IRS‐1 undergoes rapid tyrosine phosphorylation during insulin stimulation and forms a stable complex containing the 85 kDa subunit (p85) of the phosphatidylinositol (PtdIns) 3′‐kinase, but p85 is not tyrosyl phosphorylated. IRS‐1 contains nine tyrosine phosphorylation sites in YXXM (Tyr‐Xxx‐Xxx‐Met) motifs. Formation of the IRS‐1‐PtdIns 3′‐kinase complex in vitro is inhibited by synthetic peptides containing phosphorylated YXXM motifs, suggesting that the binding of PtdIns 3′‐kinase to IRS‐1 is mediated through the SH2 (src homology‐2) domains of p85. Furthermore, overexpression of IRS‐1 potentiates the activation of PtdIns 3‐kinase in insulin‐stimulated cells, and tyrosyl phosphorylated IRS‐1 or peptides containing phosphorylated YXXM motifs activate PtdIns 3′‐kinase in vitro. We conclude that the binding of tyrosyl phosphorylated IRS‐1 to the SH2 domains of p85 is the critical step that activates PtdIns 3′‐kinase during insulin stimulation.
IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It contains over 20 potential tyrosine phosphorylation sites, and we suspect that multiple insulin signals are enabled when the activated insulin receptor kinase phosphorylates several of them. Tyrosine-phosphorylated IRS-1 binds specifically to various cellular proteins containing Src homology 2 (SH2) domains (SH2 proteins). We identified some of the tyrosine residues of IRS-1 that undergo insulin-stimulated phosphorylation by the purified insulin receptor and in intact cells during insulin stimulation. Automated sequencing and manual radiosequencing revealed the phosphorylation of tyrosine residues 460, 608, 628, 895, 939, 987, 1172, and 1222; additional sites remain to be identified. Immobilized SH2 domains from the 85-kDa regulatory subunit (p85c) of the phosphatidylinositol 3'-kinase bind preferentially to tryptic phosphopeptides containing Tyr(P)-608 and Tyr(P)-939. By contrast, the SH2 domain in GRB2 and the amino-terminal SH2 domain in SHPTP2 (Syp) specifically bind to Tyr(P)-895 and Tyr(P)-1172, respectively.These results confirm that p85a recognizes YMXM motifs and suggest that GRB2 prefers a phosphorylated YVNI motif, whereas SHPTP2 (Syp) binds to a phosphorylated YIDL motif. These results extend the notion that IRS-1 is a multisite docking protein that engages various downstream regulatory elements during insulin signal transmission.IRS-1 (insulin receptor substrate 1) is a principal insulin receptor substrate that undergoes tyrosine phosphorylation during insulin stimulation. It was originally identified as a 185-kDa phosphoprotein in antiphosphotyrosine antibody (aPY) immunoprecipitates from insulin-stimulated Fao hepatoma cells (42). IRS-1 was subsequently purified and cloned from rat liver and found to be expressed in most cells and tissues (2, 28, 37). Several observations suggest that IRS-1 plays a central role in signal transduction by the insulin receptor. During differentiation of 3T3-L1 fibroblasts into insulin-sensitive adipocytes, IRS-1 expression increases coordinately with that of the insulin receptor and . Overexpression of IRS-1 in Chinese hamster ovary (CHO) cells increases insulin-stimulated mitogenesis (36), and microinjection of recombinant IRS-1 into Xenopus oocytes significantly increases their sensitivity to insulin and IGF-1 (8). Moreover, a myeloid progenitor cell (32D) which contains few insulin receptors and no detectable IRS-1 is completely insensitive to insulin even after expression of the insulin receptor; however, expression of IRS-1 together with the insulin receptor restores a normal insulin mitogenic response (41). Thus, IRS-1 may be an essential element for certain insulin responses.During ligand binding, insulin and other growth factor receptors undergo autophosphorylation on specific tyrosine residues (16). Most evidence suggests that the tyrosine kinase is essential for biological activity. In the case of the epide...
IntroductionInsulin rapidly stimulates tyrosine phosphorylation ofa protein of -185 kD in most cell types. This protein, termed insulin receptor substrate-I (IRS-1), has been implicated in insulin signal transmission based on studies with insulin receptor mutants. In the present study we have examined the levels of IRS-1 and the phosphorylation state ofinsulin receptor and IRS-1 in liver and muscle after insulin stimulation in vivo in two rat models of insulin resistance, i.e., insulinopenic diabetes and fasting, and a mouse model of non-insulin-dependent diabetes mellitus (ob/ob) by immunoblotting with anti-peptide antibodies to IRS-1 and anti-phosphotyrosine antibodies. As previously described, there was an increase in insulin binding and a parallel increase in insulin-stimulated receptor phosphorylation in muscle of fasting and streptozotocin-induced (STZ) diabetic rats. There was also a modest increase in overall receptor phosphorylation in liver in these two models, but when normalized for the increase in binding, receptor phosphorylation was decreased, in liver and muscle of STZ diabetes and in liver of 72 h fasted rats. In the hyperinsulinemic ob/ob mouse there was a decrease in insulin binding and receptor phosphorylation in both liver and muscle. The tyrosyl phosphorylation of IRS-1 after insulin stimulation reflected an amplification of the receptor phosphorylation in liver and muscle of hypoinsulinemic animals (fasting and STZ diabetes) with a twofold increase, and showed a significant reduction ('-50%) in liver and muscle of ob/ob mouse. By contrast, the levels of IRS-1 protein showed a tissue specific regulation with a decreased level in muscle and an increased level in liver in hypoinsulinemic states of insulin resistance, and decreased levels in liver in the hyperinsulinemic ob/ob mouse. These data indicate that: (a) IRS-1 protein levels are differentially regulated in liver and muscle; (b) insulin levels may play a role in this differential regulation of IRS-1; (c) IRS-1 phosphorylation depends more on insulin receptor kinase activity than IRS-1 protein levels; and (d) Insulin resistance is characteristic of many disease states including non-insulin-dependent diabetes mellitus (NIDDM),' uncontrolled insulin-dependent diabetes, obesity, and prolonged fasting (1-4). Although various defects in insulin action have been reported in these conditions, the exact mechanisms involved in the insulin resistance have not been adequately defined. Insulin initiates its metabolic and growth-promoting effects by binding to the a subunit of its tetrameric receptor (5), thereby activating the kinase in the ,B subunit, which in turn catalyzes the intramolecular autophosphorylation of specific tyrosine residues of the 13 subunit, further enhancing the tyrosine kinase activity of the receptor toward other protein substrates (5-7). Considerable evidence demonstrates that insulin receptor tyrosine kinase activity is essential for many, if not all, of the biological effects of insulin ( 8-10).Although many ...
Phosphatidylinositol 3-kinase (PI-3 kinase) is implicated in the regulation of diverse cellular processes, including insulin-stimulated glucose transport. PI-3 kinase is composed of a 110-kDa catalytic subunit and an 85-kDa regulatory subunit. Here, we describe p55 PIK , a new regulatory subunit that was isolated by screening expression libraries with tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1). p55PIK is composed of a unique 30-residue NH 2 terminus followed by a proline-rich motif and two Src homology 2 (SH2) domains with significant sequence identity to those in p85. Phosphatidylinositol 3-kinase (PI-3 kinase) is a common signaling element which plays a role in the regulation of a broad array of biological responses by activated receptors for hormones, growth factors, cytokines, and antigens (6,13,15,42,46,48,61). It is composed of a 110-kDa catalytic subunit (p110) associated with an 85-kDa regulatory subunit (p85) that contains one Src homology 3 (SH3) domain, homology to the breakpoint cluster region (bcr) gene, two proline-rich motifs, and two SH2 domains (11). Interestingly, p110␣ displays dual catalytic specificity, as it phosphorylates the D-3 position of phosphatidylinositol and its phosphorylated derivatives and serine residues in p85 and insulin receptor substrate 1 (IRS-1) (12, 32). Mammalian p110 is homologous to VPS34, a Saccharomyces cerevisiae PI-3 kinase which is involved in vacuolar protein sorting (49); however, the molecular role of PI-3 kinase in mammalian cells is unclear (22).The p85 regulatory subunit has a broad potential to couple the PI-3 kinase to multiple signaling elements by employing its SH3 domain, proline-rich motifs, bcr homology region, or SH2 domains (24). Most activated receptors with tyrosine kinase activity engage the SH2 domains in p85 through phosphorylated YXXM motifs in the receptors themselves or a closely associated subunit (52). The platelet-derived growth factor receptor, one of the best-characterized systems, associates directly with the SH2 domains in p85 at a phosphorylated YMDM motif in the kinase insert region (61). Inhibition of PI-3 kinase catalytic activity with wortmannin or disruption of p85 function by site-directed mutagenesis blocks several growth factor-stimulated processes, including mitogenesis and antiapoptosis (61, 67), differentiation (27), receptor trafficking (23), chemotaxis (31, 45), membrane ruffling (29, 64), and insulin-stimulated glucose transport (7,16,44) and Xenopus oocyte maturation (8, 9). In addition, PI-3 kinase appears to be required for the stimulation of p70S6k by platelet-derived growth factor and insulin and probably other growth factors (7,10,40). On the basis of these results, PI-3 kinase plays a central role in cellular signaling.Insulin regulates PI-3 kinase by tyrosine phosphorylation of IRS-1 and IRS-2, multipotential docking proteins which contain multiple potential tyrosine phosphorylation sites, including several YXXM motifs (41, 58). In addition to insulin and insulin-like growth factor 1 (IGF-1), ...
Aclidinium bromide is a novel potent, long-acting inhaled muscarinic antagonist in development for the treatment of chronic obstructive pulmonary disease. Aclidinium showed subnanomolar affinity for the five human muscarinic receptors (M 1 -M 5 3 H]aclidinium at the M 2 receptor was shorter than at the M 3 receptor, demonstrating kinetic selectivity for the M 3 receptor. In isolated guinea pig trachea, aclidinium showed comparable potency to ipratropium and tiotropium, faster onset of action than tiotropium, and duration of action similar to tiotropium and significantly longer than ipratropium. Nebulized aclidinium inhibited bronchoconstriction induced by acetylcholine in guinea pigs in a concentrationdependent manner with an onset of action faster than tiotropium. Duration of action of aclidinium (t 1/2 ϭ 29 h) was much longer than ipratropium (8 h) but shorter than tiotropium (64 h). In dogs, aclidinium induced a smaller and more transient increase in heart rate than tiotropium at comparable supratherapeutic doses. Therefore, under these conditions, aclidinium showed a greater therapeutic index than tiotropium (4.2 versus 1.6). These results indicate that aclidinium is a potent muscarinic antagonist with a fast onset of action, a long duration of effect, and a favorable cardiovascular safety profile.Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory disease characterized by chronic airflow obstruction attributed to long-term exposure to inhaled noxious gases and particles, most often related to cigarette smoking that is not fully reversible after bronchodilator therapy (www.goldcopd.org) (Rabe et al., 2007). Recent projections from the World Health Organization predict that COPD will become the fourth most common cause of death by 2030 and the third most common cause of chronic disability by 2020 (Lopez et al., 2006;Mathers and Loncar, 2006). Acetylcholine released by parasympathetic nerves regulates airway constriction, mucus secretion, and vasodilation through its interaction with muscarinic receptors localized in smooth muscle, mucosal glands, pulmonary vasculature, and nerve endings of the lungs (Belmonte, 2005).There are five subtypes of the muscarinic receptors, M 1 to M 5 , that are members of the superfamily of G-protein-cou-
BackgroundSensory nerves innervating the airways play an important role in regulating various cardiopulmonary functions, maintaining homeostasis under healthy conditions and contributing to pathophysiology in disease states. Hypo-osmotic solutions elicit sensory reflexes, including cough, and are a potent stimulus for airway narrowing in asthmatic patients, but the mechanisms involved are not known. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) is widely expressed in the respiratory tract, but its role as a peripheral nociceptor has not been explored.ObjectiveWe hypothesized that TRPV4 is expressed on airway afferents and is a key osmosensor initiating reflex events in the lung.MethodsWe used guinea pig primary cells, tissue bioassay, in vivo electrophysiology, and a guinea pig conscious cough model to investigate a role for TRPV4 in mediating sensory nerve activation in vagal afferents and the possible downstream signaling mechanisms. Human vagus nerve was used to confirm key observations in animal tissues.ResultsHere we show TRPV4-induced activation of guinea pig airway–specific primary nodose ganglion cells. TRPV4 ligands and hypo-osmotic solutions caused depolarization of murine, guinea pig, and human vagus and firing of Aδ-fibers (not C-fibers), which was inhibited by TRPV4 and P2X3 receptor antagonists. Both antagonists blocked TRPV4-induced cough.ConclusionThis study identifies the TRPV4-ATP-P2X3 interaction as a key osmosensing pathway involved in airway sensory nerve reflexes. The absence of TRPV4-ATP–mediated effects on C-fibers indicates a distinct neurobiology for this ion channel and implicates TRPV4 as a novel therapeutic target for neuronal hyperresponsiveness in the airways and symptoms, such as cough.
BackgroundFibroblast to myofibroblast transition is believed to contribute to airway remodelling in lung diseases such as asthma and chronic obstructive pulmonary disease. This study examines the role of aclidinium, a new long-acting muscarinic antagonist, on human fibroblast to myofibroblast transition.MethodsHuman bronchial fibroblasts were stimulated with carbachol (10−8 to 10−5 M) or transforming growth factor-β1 (TGF-β1; 2 ng/ml) in the presence or absence of aclidinium (10−9 to 10−7 M) or different drug modulators for 48 h. Characterisation of myofibroblasts was performed by analysis of collagen type I and α-smooth muscle actin (α-SMA) mRNA and protein expression as well as α-SMA microfilament immunofluorescence. ERK1/2 phosphorylation, RhoA-GTP and muscarinic receptors (M) 1, 2 and 3 protein expression were determined by western blot analysis and adenosine 3′-5′ cyclic monophosphate levels were determined by ELISA. Proliferation and migration of fibroblasts were also assessed.ResultsCollagen type I and α-SMA mRNA and protein expression, as well as percentage α-SMA microfilament-positive cells, were upregulated in a similar way by carbachol and TGF-β1, and aclidinium reversed these effects. Carbachol-induced myofibroblast transition was mediated by an increase in ERK1/2 phosphorylation, RhoA-GTP activation and cyclic monophosphate downregulation as well as by the autocrine TGF-β1 release, which were effectively reduced by aclidinium. TGF-β1 activated the non-neuronal cholinergic system. Suppression of M1, M2 or M3 partially prevented carbachol- and TGF-β1-induced myofibroblast transition. Aclidinium dose-dependently reduced fibroblast proliferation and migration.ConclusionAclidinium inhibits human lung fibroblast to myofibrobast transition.
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