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1.
Vitam Horm ; 104: 263-311, 2017.
Article in English | MEDLINE | ID: mdl-28215298

ABSTRACT

Age-related neurodegenerative diseases, such as Alzheimer's disease, will represent one of the largest future burdens on worldwide healthcare systems due to the increasing proportion of elderly in our society. As deficiencies in neurotrophins are implicated in the pathogenesis of many age-related neurodegenerative disorders, it is reasonable to consider that global neurotrophin resistance may also become a major healthcare threat. Central nervous system networks are effectively maintained through aging by neuroprotective and neuroplasticity signaling mechanisms which are predominantly controlled by neurotrophin receptor signaling. Neurotrophin receptors are single pass receptor tyrosine kinases that form dimeric structures upon ligand binding to initiate cellular signaling events that control many protective and plasticity-related pathways. Declining functionality of the neurotrophin ligand-receptor system is considered one of the hallmarks of neuropathological aging. Therefore, it is imperative to develop effective therapeutic strategies to contend with this significant issue. While the therapeutic applications of cognate ligands for neurotrophin receptors are limited, the development of nonpeptidergic, small-molecule ligands can overcome these limitations, and productively regulate this important receptor system with beneficial effects. Using our advanced knowledge of the high-dimensionality complexity of receptor systems, the future generation of precision medicines targeting these systems will be an attainable goal.


Subject(s)
Drug Design , Drugs, Investigational/therapeutic use , Nerve Growth Factors/therapeutic use , Neurodegenerative Diseases/drug therapy , Protein Precursors/therapeutic use , Receptors, Nerve Growth Factor/agonists , Animals , Central Nervous System/drug effects , Central Nervous System/growth & development , Central Nervous System/metabolism , Cognitive Dysfunction/drug therapy , Cognitive Dysfunction/metabolism , Cognitive Dysfunction/prevention & control , Dimerization , Drugs, Investigational/chemistry , Humans , Ligands , Molecular Targeted Therapy , Nerve Growth Factors/chemistry , Nerve Growth Factors/metabolism , Neurodegenerative Diseases/metabolism , Neuronal Plasticity/drug effects , Neurons/drug effects , Neurons/metabolism , Neuroprotection/drug effects , Neuroprotective Agents/chemistry , Neuroprotective Agents/metabolism , Neuroprotective Agents/therapeutic use , Nootropic Agents/chemistry , Nootropic Agents/metabolism , Nootropic Agents/therapeutic use , Protein Precursors/chemistry , Protein Precursors/metabolism , Receptors, Nerve Growth Factor/metabolism , Signal Transduction/drug effects
2.
J Mol Graph Model ; 49: 38-46, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24491483

ABSTRACT

SIRT6 is a histone deacetylase that has been proposed as a potential therapeutic target for metabolic disorders and the prevention of age-associated diseases. We have previously reported on the identification of quercetin and vitexin as SIRT6 inhibitors, and studied structurally related flavonoids including luteolin, kaempferol, apigenin and naringenin. It was determined that the SIRT6 protein remained active after immobilization and that a single frontal displacement could correctly predict the functional activity of the immobilized enzyme. The previous study generated a preliminary pharmacophore for the quercetin binding site on SIRT6, containing 3 hydrogen bond donors and one hydrogen bond acceptor. In this study, we have generated a refined pharmacophore with an additional twelve quercetin analogs. The resulting model had a positive linear behavior between the experimental elution time verses the fit values obtained from the model with a correlation coefficient of 0.8456.


Subject(s)
Quercetin/chemistry , Quercetin/metabolism , Sirtuins/chemistry , Sirtuins/metabolism , Binding Sites , Humans , Hydrogen Bonding
3.
Mini Rev Med Chem ; 12(9): 831-40, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22681251

ABSTRACT

Pharmacotherapeutic targeting of G protein-coupled receptors (GPCRs) is perhaps the most important field of drug design, as agents designed to control these receptors constitute more than half of the pharmacopeia. Initially GPCRs were considered to be unitary entities, possessing all of their potential functionality in their characteristic heptahelical core. Early models of the functional activity of GPCRs considered them to possess just a simple 'on' or 'off' status. Recent research however has allowed us to realize that GPCR functionality is dependent upon many other proteins outside of the heptahelical core, on the site of GPCR expression in a tissue or a microdomain in a cell, and, most importantly, on the formation of differential 'active' states preferentially coupled to specific signal transduction structures. The recognition of such signaling diversity has facilitated the ability to appreciate and identify ligands for GPCRs that demonstrate a bias towards one signaling form of a receptor to another. However while potentially increasing our ability for selective signal targeting, our approach to understanding the physiological ramifications of systemic signaling manipulation is underdeveloped. This explosion in the complexity of GPCR signaling is now becoming familiar territory to receptor biologists, yet the application of this knowledge to drug design is relatively limited. This review will attempt to outline potential pitfalls and unseen benefits of using signaling bias in therapeutic design as well as highlighting new applications such as Game Theory for uncovering new therapeutic applications for biased agonists.


Subject(s)
Biological Products/pharmacology , Receptors, G-Protein-Coupled/physiology , Signal Transduction/physiology , Drug Design , Feedback, Physiological/drug effects , Feedback, Physiological/physiology , Game Theory , Humans , Ligands , Molecular Targeted Therapy , Protein Conformation , Receptors, G-Protein-Coupled/agonists , Receptors, G-Protein-Coupled/antagonists & inhibitors , Signal Transduction/drug effects
4.
Int J Obes (Lond) ; 35(5): 714-27, 2011 May.
Article in English | MEDLINE | ID: mdl-20921964

ABSTRACT

BACKGROUND: The problems of adherence to energy restriction in humans are well known. OBJECTIVE: To compare the feasibility and effectiveness of intermittent continuous energy (IER) with continuous energy restriction (CER) for weight loss, insulin sensitivity and other metabolic disease risk markers. DESIGN: Randomized comparison of a 25% energy restriction as IER (∼ 2710 kJ/day for 2 days/week) or CER (∼ 6276 kJ/day for 7 days/week) in 107 overweight or obese (mean (± s.d.) body mass index 30.6 (± 5.1) kg m(-2)) premenopausal women observed over a period of 6 months. Weight, anthropometry, biomarkers for breast cancer, diabetes, cardiovascular disease and dementia risk; insulin resistance (HOMA), oxidative stress markers, leptin, adiponectin, insulin-like growth factor (IGF)-1 and IGF binding proteins 1 and 2, androgens, prolactin, inflammatory markers (high sensitivity C-reactive protein and sialic acid), lipids, blood pressure and brain-derived neurotrophic factor were assessed at baseline and after 1, 3 and 6 months. RESULTS: Last observation carried forward analysis showed that IER and CER are equally effective for weight loss: mean (95% confidence interval ) weight change for IER was -6.4 (-7.9 to -4.8) kg vs -5.6 (-6.9 to -4.4) kg for CER (P-value for difference between groups = 0.4). Both groups experienced comparable reductions in leptin, free androgen index, high-sensitivity C-reactive protein, total and LDL cholesterol, triglycerides, blood pressure and increases in sex hormone binding globulin, IGF binding proteins 1 and 2. Reductions in fasting insulin and insulin resistance were modest in both groups, but greater with IER than with CER; difference between groups for fasting insulin was -1.2 (-1.4 to -1.0) µU ml(-1) and for insulin resistance was -1.2 (-1.5 to -1.0) µU mmol(-1) l(-1) (both P = 0.04). CONCLUSION: IER is as effective as CER with regard to weight loss, insulin sensitivity and other health biomarkers, and may be offered as an alternative equivalent to CER for weight loss and reducing disease risk.


Subject(s)
Caloric Restriction , Insulin Resistance , Metabolic Syndrome/therapy , Overweight/therapy , Weight Loss , Adult , Biomarkers/metabolism , Breast Neoplasms/prevention & control , Cardiovascular Diseases/prevention & control , Feasibility Studies , Female , Humans , Metabolic Syndrome/metabolism , Middle Aged , Overweight/metabolism , Patient Compliance/statistics & numerical data , Risk Factors
5.
Genes Brain Behav ; 9(5): 512-8, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20345896

ABSTRACT

Brain-derived neurotrophic factor (BDNF) regulates synaptic plasticity and neurogenesis, and BDNF plasma and serum levels have been associated with depression, Alzheimer's disease, and other psychiatric and neurodegenerative disorders. In a relatively large community sample, drawn from the Baltimore Longitudinal Study of Aging (BLSA), we examine whether BDNF plasma concentration is associated with the Val66Met functional polymorphism of the BDNF gene (n = 335) and with depression-related personality traits assessed with the NEO-PI-R (n = 391). Plasma concentration of BDNF was not associated with the Val66Met variant in either men or women. However, in men, but not in women, BDNF plasma level was associated with personality traits linked to depression. Contrary to the notion that low BDNF is associated with negative outcomes, we found lower plasma levels in men who score lower on depression and vulnerability to stress (two facets of Neuroticism) and higher on Conscientiousness and Extraversion. These findings challenge the prevailing hypothesis that lower peripheral levels of BDNF are a marker of depression.


Subject(s)
Brain-Derived Neurotrophic Factor/blood , Brain-Derived Neurotrophic Factor/genetics , Depression/blood , Personality/genetics , Adult , Aged , Aged, 80 and over , Aging/physiology , Biomarkers/blood , Depression/genetics , Female , Genetic Variation , Humans , Male , Middle Aged , Personality/physiology , Polymorphism, Single Nucleotide , Reference Values , Sex Factors , Statistics, Nonparametric
6.
Curr Med Chem ; 16(35): 4668-78, 2009.
Article in English | MEDLINE | ID: mdl-19903142

ABSTRACT

Coordinated and constructive physical activity is correlated with the maintenance of cognitive function in humans. Voluntary running also enhances neuroplasticity in adult and aging rodents, but the molecular pathways underlying these effects are still being elucidated. Considering the multifactorial nature of the biochemical links between physical activity and neurophysiology it is likely that there are many pharmacological mechanisms by which the beneficial actions of exercise can be effectively reproduced using chemical agents. Most studies to date have focused on brain-derived neurotrophic factor (BDNF) as a signaling target for the enhancement of neuronal function by exercise. The goal of the current review is to move beyond BDNF by exploring the diversity of molecular pathways regulated by physical activity in a variety of situations. We will discuss the availability and mechanism of action for several diverse physical activity pharmacomimetics. As physical activity enhances both neuroplasticity and cognition, understanding the molecular targets for these effects may lead to the development of protent new therapeutic interventions for age-related neurodegenerative conditions such as Alzheimer's disease.


Subject(s)
Brain-Derived Neurotrophic Factor/metabolism , Exercise , Hippocampus/metabolism , Neuroprotective Agents/metabolism , Brain-Derived Neurotrophic Factor/agonists , Glucocorticoids/metabolism , Hippocampus/drug effects , Humans , Insulin-Like Growth Factor I/metabolism , Mitochondria/metabolism , Neuroprotective Agents/pharmacology , Nitric Oxide/metabolism , Signal Transduction , Wnt Proteins/metabolism
7.
J Neuroendocrinol ; 15(9): 831-9, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12899677

ABSTRACT

Gonadotropin-releasing hormone (GnRH)-II stimulates luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion when administered at high doses in mammals, and this effect has been assumed to be mediated through the GnRH-II receptor expressed on gonadotropes. This study used two selective GnRH-I receptor antagonists to test the alternative hypothesis that GnRH-II acts through the GnRH-I receptor to elicit gonadotropin secretion. The antagonist, antide, was used to characterize the receptor-relay because it was a pure antagonist in vitro based on inositol phosphate responses in COS-7 cells transfected with either mammalian GnRH-I and GnRH-II receptors and, in vivo, potently antagonized the gonadotropin-releasing effect of a single injection of 250 ng GnRH-I in our sexually inactive sheep model. In a series of studies in sheep, antide (i). blocked the acute LH response to a single injection of GnRH-II (20 microg antide: 10 microg GnRH-II); (ii). blocked both the acute, pulsatile LH response and the FSH priming response to 2-hourly injections of GnRH-II over 36 h (100 microg antide/8 h: 4 microg GnRH-II/2 h); and (iii). chronically blocked both the pulsatile LH response and the marked FSH priming response to 4-hourly injections of GnRH-II over 10 days (75 microg antide/8 h: 4 microg GnRH-II/4 h). In two final experiments, the GnRH-I antagonist 135-18, shown previously to agonize the mammalian GnRH-II receptor, blocked the gonadotropin-releasing effects of GnRH-I (250 ng) but failed to elicit an LH response when given alone, and simultaneous administration of GnRH-II (250 ng) failed to alter the LH-releasing effect of GnRH-I (50-500 ng). These data thus support our hypothesis. Based on additional literature, it is unlikely that the GnRH-II decapeptide is a native regulator of the gonadotrope in mammals.


Subject(s)
Gonadotropin-Releasing Hormone/physiology , Gonadotropins/metabolism , Animals , COS Cells , Drug Interactions , Gonadotropin-Releasing Hormone/antagonists & inhibitors , Gonadotropin-Releasing Hormone/pharmacology , Hormone Antagonists/pharmacology , Male , Oligopeptides/pharmacology , Protein Isoforms/antagonists & inhibitors , Protein Isoforms/pharmacology , Protein Isoforms/physiology , Receptors, LHRH/antagonists & inhibitors , Sheep
8.
Proc Natl Acad Sci U S A ; 98(17): 9636-41, 2001 Aug 14.
Article in English | MEDLINE | ID: mdl-11493674

ABSTRACT

Mammalian gonadotropin-releasing hormone (GnRH I: pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) stimulates pituitary gonadotropin secretion, which in turn stimulates the gonads. Whereas a hypothalamic form of GnRH of variable structure (designated type I) had been shown to regulate reproduction through a cognate type I receptor, it has recently become evident that most vertebrates have one or two other forms of GnRH. One of these, designated type II GnRH (GnRH II: pGlu-His-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2), is conserved from fish to man and is widely distributed in the brain, suggesting important neuromodulatory functions such as regulating K+ channels and stimulating sexual arousal. We now report the cloning of a type II GnRH receptor from marmoset cDNA. The receptor has only 41% identity with the type I receptor and, unlike the type I receptor, has a carboxyl-terminal tail. The receptor is highly selective for GnRH II. As with the type I receptor, it couples to G(alpha)q/11 and also activates extracellular signal-regulated kinase (ERK1/2) but differs in activating p38 mitogen activated protein (MAP) kinase. The type II receptor is more widely distributed than the type I receptor and is expressed throughout the brain, including areas associated with sexual arousal, and in diverse non-neural and reproductive tissues, suggesting a variety of functions. Surprisingly, the type II receptor is expressed in the majority of gonadotropes. The presence of two GnRH receptors in gonadotropes, together with the differences in their signaling, suggests different roles in gonadotrope functioning.


Subject(s)
Gonadotropin-Releasing Hormone/analogs & derivatives , Gonadotropin-Releasing Hormone/physiology , Receptors, LHRH/isolation & purification , Amino Acid Sequence , Animals , COS Cells , Callithrix , Chlorocebus aethiops , Cloning, Molecular , Evolution, Molecular , Expressed Sequence Tags , Female , Follicle Stimulating Hormone/metabolism , Haplorhini , Humans , Inositol Phosphates/metabolism , Luteinizing Hormone/metabolism , Male , Mice , Molecular Sequence Data , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Nerve Tissue Proteins/physiology , Nervous System/embryology , Polymerase Chain Reaction , Protein Structure, Tertiary , Receptors, LHRH/drug effects , Receptors, LHRH/genetics , Receptors, LHRH/physiology , Recombinant Fusion Proteins/metabolism , Reproduction/physiology , Sequence Alignment , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , Sexual Behavior, Animal/physiology , Sheep , Signal Transduction , Species Specificity
9.
Mol Cell Biol ; 20(22): 8352-63, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11046132

ABSTRACT

Platelet-derived growth factor (PDGF) is a potent mitogen for many cell types. The PDGF receptor (PDGFR) is a receptor tyrosine kinase that mediates the mitogenic effects of PDGF by binding to and/or phosphorylating a variety of intracellular signaling proteins upon PDGF-induced receptor dimerization. We show here that the Na(+)/H(+) exchanger regulatory factor (NHERF; also known as EBP50), a protein not previously known to interact with the PDGFR, binds to the PDGFR carboxyl terminus (PDGFR-CT) with high affinity via a PDZ (PSD-95/Dlg/Z0-1 homology) domain-mediated interaction and potentiates PDGFR autophosphorylation and extracellular signal-regulated kinase (ERK) activation in cells. A point-mutated version of the PDGFR, with the terminal leucine changed to alanine (L1106A), cannot bind NHERF in vitro and is markedly impaired relative to the wild-type receptor with regard to PDGF-induced autophosphorylation and activation of ERK in cells. NHERF potentiation of PDGFR signaling depends on the capacity of NHERF to oligomerize. NHERF oligomerizes in vitro when bound with PDGFR-CT, and a truncated version of the first NHERF PDZ domain that can bind PDGFR-CT but which does not oligomerize reduces PDGFR tyrosine kinase activity when transiently overexpressed in cells. PDGFR activity in cells can also be regulated in a NHERF-dependent fashion by stimulation of the beta(2)-adrenergic receptor, a known cellular binding partner for NHERF. These findings reveal that NHERF can directly bind to the PDGFR and potentiate PDGFR activity, thus elucidating both a novel mechanism by which PDGFR activity can be regulated and a new cellular role for the PDZ domain-containing adapter protein NHERF.


Subject(s)
Phosphoproteins/metabolism , Receptors, Platelet-Derived Growth Factor/metabolism , Amino Acid Motifs , Animals , CHO Cells , Cricetinae , Genes, Dominant , Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/genetics , Phosphorylation , Platelet-Derived Growth Factor/metabolism , Platelet-Derived Growth Factor/pharmacology , Point Mutation , Receptors, Adrenergic, beta-2/metabolism , Receptors, Platelet-Derived Growth Factor/drug effects , Receptors, Platelet-Derived Growth Factor/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sodium-Hydrogen Exchangers
10.
J Biol Chem ; 275(29): 22583-9, 2000 Jul 21.
Article in English | MEDLINE | ID: mdl-10807918

ABSTRACT

The receptor for insulin-like growth factor 1 (IGF-1) mediates multiple cellular responses, including stimulation of both proliferative and anti-apoptotic pathways. We have examined the role of cross talk between the IGF-1 receptor (IGF-1R) and the epidermal growth factor receptor (EGFR) in mediating responses to IGF-1. In COS-7 cells, IGF-1 stimulation causes tyrosine phosphorylation of the IGF-1R beta subunit, the EGFR, insulin receptor substrate-1 (IRS-1), and the Shc adapter protein. Shc immunoprecipitates performed after IGF-1 stimulation contain coprecipitated EGFR, suggesting that IGF-1R activation induces the assembly of EGFR.Shc complexes. Tyrphostin AG1478, an inhibitor of the EGFR kinase, markedly attenuates IGF-1-stimulated phosphorylation of EGFR, Shc, and ERK1/2 but has no effect on phosphorylation of IGF-1R, IRS-1, and protein kinase B (Akt). Cross talk between IGF-1 and EGF receptors is mediated through an autocrine mechanism involving matrix metalloprotease-dependent release of heparin-binding EGF (HB-EGF), because IGF-1-mediated ERK activation is inhibited both by [Glu(52)]Diphtheria toxin, a specific inhibitor of HB-EGF, and the metalloprotease inhibitor 1,10-phenanthroline. These data demonstrate that IGF-1 stimulation of the IRS-1/PI3K/Akt pathway and the EGFR/Shc/ERK1/2 pathway occurs by distinct mechanisms and suggest that IGF-1-mediated "transactivation" of EGFR accounts for the majority of IGF-1-stimulated Shc phosphorylation and subsequent activation of the ERK cascade.


Subject(s)
Adaptor Proteins, Signal Transducing , Adaptor Proteins, Vesicular Transport , ErbB Receptors/genetics , Insulin-Like Growth Factor I/genetics , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinases/genetics , Proteins/genetics , Transcriptional Activation , Animals , COS Cells , Enzyme Activation , ErbB Receptors/metabolism , Insulin-Like Growth Factor I/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Proteins/metabolism , Shc Signaling Adaptor Proteins , Signal Transduction/genetics , src Homology Domains
11.
Proc Natl Acad Sci U S A ; 97(4): 1489-94, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10677489

ABSTRACT

Acting through a number of distinct pathways, many G protein-coupled receptors (GPCRs) activate the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. Recently, it has been shown that in some cases, clathrin-mediated endocytosis is required for GPCR activation of the ERK/MAPK cascade, whereas in others it is not. Accordingly, we compared ERK activation mediated by a GPCR that does not undergo agonist-stimulated endocytosis, the alpha(2A) adrenergic receptor (alpha(2A) AR), with ERK activation mediated by the beta(2) adrenergic receptor (beta(2) AR), which is endocytosed. Surprisingly, we found that in COS-7 cells, ERK activation by the alpha(2A) AR, like that mediated by both the beta(2) AR and the epidermal growth factor receptor (EGFR), is sensitive to mechanistically distinct inhibitors of clathrin-mediated endocytosis, including monodansylcadaverine, a mutant dynamin I, and a mutant beta-arrestin 1. Moreover, we determined that, as has been shown for many other GPCRs, both alpha(2A) and beta(2) AR-mediated ERK activation involves transactivation of the EGFR. Using confocal immunofluorescence microscopy, we found that stimulation of the beta(2) AR, the alpha(2A) AR, or the EGFR each results in internalization of a green fluorescent protein-tagged EGFR. Although beta(2) AR stimulation leads to redistribution of both the beta(2) AR and EGFR, activation of the alpha(2A) AR leads to redistribution of the EGFR but the alpha(2A) AR remains on the plasma membrane. These findings separate GPCR endocytosis from the requirement for clathrin-mediated endocytosis in EGFR transactivation-mediated ERK activation and suggest that it is the receptor tyrosine kinase or another downstream effector that must engage the endocytic machinery.


Subject(s)
Endocytosis , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Receptors, Cell Surface/metabolism , Adrenergic alpha-Agonists/pharmacology , Animals , Brimonidine Tartrate , COS Cells , Clathrin/metabolism , Enzyme Inhibitors , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Fluorescent Antibody Technique , Isoproterenol/pharmacology , Phosphorylation , Quinazolines , Quinoxalines/pharmacology , Receptors, Adrenergic, alpha/metabolism , Receptors, Adrenergic, beta/metabolism , Transfection , Tyrphostins/pharmacology
12.
J Biol Chem ; 275(13): 9572-80, 2000 Mar 31.
Article in English | MEDLINE | ID: mdl-10734107

ABSTRACT

Many G protein-coupled receptors (GPCRs) activate MAP kinases by stimulating tyrosine kinase signaling cascades. In some systems, GPCRs stimulate tyrosine phosphorylation by inducing the "transactivation" of a receptor tyrosine kinase (RTK). The mechanisms underlying GPCR-induced RTK transactivation have not been clearly defined. Here we report that GPCR activation mimics growth factor-mediated stimulation of the epidermal growth factor receptor (EGFR) with respect to many facets of RTK function. beta(2)-Adrenergic receptor (beta(2)AR) stimulation of COS-7 cells induces EGFR dimerization, tyrosine autophosphorylation, and EGFR internalization. Coincident with EGFR transactivation, isoproterenol exposure induces the formation of a multireceptor complex containing both the beta(2)AR and the "transactivated" EGFR. beta(2)AR-mediated EGFR phosphorylation and subsequent beta(2)AR stimulation of extracellular signal-regulated kinase (ERK) 1/2 are sensitive to selective inhibitors of both EGFR and Src kinases, indicating that both kinases are required for EGFR transactivation. beta(2)AR-dependent signaling to ERK1/2, like direct EGF stimulation of ERK1/2 activity, is sensitive to inhibitors of clathrin-mediated endocytosis, suggesting that signaling downstream of both the EGF-activated and the GPCR-transactivated EGFRs requires a productive engagement of the complex with the cellular endocytic machinery. Thus, RTK transactivation is revealed to be a process involving both association of receptors of distinct classes and the interaction of the transactivated RTK with the cells endocytic machinery.


Subject(s)
ErbB Receptors/metabolism , Mitogen-Activated Protein Kinases/metabolism , Receptors, Adrenergic, beta-2/physiology , Animals , COS Cells , Clathrin/physiology , Endocytosis/physiology , Enzyme Activation/physiology , ErbB Receptors/genetics , Ligands , Phosphorylation , Protein Binding , Receptors, Adrenergic, beta-2/metabolism , Transcriptional Activation
13.
J Biol Chem ; 275(3): 2239-45, 2000 Jan 21.
Article in English | MEDLINE | ID: mdl-10636931

ABSTRACT

The dermatonecrotic toxin produced by Pasteurella multocida is one of the most potent mitogenic substances known for fibroblasts in vitro. Exposure to recombinant P. multocida toxin (rPMT) causes phospholipase C-mediated hydrolysis of inositol phospholipids, calcium mobilization, and activation of protein kinase C via a poorly characterized mechanism involving G(q/11) family heterotrimeric G proteins. To determine whether the regulation of G protein pathways contributes to the mitogenic effects of rPMT, we have examined the mechanism whereby rPMT stimulates the Erk mitogen-activated protein kinase cascade in cultured HEK-293 cells. Treatment with rPMT resulted in a dose and time-dependent increase in Erk 1/2 phosphorylation that paralleled its stimulation of inositol phospholipid hydrolysis. Both rPMT- and alpha-thrombin receptor- stimulated Erk phosphorylation were selectively blocked by cellular expression of two peptide inhibitors of G(q/11) signaling, the dominant negative mutant G protein-coupled receptor kinase, GRK2(K220R), and the Galpha(q) carboxyl-terminal peptide, Galpha(q)-(305-359). Like alpha-thrombin receptor-mediated Erk activation, the effect of rPMT was insensitive to the protein kinase C inhibitor GF109203X, but was blocked by the epidermal growth factor receptor-specific tyrphostin, AG1478 and by dominant negative mutants of mSos1 and Ha-Ras. These data indicate that rPMT employs G(q/11) family heterotrimeric G proteins to induce Ras-dependent Erk activation via protein kinase C-independent "transactivation" of the epidermal growth factor receptor.


Subject(s)
Bacterial Proteins , Bacterial Toxins/metabolism , Enzyme Activation , ErbB Receptors/metabolism , GTP-Binding Proteins/metabolism , Mitogen-Activated Protein Kinase Kinases/metabolism , Mitogens/metabolism , Cell Division/drug effects , Cell Line , Cyclic AMP-Dependent Protein Kinases/pharmacology , Dose-Response Relationship, Drug , Epidermal Growth Factor/pharmacology , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Gene Expression Regulation, Enzymologic , Humans , Hydrolysis , Lysophospholipids/pharmacology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinase Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Mutation , Phosphatidylinositols/metabolism , Receptors for Activated C Kinase , Receptors, Cell Surface/antagonists & inhibitors , Recombinant Proteins/metabolism , Thrombin/pharmacology , Time Factors , Transcriptional Activation , Transfection , Virulence Factors, Bordetella/metabolism , beta-Adrenergic Receptor Kinases
14.
J Biol Chem ; 275(15): 11312-9, 2000 Apr 14.
Article in English | MEDLINE | ID: mdl-10753943

ABSTRACT

beta-Arrestins can act as adapter molecules, coupling G-protein-coupled receptors to proteins involved in mitogenic as well as endocytic pathways. We have previously identified c-SRC as a molecule that is rapidly recruited to the beta2-adrenergic receptor in a beta-arrestin1-dependent manner. Recruitment of c-SRC to the receptor appears to be involved in pathways leading to receptor internalization and mitogen-activated protein kinase activation. This recruitment of c-SRC to the receptor involves an interaction between the amino-terminal proline-rich region of beta-arrestin1 and the Src homology 3 (SH3) domain of c-SRC, but deletion of the proline-rich domain does not totally ablate the interaction. We have found that a major interaction also exists between beta-arrestin1 and the catalytic or kinase domain (SH1) of c-SRC. We therefore hypothesized that a catalytically inactive mutant of the isolated catalytic subunit, SH1(kinase dead) (SH1(KD)), would specifically block those cellular actions of c-SRC that are mediated by beta-arrestin1 recruitment to the G-protein-coupled receptor. In contrast, the majority of cellular phosphorylations catalyzed by c-SRC, which do not involve interaction with the SH1 domain, would be predicted to be unaffected. The SH1(KD) mutant did indeed block beta2-adrenergic receptor internalization and receptor-stimulated tyrosine phosphorylation of dynamin, actions previously shown to be c-SRC-dependent. In contrast, SAM-68 and whole cell tyrosine phosphorylation by c-SRC was unaffected, indicating that the SH1(KD) mutant did not inhibit c-SRC tyrosine kinase activity in general. These results not only clarify the nature of the beta-arrestin1/c-SRC interaction but also implicate beta-arrestin1 as an important mediator of receptor internalization by recruiting tyrosine kinase activity to the cell surface to phosphorylate key endocytic intermediates, such as dynamin.


Subject(s)
Arrestins/physiology , Endocytosis , Protein-Tyrosine Kinases/physiology , Receptors, Adrenergic, beta-2/metabolism , CSK Tyrosine-Protein Kinase , Catalytic Domain , Cell Line , Dynamins , ErbB Receptors/physiology , GTP Phosphohydrolases/metabolism , Humans , Phosphorylation , Protein-Tyrosine Kinases/chemistry , Structure-Activity Relationship , Tyrosine/metabolism , beta-Arrestins , src Homology Domains , src-Family Kinases
15.
J Biol Chem ; 274(20): 13978-84, 1999 May 14.
Article in English | MEDLINE | ID: mdl-10318809

ABSTRACT

G protein-coupled receptors (GPCRs) initiate Ras-dependent activation of the Erk 1/2 mitogen-activated protein kinase cascade by stimulating recruitment of Ras guanine nucleotide exchange factors to the plasma membrane. Both integrin-based focal adhesion complexes and receptor tyrosine kinases have been proposed as scaffolds upon which the GPCR-induced Ras activation complex may assemble. Using specific inhibitors of focal adhesion complex assembly and receptor tyrosine kinase activation, we have determined the relative contribution of each to activation of the Erk 1/2 cascade following stimulation of endogenous GPCRs in three different cell types. The tetrapeptide RGDS, which inhibits integrin dimerization, and cytochalasin D, which depolymerizes the actin cytoskeleton, disrupt the assembly of focal adhesions. In PC12 rat pheochromocytoma cells, both agents block lysophosphatidic acid (LPA)- and bradykinin-stimulated Erk 1/2 phosphorylation, suggesting that intact focal adhesion complexes are required for GPCR-induced mitogen-activated protein kinase activation in these cells. In Rat 1 fibroblasts, Erk 1/2 activation via LPA and thrombin receptors is completely insensitive to both agents. Conversely, the epidermal growth factor receptor-specific tyrphostin AG1478 inhibits GPCR-mediated Erk 1/2 activation in Rat 1 cells but has no effect in PC12 cells. In HEK-293 human embryonic kidney cells, LPA and thrombin receptor-mediated Erk 1/2 activation is partially sensitive to both the RGDS peptide and tyrphostin AG1478, suggesting that both focal adhesion and receptor tyrosine kinase scaffolds are employed in these cells. The dependence of GPCR-mediated Erk 1/2 activation on intact focal adhesions correlates with expression of the calcium-regulated focal adhesion kinase, Pyk2. In all three cell types, GPCR-stimulated Erk 1/2 activation is significantly inhibited by the Src kinase inhibitors, herbimycin A and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-D-3,4-pyrimidine (PP1), suggesting that Src family nonreceptor tyrosine kinases represent a point of convergence for signals originating from either scaffold.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , ErbB Receptors/physiology , Mitogen-Activated Protein Kinases , Protein-Tyrosine Kinases/metabolism , Receptor Protein-Tyrosine Kinases/physiology , Animals , Enzyme Activation , Focal Adhesion Kinase 2 , Humans , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3 , Oligopeptides/metabolism , PC12 Cells , Peptide Fragments/metabolism , Phosphorylation , Quinazolines , Rats , Tyrphostins/metabolism , src-Family Kinases/antagonists & inhibitors
16.
Biochem J ; 339 ( Pt 1): 55-61, 1999 Apr 01.
Article in English | MEDLINE | ID: mdl-10085227

ABSTRACT

We have studied the effects of agonist and antagonist binding, agonist-induced activation and agonist-induced desensitization of the human tachykinin NK2 receptor mutated at polar residues Asn-51 [in transmembrane helix 1 (TM1)], Asp-79 (TM2) and Asn-303 (TM7), which are highly conserved in the transmembrane domain in the rhodopsin family of G-protein-coupled receptors. Wild-type and mutant receptors were expressed in both COS-1 cells and Xenopus oocytes. The results show that the N51D mutation results in a receptor which, in contrast with the wild-type receptor, is desensitized by the application of a concentration of 1 microM of the partial agonist GR64349, indicating that the mutant is more sensitive to agonist activation than is the wild-type receptor. In addition, we show that, whereas the D79E mutant displayed activation properties similar to those of the wild-type receptor, the D79N and D79A mutants displayed a severely impaired ability to activate the calcium-dependent chloride current. This suggests that it is the negative charge at Asn-79, rather than the ability of this residue to hydrogen-bond, that is critical for the activity of the receptor. Interestingly, the placement of a negative charge at position 303 could compensate for the removal of the negative charge at position 79, since the double mutant D79N/N303D displayed activation properties similar to those of the wild-type receptor. This suggests that these two residues are functionally coupled, and may even be in close proximity in the three-dimensional structure of the human tachykinin NK2 receptor. A three-dimensional model of the receptor displaying this putative interaction is presented.


Subject(s)
Receptors, Neurokinin-2/metabolism , Animals , Asparagine/chemistry , Asparagine/metabolism , Aspartic Acid/chemistry , Aspartic Acid/metabolism , COS Cells , Humans , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation , Receptors, Neurokinin-2/agonists , Receptors, Neurokinin-2/chemistry , Recombinant Proteins/agonists , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Xenopus
17.
Science ; 283(5402): 655-61, 1999 Jan 29.
Article in English | MEDLINE | ID: mdl-9924018

ABSTRACT

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.


Subject(s)
Arrestins/metabolism , Mitogen-Activated Protein Kinases , Proto-Oncogene Proteins pp60(c-src)/metabolism , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction , Adrenergic beta-Agonists/metabolism , Adrenergic beta-Agonists/pharmacology , Animals , Arrestins/genetics , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Line , Cell Membrane/metabolism , Enzyme Activation , GTP-Binding Proteins/metabolism , Humans , Isoproterenol/metabolism , Isoproterenol/pharmacology , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Models, Biological , Phosphorylation , Point Mutation , Precipitin Tests , Receptor Cross-Talk , Receptors, Cell Surface/metabolism , Transfection , beta-Arrestin 1 , beta-Arrestins , src Homology Domains
18.
J Biol Chem ; 274(3): 1185-8, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9880482

ABSTRACT

Some forms of G protein-coupled receptor signaling, such as activation of mitogen-activated protein kinase cascade as well as resensitization of receptors after hormone-induced desensitization, require receptor internalization via dynamin-dependent clathrin-coated pit mechanisms. Here we demonstrate that activation of beta2-adrenergic receptors (beta2-ARs) leads to c-Src-mediated tyrosine phosphorylation of dynamin, which is required for receptor internalization. Two tyrosine residues, Tyr231 and Tyr597, are identified as the major phosphorylation sites. Mutation of these residues to phenylalanine dramatically decreases the c-Src-mediated phosphorylation of dynamin following beta2-AR stimulation. Moreover, expression of Y231F/Y597F dynamin inhibits beta2-AR internalization and the isoproterenol-stimulated mitogen-activated protein kinase activation. Thus, agonist-induced, c-Src-mediated tyrosine phosphorylation of dynamin is essential for its function in clathrin mediated G protein-coupled receptor endocytosis.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , GTP Phosphohydrolases/metabolism , Microtubules/enzymology , Protein-Tyrosine Kinases/metabolism , Receptors, Adrenergic, beta-2/metabolism , Signal Transduction , Tyrosine/metabolism , src Homology Domains , CSK Tyrosine-Protein Kinase , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Cell Line , Chromatography, High Pressure Liquid , Dynamins , Enzyme Activation , Enzyme Inhibitors/pharmacology , Humans , Phosphorylation , Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effects , src-Family Kinases
19.
Br J Pharmacol ; 124(4): 675-84, 1998 Jun.
Article in English | MEDLINE | ID: mdl-9690859

ABSTRACT

1. Repeated applications of neurokinin A (NKA) to oocytes injected with 25 ng wild-type hNK2 receptor cRNA caused complete attenuation of second and subsequent NKA-induced responses while analogous experiments using repeated applications of GR64349 and [Nle10]NKA(4-10) resulted in no such desensitization. This behaviour has been previously attributed to the ability of the different ligands to stabilize different active conformations of the receptor that have differing susceptibilities to receptor kinases (Nemeth & Chollet. 1995). 2. However, for Xenopus oocytes injected (into the nucleus) with 10 ng wild-type hNK2 receptor cDNA, a single 100 nM concentration of any of the three ligands resulted in complete desensitization to further concentrations. 3. On the other hand, none of the ligands caused any desensitization in oocytes injected with 0.25 ng wild-type hNK2 receptor cRNA. even at concentrations up to 10 microM. 4. The two N-terminally truncated analogues of neurokinin A have a lower efficacy than NKA and it is likely that it is this property which causes the observed differences in desensitization, rather than the formation of alternative active states of the receptor. 5. The peak calcium-dependent chloride current is not a reliable measure of maximal receptor stimulation and efficacy is better measured in this system by studying agonist-induced desensitization. 6. The specific adenylyl cyclase inhibitor SQ22536 can enhance NKA and GR64349-mediated desensitization which suggests that agonist-induced desensitization involves the inhibition of adenylyl cyclase and the subsequent down-regulation of the cyclic AMP-dependent protein kinase, possibly by cross-talk to a second signalling pathway.


Subject(s)
Receptors, Neurokinin-2/agonists , Adenylyl Cyclase Inhibitors , Animals , Calcium/metabolism , Chloride Channels/physiology , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Female , Humans , In Vitro Techniques , Neurokinin A/analogs & derivatives , Neurokinin A/pharmacology , Oocytes , Patch-Clamp Techniques , Peptide Fragments/pharmacology , Receptors, Neurokinin-2/biosynthesis , Receptors, Neurokinin-2/physiology , Signal Transduction/drug effects , Xenopus laevis
20.
Br J Pharmacol ; 119(2): 315-20, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8886415

ABSTRACT

1. The whole-cell patch-clamp technique was used to investigate the actions of substance P and other agonists at neurokinin (NK) receptors on voltage-gated K+ and Ca+ channel currents in undifferentiated mouse neuroblastoma x rat glioma NG 108-15 cells. 2. Both substance P (0.3-30 microM) and the NK1 receptor selective agonist GR73632 (10 nM-10 microM) caused concentration-dependent inhibition of K+ currents. GR64349 and senktide (agonists at NK2 and NK3 receptors respectively) also inhibited K+ currents, but only at concentrations which were several orders of magnitude greater than GR73632, suggesting that current inhibition was mediated via NK1 receptors. 3. Substance P and GR73632 were without effect on residual K+ currents recorded in the presence of extracellular Co2+ (4 mM) to abolish the Ca(2+)-sensitive component (IKca) of the K+ current. Ca2+ channel currents, recorded with either Ba2+ or Ca2+ as charge carrier, were unaffected by NK1, NK2 and NK3 receptor ligands. 4. Iontophoretic application of GR73632 produced a current-dependent reduction of K+ currents. In the presence of the non-peptide NK1 antagonists, CP-99,994 and RP67580, and the peptide antagonist, GR82334, the current-response relationship was reversibly shifted to the right. This indicates that the response is mediated by NK1 receptors. 5. Our results indicate that activation of NK1 receptors leads to the selective inhibition of IKca in undifferentiated NG 108-15 cells.


Subject(s)
Calcium/pharmacology , Potassium Channel Blockers , Substance P/metabolism , Tachykinins/pharmacology , Animals , Calcium Channels/drug effects , Calcium Channels/physiology , Drug Interactions , Glioma/metabolism , Kinetics , Mice , Neuroblastoma/metabolism , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/pharmacology , Potassium/metabolism , Potassium Channels/physiology , Rats , Receptors, Neurokinin-1/agonists , Receptors, Neurokinin-1/physiology , Sensitivity and Specificity , Substance P/analogs & derivatives , Substance P/antagonists & inhibitors , Substance P/pharmacology , Tumor Cells, Cultured
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