the highly conserved expression of oxytocin in specific hypothalamic nuclei, the expression of its receptor in the brain is highly diverse among different mammalian species or even within individuals of the same species. The diversity in the pattern of brain Oxtr expression among mammals is thought to contribute to the broad range of social systems and organizations. Yet, the mechanisms underlying this diversity are poorly understood. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression levels of the Oxtr in individuals with autism. Here we hypothesize that DNA methylation is involved in the expression regulation of Oxtr in the mouse brain. By combining bisulfite DNA conversion and Next-Generation Sequencing we found that specific CpG sites are differentially methylated between distinct brain regions expressing different levels of Oxtr mRNA. Some of these CpG sites are located within putative binding sites of transcription factors known to regulate Oxtr expression, including estrogen receptor a (ERa) and SP1. Specifically, methylation of the SP1 site was found to positively correlate with Oxtr expression. Furthermore, we revealed that the methylation levels of these sites in the various brain regions predict the relationship between ERa and Oxtr mRNA levels. Collectively, our results suggest that brain region-specific expression of the mouse Oxtr gene is epigenetically regulated by DNA methylation of its promoter. #

Research paper thumbnail of Upregulation of Prostaglandin Receptor EP1 Expression Involves Its Association with Cyclooxygenase-2

PLoS ONE, 2014

While many signals cause upregulation of the pro-inflammatory enzyme cyclooxygenase -2 (COX-2), m... more While many signals cause upregulation of the pro-inflammatory enzyme cyclooxygenase -2 (COX-2), much less is known about mechanisms that actively downregulate its expression. We have recently shown that the prostaglandin EP1 receptor reduces the expression of COX-2 in a pathway that facilitates its ubiquitination and degradation via the 26S proteasome. Here we show that an elevation of COX-2 intracellular levels causes an increase in the endogenous expression of prostaglandin EP1. The increase in EP1 levels does not occur at the transcriptional level, but is rather associated with complex formation between the receptor and COX-2, which occurs both in vitro and in mammalian tissues. The EP1-COX-2 complex is disrupted following binding of arachidonic acid to COX-2 and accompanied by a parallel reduction in EP1 levels. We propose that a transient interaction between COX-2 and EP1 constitutes a feedback loop whereby an increase in COX-2 expression elevates EP1, which ultimately acts to downregulate COX-2 by expediting its proteasomal degradation. Such a post translational mechanism may serve to control both the ligand-generating system of COX-2 and its reception system.

Research paper thumbnail of  -Arrestins: Multifunctional Cellular Mediators

Physiology, 2008

Initially thought to play a role only in G-protein-coupled receptor desensitization, ␤-arrestins ... more Initially thought to play a role only in G-protein-coupled receptor desensitization, ␤-arrestins are ascribed with new roles such as scaffolding and signaling proteins by their own right. This review explores the many functions of ␤-arrestins, with an emphasis on their recently identified role as regulators of receptor signaling.

Research paper thumbnail of ERK-dependent PSD-95 induction in the gustatory cortex is necessary for taste learning, but not retrieval

Nature Neuroscience, 2008

The processes underlying long-term memory formation in the neocortex are poorly understood. Using... more The processes underlying long-term memory formation in the neocortex are poorly understood. Using taste learning, we found learning-related induction of PSD-95 in the gustatory cortex, which was temporally restricted, coupled to the learning of a novel, but not familiar, taste and controlled by ERK. Using temporally and spatially restricted RNA interference knockdown of PSD-95 in vivo, we found that PSD-95 induction is necessary for learning novel tastes, but not for the recollection of familiar ones.

Research paper thumbnail of Sensing heart stress

Research paper thumbnail of Ectodomain Shedding-Dependent Transactivation of Epidermal Growth Factor Receptors in Response to Insulin-Like Growth Factor Type I

Molecular Endocrinology, 2004

Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growt... more Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R ؊ cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R ؉ cells), stimulation of R ؉ cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R ؊ cells. The R ؊ cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R ؉ cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner. (Molecular Endo-

Research paper thumbnail of Bradykinin Induced Mitogenesis of Androgen Independent Prostate Cancer Cells

The Journal of Urology, 2001

We investigated the mitogenic role of bradykinin (BK) in the regulation of the extracellular sign... more We investigated the mitogenic role of bradykinin (BK) in the regulation of the extracellular signal regulated kinase 1 and 2 (ERK), and the growth of androgen insensitive prostate cancer cells. Androgen insensitive PC3 cells were used in these studies. BK and epidermal growth factor were used as mitogens. The chemical inhibitors tyrphostin AG1478 (epidermal growth factor receptor inhibitor), bisindolylmaliemide (protein kinase C inhibitor) and PD98059 (MEK inhibitor) were applied 30 minutes before stimulation with agonist. Down-regulation of protein kinase C was accomplished by incubating cells overnight with phorbol ester. Cell proliferation was measured using WST-1 reagent and the trypan blue exclusion assay. ERK expression and activation were assayed by immunoblotting for total and phosphorylated ERK. Bradykinin induced the proliferation of PC3 cells in a pathway that requires the activation of ERK. The BK regulated activation of ERK was time and dose dependent, yielding a maximal response at the same concentration range that elicits cellular growth. BK exerted its effect on ERK activation via a protein kinase C and epidermal growth factor receptor dependent pathway. Inhibition of the kinase activity of protein kinase C or epidermal growth factor receptor eliminated BK induced ERK activation. Furthermore, the inhibition of epidermal growth factor receptor transactivation abolished BK induced cell proliferation. Our results show that BK induces the proliferation of androgen insensitive prostate cancer cells and suggest a possible pathophysiological role for BK in prostate cancer.

Research paper thumbnail of Guanosine Phosphate Binding Protein Coupled Receptors in Prostate Cancer: A Review

The Journal of Urology, 2002

Androgens are the primary growth promoters of the prostate gland and yet prostate tumors progress... more Androgens are the primary growth promoters of the prostate gland and yet prostate tumors progress despite androgen ablation. This progression suggests a role for additional cellular factors in the progression to androgen independent disease. We examined the role of a family of extracellular signal regulators, namely the guanosine phosphate binding (G) protein coupled receptor (GPCR) family, in prostate cancer. A comprehensive review of the literature was performed on GPCRs and prostate cancer, and supplemented with published and unpublished observations made at our laboratory. Emphasis was placed on the mechanistic aspects of mitogenic signaling pathways involved to identify potential targets for therapy. Expression of some GPCRs and GPCR ligands is elevated in prostate cancer cells and adjacent prostatic stromal tissue. In vitro studies demonstrate that activation of GPCRs confers a distinct growth and survival advantage on prostate cancer cells, including enhanced proliferation and decreased programmed cell death (apoptosis). Specifically stimulation of GPCRs for lysophosphatidic acid and bradykinin induces proliferation of androgen independent prostate cancer cells via the activation of the extracellular signal regulated kinase (ERK) pathway. Induction of ERK by the bradykinin and lysophosphatidic acid in prostate cells proceeds via distinct pathways and involves Galphaq and Gbetagamma subunits, respectively. The Gbetagamma dependent activation of ERK requires tyrosine kinases, including epidermal growth factor receptor and c-Src. Furthermore, stimulation with LPA enhances the survival of prostate cancer cells via activation of the inducible transcription factor nuclear factor-kappaB. GPCR stimulation induces proliferation and prevents apoptosis of hormone independent prostate cancer cells, indicating their important role in the progression of prostate cancer. While further confirmatory studies are required to verify the role of GPCRs in disease progression, the therapeutic implications of these studies may enhance the armamentarium in the fight against prostate cancer.

Research paper thumbnail of Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin

Journal of Neurochemistry, 2012

Synaptic vesicles are pre-synaptic organelles which release their neurotransmitter content onto t... more Synaptic vesicles are pre-synaptic organelles which release their neurotransmitter content onto the post-synaptic neuron by exocytosis, followed by their local reconstitution by endocytosis, in what is known as the synaptic vesicle cycle . A minority of vesicles constitute the readily releasable pool (RRP) of vesicles which are immediately ready for exocytosis. The rest are either recycling vesicles, which can be recruited rapidly, or they belong to the reserve or resting pool, meaning that mobilizing them for eventual use requires the investment of substantially more time and effort (Denker and Rizzoli 2010). The interplay between the pools and the kinetics of the cycle determine the overall release dynamics of the terminal (Pan and

Research paper thumbnail of β-Arrestin–mediated β1-adrenergic receptor transactivation of the EGFR confers cardioprotection

Journal of Clinical Investigation, 2007

Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), membe... more Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from G s -dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β 1 AR signaling to the EGFR. This β-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

Research paper thumbnail of Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediates  -1 Adrenergic Receptor Endocytosis through Different Pathways

Journal of Biological Chemistry, 2003

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled rece... more Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.

Research paper thumbnail of Oligomerisation of angiotensin receptors: novel aspects in disease and drug therapy

Journal of Renin-Angiotensin-Aldosterone …, 2004

The concept that 7 transmembrane receptors (7TMRs) exist and function as independent monomers has... more The concept that 7 transmembrane receptors (7TMRs) exist and function as independent monomers has facilitated a therapeutic approach of selective targeting of receptors. However, oligomerisation of 7TMRs is now being recognised as an important biological phenomenon that adds a level of complexity to their signalling. In vitro, many 7TMR heterodimers display altered binding, signalling, and trafficking properties compared to their constituent monomeric units. This review discusses an emerging concept regarding the role of 7TMR heterodimerisation in vivo, and its significance to drug therapy. Studies of angiotensin receptor signalling indicate a pivotal role for heterodimerisation in the pathogenesis of human disorders. Furthermore, the occurrence of angiotensin receptor heterodimers has a profound effect on the responsiveness to treatment with 7TMR blockers. Global assessment of the prevalence of different heterodimers during disease and their responsiveness to drug therapy is likely to optimise patient treatment and reduce side-effects associated with 7TMR blockers.

Research paper thumbnail of Functional and quantitative measures of receptor-coupled G proteins in human mononuclear leukocytes: No change with age

Experimental …, 1996

Aging has been associated with alterations in signal transduction for a number of neurotransmitte... more Aging has been associated with alterations in signal transduction for a number of neurotransmitter receptors in human tissues. Heterotrimeric G proteins play a pivotal role in postreceptor information transduction, by coupling a variety of hormone and neurotransmitter receptors to several ...

Research paper thumbnail of Down-regulation of cyclooxygenase-2 by the carboxyl tail of the angiotensin II type 1 receptor

The Journal of biological chemistry, Jan 7, 2014

The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the pr... more The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT1) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT1 also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or β-arrestin-dependent signaling. Instead, AT1 enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequen...

Research paper thumbnail of Underwater trauma causes a long-term specific increase in the expression of cyclooxygenase-2 in the ventral CA1 of the hippocampus

Psychoneuroendocrinology, 2014

The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal ne... more The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal neurons, but its role in emotional trauma is not known. Here we show that a single acute stress caused by a near-drowning experience results in heightened anxiety-like behavior one month after the trauma. Biochemical analyses of dorsal and ventral hippocampal CA1, CA3 and dentate gyrus revealed decreased ubiquitination and elevated levels of COX-2 in the traumatized animals only in the ventral CA1. To reveal the identity of the ubiquitin E3 ligase that targets COX-2, we tested the effect of several representative E3 ligases on COX-2 expression in vitro. We found that while AIP4 and Nedd4 had no effect, Mdm2 lowered COX-2 expression by nearly 50%, an effect that was not observed by its dominant negative form. To test whether this also occurs in the hippocampus, we immunoprecipitated Mdm2 from dorsal and ventral CA1 of traumatized and control animals and probed for the presence of COX-2. Our results showed that the levels of Mdm2 were not affected by the trauma but there was significantly less COX-2 associated with Mdm2 in the ventral but not dorsal CA1 of the traumatized animals. Together these data propose that an increase in COX-2 expression in ventral CA1 following trauma is likely due to its attenuated degradation. Unraveling the pathways and mechanisms that control hippocampal COX-2 degradation is important to boost the development of novel therapeutic approaches designed to treat stress-related pathologies.

Research paper thumbnail of Receptor-Signaling Pathways in Heart Failure

Principles of Molecular Cardiology, 2005

Heart failure, a progressive disorder characterized by deterioration of cardiac function and prem... more Heart failure, a progressive disorder characterized by deterioration of cardiac function and premature myocardial cell death, results from several common heart diseases such as coronary atherosclerosis, hypertension, and valvular diseases (1,2). With almost 550,000 new cases diagnosed each year, heart failure affects an estimated 4.7 million Americans, and costs associated with the disease range from 10 billion to10 billion to 40 billion per year (3). The aggregate 5-year mortality of patients with heart failure is about 50%, while the 1-year mortality of patients with advanced disease may exceed 50% (3). To maintain adequate myocardial contractility in response to injury, the heart uses several mechanisms, including hypertrophy of myocardial cells, changing the energetics of myocardial cell contraction, and upregulating transcription of several genes (4,5).

Research paper thumbnail of β1-Adrenergic receptor downregulates the expression of cyclooxygenase-2

Biochemical and Biophysical Research Communications, 2014

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the generation of prostanoids, and i... more Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the generation of prostanoids, and is thus one of the key players in the inflammatory process. Contrary to the constitutively expressed isoform COX-1, the expression of COX-2 is rapidly and transiently upregulated following pathological stimuli but little is known about pathways that mediate its degradation. Here we show that co-expression of COX-2 together with the β1 adrenergic receptor (β1AR) specifically lowers the expression of COX-2 in a dose-dependent manner. We further find that stimulation of the receptor for prolonged periods of time does not reverse the β1AR-induced decrease in COX-2, suggesting that this effect does not occur via classical β1-mediated signaling pathways. Rather we find that the half-life of COX-2 is significantly decreased in the presence of β1AR and that inhibition of the proteasome reverses the effect of the receptor on COX-2. Together these findings ascribe a new role for β1AR in the downregulation of COX-2.

Research paper thumbnail of Lessons From Overexpressed Mouse Models

The Adrenergic Receptors, 2006

ABSTRACT

Research paper thumbnail of Tyrosine Phosphorylation of the 2B Subunit of the NMDA Receptor Is Necessary for Taste Memory Formation

Journal of Neuroscience, 2009

We aimed to test whether tyrosine phosphorylation of the NMDA receptor (NMDAR) in the insular cor... more We aimed to test whether tyrosine phosphorylation of the NMDA receptor (NMDAR) in the insular cortex is necessary for novel taste learning. We found that in rats, novel taste learning leads to elevated phosphorylation of tyrosine 1472 of the NR2B subunit of the NMDAR and increases the interaction of phosphorylated NR2B with the major postsynaptic scaffold protein PSD-95. Injection of the tyrosine kinase inhibitor genistein directly into the insular cortex of rats before novel taste exposure prevented the increase in NR2B tyrosine phosphorylation and behaviorally attenuated taste-memory formation. Functionally, tyrosine phosphorylation of NR2B after learning was found to determine the synaptic distribution of the NMDAR, since microinjection of genistein to the insular cortex altered the distribution pattern of NMDAR caused by novel taste learning.

Research paper thumbnail of Elevated dopamine receptor-coupled Gs protein measures in mononuclear leukocytes of patients with schizophrenia

Schizophrenia Research, 2001

Heterotrimeric G proteins play a pivotal role in post-receptor information transduction and were ... more Heterotrimeric G proteins play a pivotal role in post-receptor information transduction and were previously implicated in the pathophysiology and treatment of mood disorders. Changes previously detected in G protein levels in post-mortem brain of patients with schizophrenia could reflect effects of antipsychotic medication. The present study aims at quantitatively and functionally evaluating receptor-coupled G proteins in mononuclear leukocytes obtained from 23 untreated patients with schizophrenia and 30 healthy subjects in an attempt to unravel a pattern of G protein measures in schizophrenia distinctive from patterns previously obtained in mood disorders. Dopamineenhanced guanine nucleotide binding capacity to G s protein through D 1 /D 5 receptor in mononuclear leukocytes of untreated patients with schizophrenia was significantly increased in comparison with healthy subjects, and positively correlated with both the total PANSS score and the positive subscale. b-Adrenergic and muscarinic receptor-coupled G protein functions, as well as G s a, G i a and Gb immunoreactivities, were similar to healthy subjects. These findings, distinctive for schizophrenia, unrelated to drug treatment, and differential from previous findings in mania and depression, may potentially help to differentially diagnose, after the first psychotic episode, between the major psychoses: schizophrenia and manic-depressive illness.

Research paper thumbnail of Brain region-specific methylation in the promoter of the murine oxytocin receptor gene is involved in its expression regulation

Psychoneuroendocrinology, 2014

Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsi... more Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsion and breast-feeding, and has been recently implicated in mammalian social behavior. The actions of both central and peripheral oxytocin are mediated through the oxytocin receptor (Oxtr), which is encoded by a single gene. In contrast to the highly conserved expression of oxytocin in specific hypothalamic nuclei, the expression of its receptor in the brain is highly diverse among different mammalian species or even within individuals of the same species. The diversity in the pattern of brain Oxtr expression among mammals is thought to contribute to the broad range of social systems and organizations. Yet, the mechanisms underlying this diversity are poorly understood. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression levels of the Oxtr in individuals with autism. Here we hypothesize that DNA methylation is involved in the expression regulation of Oxtr in the mouse brain. By combining bisulfite DNA conversion and Next-Generation Sequencing we found that specific CpG sites are differentially methylated between distinct brain regions expressing different levels of Oxtr mRNA. Some of these CpG sites are located within putative binding sites of transcription factors known to regulate Oxtr expression, including estrogen receptor a (ERa) and SP1. Specifically, methylation of the SP1 site was found to positively correlate with Oxtr expression. Furthermore, we revealed that the methylation levels of these sites in the various brain regions predict the relationship between ERa and Oxtr mRNA levels. Collectively, our results suggest that brain region-specific expression of the mouse Oxtr gene is epigenetically regulated by DNA methylation of its promoter. #

Research paper thumbnail of Upregulation of Prostaglandin Receptor EP1 Expression Involves Its Association with Cyclooxygenase-2

PLoS ONE, 2014

While many signals cause upregulation of the pro-inflammatory enzyme cyclooxygenase -2 (COX-2), m... more While many signals cause upregulation of the pro-inflammatory enzyme cyclooxygenase -2 (COX-2), much less is known about mechanisms that actively downregulate its expression. We have recently shown that the prostaglandin EP1 receptor reduces the expression of COX-2 in a pathway that facilitates its ubiquitination and degradation via the 26S proteasome. Here we show that an elevation of COX-2 intracellular levels causes an increase in the endogenous expression of prostaglandin EP1. The increase in EP1 levels does not occur at the transcriptional level, but is rather associated with complex formation between the receptor and COX-2, which occurs both in vitro and in mammalian tissues. The EP1-COX-2 complex is disrupted following binding of arachidonic acid to COX-2 and accompanied by a parallel reduction in EP1 levels. We propose that a transient interaction between COX-2 and EP1 constitutes a feedback loop whereby an increase in COX-2 expression elevates EP1, which ultimately acts to downregulate COX-2 by expediting its proteasomal degradation. Such a post translational mechanism may serve to control both the ligand-generating system of COX-2 and its reception system.

Research paper thumbnail of  -Arrestins: Multifunctional Cellular Mediators

Physiology, 2008

Initially thought to play a role only in G-protein-coupled receptor desensitization, ␤-arrestins ... more Initially thought to play a role only in G-protein-coupled receptor desensitization, ␤-arrestins are ascribed with new roles such as scaffolding and signaling proteins by their own right. This review explores the many functions of ␤-arrestins, with an emphasis on their recently identified role as regulators of receptor signaling.

Research paper thumbnail of ERK-dependent PSD-95 induction in the gustatory cortex is necessary for taste learning, but not retrieval

Nature Neuroscience, 2008

The processes underlying long-term memory formation in the neocortex are poorly understood. Using... more The processes underlying long-term memory formation in the neocortex are poorly understood. Using taste learning, we found learning-related induction of PSD-95 in the gustatory cortex, which was temporally restricted, coupled to the learning of a novel, but not familiar, taste and controlled by ERK. Using temporally and spatially restricted RNA interference knockdown of PSD-95 in vivo, we found that PSD-95 induction is necessary for learning novel tastes, but not for the recollection of familiar ones.

Research paper thumbnail of Sensing heart stress

Research paper thumbnail of Ectodomain Shedding-Dependent Transactivation of Epidermal Growth Factor Receptors in Response to Insulin-Like Growth Factor Type I

Molecular Endocrinology, 2004

Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growt... more Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R ؊ cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R ؉ cells), stimulation of R ؉ cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R ؊ cells. The R ؊ cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R ؉ cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner. (Molecular Endo-

Research paper thumbnail of Bradykinin Induced Mitogenesis of Androgen Independent Prostate Cancer Cells

The Journal of Urology, 2001

We investigated the mitogenic role of bradykinin (BK) in the regulation of the extracellular sign... more We investigated the mitogenic role of bradykinin (BK) in the regulation of the extracellular signal regulated kinase 1 and 2 (ERK), and the growth of androgen insensitive prostate cancer cells. Androgen insensitive PC3 cells were used in these studies. BK and epidermal growth factor were used as mitogens. The chemical inhibitors tyrphostin AG1478 (epidermal growth factor receptor inhibitor), bisindolylmaliemide (protein kinase C inhibitor) and PD98059 (MEK inhibitor) were applied 30 minutes before stimulation with agonist. Down-regulation of protein kinase C was accomplished by incubating cells overnight with phorbol ester. Cell proliferation was measured using WST-1 reagent and the trypan blue exclusion assay. ERK expression and activation were assayed by immunoblotting for total and phosphorylated ERK. Bradykinin induced the proliferation of PC3 cells in a pathway that requires the activation of ERK. The BK regulated activation of ERK was time and dose dependent, yielding a maximal response at the same concentration range that elicits cellular growth. BK exerted its effect on ERK activation via a protein kinase C and epidermal growth factor receptor dependent pathway. Inhibition of the kinase activity of protein kinase C or epidermal growth factor receptor eliminated BK induced ERK activation. Furthermore, the inhibition of epidermal growth factor receptor transactivation abolished BK induced cell proliferation. Our results show that BK induces the proliferation of androgen insensitive prostate cancer cells and suggest a possible pathophysiological role for BK in prostate cancer.

Research paper thumbnail of Guanosine Phosphate Binding Protein Coupled Receptors in Prostate Cancer: A Review

The Journal of Urology, 2002

Androgens are the primary growth promoters of the prostate gland and yet prostate tumors progress... more Androgens are the primary growth promoters of the prostate gland and yet prostate tumors progress despite androgen ablation. This progression suggests a role for additional cellular factors in the progression to androgen independent disease. We examined the role of a family of extracellular signal regulators, namely the guanosine phosphate binding (G) protein coupled receptor (GPCR) family, in prostate cancer. A comprehensive review of the literature was performed on GPCRs and prostate cancer, and supplemented with published and unpublished observations made at our laboratory. Emphasis was placed on the mechanistic aspects of mitogenic signaling pathways involved to identify potential targets for therapy. Expression of some GPCRs and GPCR ligands is elevated in prostate cancer cells and adjacent prostatic stromal tissue. In vitro studies demonstrate that activation of GPCRs confers a distinct growth and survival advantage on prostate cancer cells, including enhanced proliferation and decreased programmed cell death (apoptosis). Specifically stimulation of GPCRs for lysophosphatidic acid and bradykinin induces proliferation of androgen independent prostate cancer cells via the activation of the extracellular signal regulated kinase (ERK) pathway. Induction of ERK by the bradykinin and lysophosphatidic acid in prostate cells proceeds via distinct pathways and involves Galphaq and Gbetagamma subunits, respectively. The Gbetagamma dependent activation of ERK requires tyrosine kinases, including epidermal growth factor receptor and c-Src. Furthermore, stimulation with LPA enhances the survival of prostate cancer cells via activation of the inducible transcription factor nuclear factor-kappaB. GPCR stimulation induces proliferation and prevents apoptosis of hormone independent prostate cancer cells, indicating their important role in the progression of prostate cancer. While further confirmatory studies are required to verify the role of GPCRs in disease progression, the therapeutic implications of these studies may enhance the armamentarium in the fight against prostate cancer.

Research paper thumbnail of Inhibition of exocytosis or endocytosis blocks activity-dependent redistribution of synapsin

Journal of Neurochemistry, 2012

Synaptic vesicles are pre-synaptic organelles which release their neurotransmitter content onto t... more Synaptic vesicles are pre-synaptic organelles which release their neurotransmitter content onto the post-synaptic neuron by exocytosis, followed by their local reconstitution by endocytosis, in what is known as the synaptic vesicle cycle . A minority of vesicles constitute the readily releasable pool (RRP) of vesicles which are immediately ready for exocytosis. The rest are either recycling vesicles, which can be recruited rapidly, or they belong to the reserve or resting pool, meaning that mobilizing them for eventual use requires the investment of substantially more time and effort (Denker and Rizzoli 2010). The interplay between the pools and the kinetics of the cycle determine the overall release dynamics of the terminal (Pan and

Research paper thumbnail of β-Arrestin–mediated β1-adrenergic receptor transactivation of the EGFR confers cardioprotection

Journal of Clinical Investigation, 2007

Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), membe... more Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from G s -dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β 1 AR signaling to the EGFR. This β-arrestin-dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein-coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin-mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

Research paper thumbnail of Protein Kinase A and G Protein-coupled Receptor Kinase Phosphorylation Mediates  -1 Adrenergic Receptor Endocytosis through Different Pathways

Journal of Biological Chemistry, 2003

Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled rece... more Agonist-induced phosphorylation of beta-adrenergic receptors (beta ARs) by G protein-coupled receptor kinases (GRKs) results in their desensitization followed by internalization. Whether protein kinase A (PKA)-mediated phosphorylation of beta ARs, particularly the beta 1AR subtype, can also trigger internalization is currently not known. To test this, we cloned the mouse wild type beta 1AR (WT beta 1AR) and created 3 mutants lacking, respectively: the putative PKA phosphorylation sites (PKA-beta 1AR), the putative GRK phosphorylation sites (GRK-beta 1AR), and both sets of phosphorylation sites (PKA-/GRK-beta 1AR). Following agonist stimulation, both PKA-beta 1AR and GRK-beta 1AR mutants showed comparable increases in phosphorylation and desensitization. Saturating concentrations of agonist induced only 50% internalization of either mutant compared with wild type, suggesting that both PKA and GRK phosphorylation of the receptor contributed to receptor sequestration in an additive manner. Moreover, in contrast to the WT beta 1AR and PKA-beta 1AR, sequestration of the GRK-beta 1AR and PKA-/GRK-beta 1AR was independent of beta-arrestin recruitment. Importantly, clathrin inhibitors abolished agonist-dependent internalization for both the WT beta 1AR and PKA-beta 1AR, whereas caveolae inhibitors prevented internalization only of the GRK-beta 1AR mutant. Taken together, these data demonstrate that: 1) PKA-mediated phosphorylation can trigger agonist-induced internalization of the beta 1AR and 2) the pathway selected for beta 1AR internalization is primarily determined by the kinase that phosphorylates the receptor, i.e. PKA-mediated phosphorylation directs internalization via a caveolae pathway, whereas GRK-mediated phosphorylation directs it through clathrin-coated pits.