RESUMEN
An analysis of the functional role of a diacidic motif (Asp236-Asp237) in the third intracellular loop of the AT1A angiotensin II (Ang II) receptor (AT1-R) revealed that substitution of both amino acids with alanine (DD-AA) or asparagine (DD-NN) residues diminished Ang II-induced receptor phosphorylation in COS-7 cells. However, Ang II-stimulated inositol phosphate production, mitogen-activated protein kinase, and AT1 receptor desensitization and internalization were not significantly impaired. Overexpression of dominant negative G protein-coupled receptor kinase 2 (GRK2)K220M decreased agonist-induced receptor phosphorylation by approximately 40%, but did not further reduce the impaired phosphorylation of DD-AA and DD-NN receptors. Inhibition of protein kinase C by bisindolylmaleimide reduced the phosphorylation of both the wild-type and the DD mutant receptors by approximately 30%. The inhibitory effects of GRK2K220M expression and protein kinase C inhibition by bisindolylmaleimide on agonist-induced phosphorylation were additive for the wild-type AT1-R, but not for the DD mutant receptor. Agonist-induced internalization of the wild-type and DD mutant receptors was similar and was unaltered by coexpression of GRK2K220M. These findings demonstrate that an acidic motif at position 236/237 in the third intracellular loop of the AT1-R is required for optimal Ang II-induced phosphorylation of its carboxyl-terminal tail by GRKs. Furthermore, the properties of the DD mutant receptor suggest that not only Ang II-induced signaling, but also receptor desensitization and internalization, are independent of agonist-induced GRK-mediated phosphorylation of the AT1 receptor.
Asunto(s)
Angiotensina I/metabolismo , Endocitosis , Receptores de Angiotensina/agonistas , Transducción de Señal , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , Humanos , Indoles/farmacología , Fosfatos de Inositol/metabolismo , Maleimidas/farmacología , Mutagénesis Sitio-Dirigida , Fosforilación , Ratas , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Receptores de Angiotensina/metabolismoRESUMEN
Desensitization and phosphorylation of the endogenous angiotensin II AT(1) receptor were studied in clone 9 liver cells. Agonist activation of AT(1) receptors blunted the response to subsequent addition of angiotensin II. Partial inhibition of the angiotensin II-induced calcium response was observed when cells were pretreated with dibutyryl cyclic AMP, tetradecanoyl phorbol acetate (TPA), vasopressin, or lysophosphatidic acid. All of these desensitization processes were associated with receptor phosphorylation. Angiotensin II-induced AT(1) receptor phosphorylation was partially blocked by the protein kinase C inhibitor bisindolylmaleimide I and by phosphoinositide 3-kinase inhibitors (wortmannin and LY294002); the actions of these inhibitors were not additive. Pertussis toxin pretreatment of cells also partially inhibited angiotensin II-induced AT(1) receptor phosphorylation. TPA-induced AT(1) receptor phosphorylation was completely blocked by bisindolylmaleimide I. AT(1) receptor phosphorylation was also induced by vasopressin and lysophosphatidic acid, and these effects were partially inhibited by bisindolylmaleimide I. Angiotensin II increased Akt/PKB (protein kinase B) phosphorylation and protein kinase C membrane association. The effect on Akt/PKB phosphorylation was blocked by phosphoinositide 3-kinase inhibitors. These findings indicate that clone 9 cells exhibit both homologous and heterologous desensitization in association with AT(1) receptor phosphorylation. In these hepatic cells, angiotensin II-induced receptor phosphorylation involves pertussis toxin-sensitive and -insensitive G proteins, and is mediated in part through protein kinase C and phosphoinositide 3-kinase.
Asunto(s)
Hepatocitos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , Receptores de Angiotensina/metabolismo , Angiotensina II/metabolismo , Animales , Calcio/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Toxina del Pertussis , Fosforilación , Ratas , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The angiotensin AT(2) receptor is an atypical seven transmembrane domain receptor that is coupled to activation of tyrosine phosphatase and inhibition of MAP kinase, and does not undergo agonist-induced internalization. An investigation of the occurrence and nature of AT(2) receptor phosphorylation revealed that phorbol ester-induced activation of protein kinase C (PKC) in HA-AT(2) receptor-expressing COS-7 cells caused rapid and specific phosphorylation of a single residue (Ser(354)) located in the cytoplasmic tail of the receptor. Agonist activation of AT(2) receptors by angiotensin II (Ang II) also caused rapid PKC-dependent phosphorylation of Ser(354) that was prevented by the AT(2) antagonist, PD123177, and by inhibitors of PKC. In cells coexpressing AT(1) and AT(2) receptors, Ang II-induced phosphorylation of the AT(2) receptor was reduced by either PD123177 or the AT(1) receptor antagonist, DuP753, and was abolished by treatment with both antagonists or with PKC inhibitors. These findings indicate that the AT(2) receptor is rapidly phosphorylated via PKC during homologous activation by Ang II, and also undergoes heterologous PKC-dependent phosphorylation during activation of the AT(1) receptor. The latter process may regulate the counteracting effects of AT(2) receptors on growth responses to AT(1) receptor activation.
Asunto(s)
Proteína Quinasa C/metabolismo , Receptores de Angiotensina/metabolismo , Animales , Células COS , Glicosilación , Oligopéptidos/farmacología , Fosforilación/efectos de los fármacos , Ratas , Receptor de Angiotensina Tipo 1 , Receptor de Angiotensina Tipo 2 , Receptores de Angiotensina/agonistasRESUMEN
The agonist-induced phosphorylation sites of the rat AT1a angiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Delta342) but was abolished by truncation at Ser325 (Delta325). Truncation at Ser335 (Delta335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by approximately 50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Delta335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Delta325 < Delta335 < ST-AA < Delta342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Delta325 > Delta335 > ST-AA = Delta342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.
Asunto(s)
Citoplasma/metabolismo , Receptores de Angiotensina/agonistas , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Células COS , Citoplasma/química , Fosfatos de Inositol/biosíntesis , Datos de Secuencia Molecular , Fosforilación , Mutación Puntual , Receptor de Angiotensina Tipo 1 , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Serina/análisis , Treonina/análisis , TransfecciónRESUMEN
In catfish (Ictalurus punctatus) hepatocytes angiotensin II induced an immediate increase in cytosolic Ca2+ concentration. Other angiotensin analogues also induced this effect including: human angiotensin II, fish angiotensin II, human angiotensin III, human angiotensin I, fish angiotensin I and saralasin. CGP 42112A induced a very small effect at the highest concentration tested and angiotensin IV was without effect. Angiotensin II also increased the resynthesis of phosphatidylinositol and the production of IP3. These physiological effects were not blocked by losartan (AT1-selective antagonist) or PD 123177 (AT2-selective antagonist). [125I]Angiotensin II bound to liver plasma membranes in a saturable fashion with high affinity (K(D) 2.7 nM) and a B(max) of 185 fmol/mg of protein. Binding competition experiments showed the following order of potency: human angiotensin II = fish angiotensin II > human angiotensin III > or = human angiotensin I = fish angiotensin I. These sites were insensitive to losartan or PD 123177. The data indicate that the angiotensin II receptors expressed in catfish hepatocytes are coupled to the phosphoinositide turnover/calcium mobilization signal transduction pathway and are atypical receptors, i.e., pharmacologically distinct from mammalian AT1 and AT2 receptors.
Asunto(s)
Calcio/metabolismo , Ictaluridae , Hígado/metabolismo , Fosfatidilinositoles/metabolismo , Receptores de Angiotensina/metabolismo , Transducción de Señal , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacología , Antagonistas de Receptores de Angiotensina , Animales , Unión Competitiva , Compuestos de Bifenilo/farmacología , Membrana Celular/metabolismo , Ácido Egtácico/farmacología , Humanos , Imidazoles/farmacología , Hígado/efectos de los fármacos , Losartán , Piridinas/farmacología , Tetrazoles/farmacologíaRESUMEN
Gly-His-Lys, a tripeptide isolated from human plasma that increases the growth rate of many cells, stimulated in dose-dependent fashion the activity of phosphorylase a in isolated rat hepatocytes. Such effect was associated to increases in both IP3 production and [Ca++]i. Interestingly, these effects of Gly-His-Lys were antagonized by losartan, a nonpeptide angiotensin II receptor antagonist (AT1 selective), which suggested that these receptors were involved in its effect. Binding competition experiments using the radioligand [125I][Sar1-Ile8]angiotensin II clearly indicated that Gly-His-Lys interacts with AT1 receptors. It was also observed that other histidine-containing tripeptides were also capable of interacting with these receptors.
Asunto(s)
Sustancias de Crecimiento/metabolismo , Hígado/metabolismo , Oligopéptidos/metabolismo , Receptores de Angiotensina/metabolismo , Transducción de Señal , 1-Sarcosina-8-Isoleucina Angiotensina II/metabolismo , Secuencia de Aminoácidos , Antagonistas de Receptores de Angiotensina , Animales , Unión Competitiva , Compuestos de Bifenilo/farmacología , Calcio/metabolismo , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Imidazoles/farmacología , Fosfatos de Inositol/metabolismo , Hígado/citología , Hígado/enzimología , Losartán , Datos de Secuencia Molecular , Fosforilasa a/análisis , Ratas , Ratas Wistar , Tetrazoles/farmacología , Vasopresinas/farmacologíaRESUMEN
In catfish hepatocytes (Ictalurus punctatus) alpha 1-adrenergic activation by epinephrine or norepinephrine increased cytosol Ca2+ concentration. This effect was inhibited by alpha 1-adrenergic antagonists with the potency order WB4101 > benoxathian > or = 5-methylurapidil > phentolamine > (+)niguldipine. Pretreatment with the irreversible antagonist, chloroethylclonidine, reduced the alpha 1-adrenergic effect. alpha 1-Adrenergic stimulation also increased the resynthesis of phosphatidylinositol in whole cells. In liver membranes, a relatively small (33 fmol/mg of protein) number of sites with high affinity (Kd 100 pM) for the radioligand [125I]HEAT was detected. Binding competition experiments showed the following orders of potency: (1) for agonists, oxymetazoline > epinephrine > or = norepinephrine > methoxamine; (2) for antagonists, prazosin > WB4101 > benoxathian > or = 5-methylurapidil > phentolamine > (+)niguldipine. Membrane pretreatment with chloroethylclonidine markedly reduced [125I]HEAT binding. Good correlation was observed between the radioligand binding studies and the functional data with isolated cells. The present data suggest that the alpha 1-adrenoceptor present in catfish liver cells belongs to the alpha 1B subtype.
Asunto(s)
Calcio/metabolismo , Hígado/metabolismo , Receptores Adrenérgicos alfa 1/metabolismo , Agonistas alfa-Adrenérgicos/metabolismo , Antagonistas Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Animales , Unión Competitiva , Membrana Celular/metabolismo , Epinefrina/farmacología , Ictaluridae , Cinética , Hígado/citología , Hígado/efectos de los fármacos , Norepinefrina/farmacología , Oximetazolina/farmacología , Fosfatidilinositoles/metabolismo , Propranolol/farmacología , Receptores Adrenérgicos alfa 1/efectos de los fármacosRESUMEN
Angiotensin II stimulates phosphorylase a activity and c-fos expression in isolated rat hepatocytes. Both effects are mediated through AT1 receptors. However, the time-courses and the dose-dependencies of these responses were different. These effects of angiotensin II were blocked by Losartan when the antagonist was added to the cells before the hormone or when both the hormone and the antagonist were added simultaneously. Interestingly, when the antagonist was added 2 or 3 min after the peptide hormone, the action on phosphorylase a activity was markedly decreased but, in contrast, that on c-fos expression was not affected. The data suggest that angiotensin II-mediated activation of phosphorylase a was reversible and dependent on continuous receptor activation by the hormone whereas c-fos expression did not seem to have these characteristics, i.e., our data suggest that once protooncogene expression is triggered, the process becomes independent of the hormone-receptor interaction.
Asunto(s)
Angiotensina II/farmacología , Hígado/efectos de los fármacos , Fosforilasa a/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Animales , Células Cultivadas , Femenino , Hígado/citología , Hígado/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Activation of guinea pig hepatocyte alpha 1-adrenoceptors increases phosphatidylinositol (PI) labeling, [3H]inositol phosphate production and phosphorylase activity. These adrenergic actions were not altered by pretreatment with chlorethylclonidine but were blocked by 5-methyl urapidil and prazosin (the former being 3- to 10-fold more potent than the latter), indicating that alpha 1A-adrenoceptors were involved. When the cells were incubated in buffer without calcium and containing EGTA, the alpha 1A-adrenergic stimulation of PI labeling was diminished but not abolished and that of phosphorylase was not affected. The alpha 1A-adrenergic effects were insensitive to pertussis toxin treatment. Phorbol myristate acetate inhibited the alpha 1A-adrenergic actions, although at relatively large concentrations, and also those of other agents such as angiotensin II and NaF. Our data clearly indicate that guinea pig hepatocytes express alpha 1A-adrenoceptors whose activation stimulates phosphoinositide turnover, via a pertussis toxin-insensitive process; the alpha 1A-adrenergic effects were at least partially independent of extracellular calcium.