ma teresa romero avila

Universidad Nacional Autónoma de México | UNAM · Institute of Cellular Physiology

LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling, the agonists...

Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were exp...

Lysophosphatidic acid (LPA) type 3 (LPA3) receptor mutants were generated in which the sites detected phosphorylated were substituted by non-phosphorylatable amino acids. Substitutions were made in the intracellular loop 3 (IL3 mutant), the carboxyl terminus (Ctail), and both domains (IL3/Ctail). The wild-type (WT) receptor and the mutants were exp...

LPA3 receptors were expressed in TREx HEK 293 cells, and their signaling and phosphorylation were studied. The agonist, lysophosphatidic acid (LPA), increased intracellular calcium and ERK phosphorylation through pertussis toxin-insensitive processes. Phorbol myristate acetate, but not LPA, desensitizes LPA3-mediated calcium signaling. The agonists...

LPA1 internalization to endosomes was studied employing Förster Resonance Energy Transfer (FRET) in cells coexpressing the mCherry-lysophosphatidic acid LPA1 receptors and distinct eGFP-tagged Rab proteins. Lysophosphatidic acid (LPA)-induced internalization was rapid and decreased afterward: phorbol myristate acetate (PMA) action was slower and su...

The G protein-coupled receptors (GPCRs) are plasma membrane proteins that function as sensors of changes in the internal and external milieux and play essential roles in health and disease. They are targets of hormones, neurotransmitters, local hormones (autacoids), and a large proportion of the drugs currently used as therapeutics and for “recreat...

Free fatty acid receptor 1 phosphorylation sites were studied using mutants, including a) a mutant with T215V in the third intracellular loop (3IL), b) another with changes in the carboxyl terminus (C-term): T287V, T293V, S298A, and c) a mutant with all of these changes (3IL/C-term). Agonist-induced increases in intracellular calcium were similar b...

The G protein-coupled receptors form the most abundant family of membrane proteins and are crucial physiologic players in the homeostatic equilibrium, which we define as health. They also participate in the pathogenesis of many diseases and are frequent targets of therapeutic intervention. Considering their importance, it is not surprising that dif...

La esfingosina1-fosfato (S1P) es un metabolito intermediario en el catabolismo de la esfingomielina y un “lípido bioactivo”, con la capacidad de funcionar como hormona local al modular las funciones de distintas células y tejidos; en las células regula una gran variedad de respuestas, como la migración, la diferenciación y la sobrevivencia y en los...

The lysophosphatidic acid 3 receptor (LPA3) participates in different physiological actions and in the pathogenesis of many diseases through the activation of different signal pathways. Knowledge of the regulation of the function of the LPA3 receptor is a crucial element for defining its roles in health and disease. This review describes what is kn...

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab pro...

Sphingosine 1-phosphate and FTY720-phosphate increased intracellular calcium in cells expressing S1P1 mCherry-tagged receptors; the synthetic agonist was considerably less potent. Activation of protein kinase C by phorbol myristate acetate blocked these effects. The three agents induced receptor phosphorylation and internalization, with the action...

The crosstalk between the free fatty acid receptor FFA4 and the lysophosphatidic acid receptor LPA1 seems to be of pathophysiological importance. We explored this crosstalk employing co‐expression of fluorescent protein‐tagged receptors. FFA4 activation induces functional desensitization of LPA1 receptors and phosphorylation of both receptors. LPA1...

Human α1D-adrenoceptors (α1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely...

In LNCaP cells that stably express α1A-adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects th...

Lysophosphatidic acid (LPA) modulates the function of many organs, including the lung. A549 is a lung carcinoma-derived cell line, frequently used as a model for type II pneumocytes. Here we show that these cells expressed messenger RNA coding for LPA1-3 receptors with the following order of abundance: LPA1 > LPA2 > LPA3 and that LPA was able to in...

Upon agonist stimulation, α1B-adrenergic receptors (α1B-ARs) couple to Gq, calcium signaling and protein kinase C activation; subsequently, the receptors are phosphorylated, desensitized and internalized. Internalization seems to involve scaffolding proteins, such as α-arrestin and clathrin. However, the fine mechanisms that participate remain unso...

Human α1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human α1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-reg...

Human alpha‐1D‐adrenoceptors are G protein‐coupled receptors that mediate the actions of adrenaline and noradrenaline participating in the maintenance of homeostasis as well as in the physiopathology of some diseases, such as hypertension and benign prostatic hyperplasia. There is a growing interest in investigating the function of their regulatory...

FFA1 (previously known as GPR40) is a free fatty acid receptor involved in the regulation of inflammatory processes and insulin secretion. The cellular actions resulting from FFA1 activation have received considerable attention. However, little is known on the regulation of the receptor function. In the present work, using cells transfected with th...

Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution...

The role of protein kinase c (PKC) isozymes in phorbol myristate acetate (PMA)-induced sphingosine 1-phosphate (S1P) receptor 1 (S1P1) phosphorylation was studied. Activation of S1P1 receptors induced an immediate increase in intracellular calcium, which was blocked by preincubation with PMA. Both S1P and PMA were able to increase S1P1 phosphorylat...

GPR120, Free Fatty Acid Receptor 4, is a recently deorphanized G protein-coupled receptor that seems to play cardinal roles in the regulation of metabolism and in the pathophysiology of inflammatory and metabolic disorders. In the present work a GPR120-Venus fusion protein was expressed in HEK293 Flp-In T-REx cells and its function (increase in int...

Sphingosine‐1 phosphate (S1P) is important in the regulation of angiogenesis, vascular permeability and stability, development of new blood vessels, vascular maturation, cardiac development, immunity and cellular mobility routing. A deficiency in the regulation of sphingosine phosphate receptor type 1 (S1PR1) can cause health disorders, such as dev...

Sphingosine‐1‐phosphate (S1P) is a bioactive molecule that has been implicated in regulating cellular processes. S1P exerts its extracellular effects as a ligand of a GPCR family. S1P 1 receptors are coupled to heterotrimeric G i/o proteins. The importance of S1P and its receptor S1P 1 in multiple cellular and physiological processes (vascular tone...

GPR120 is now recognized as an important therapeutic target for diabetes, due to of its anti‐inflammatory and insulin‐sensitizing effects after activation with Ω3 fatty acids. Phosphorylation of both receptor splicing variants dependent of Ω3 fatty acids, DHA and α‐LA, has been previously reported, nevertheless protein kinases involved in this regu...

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase...

α(1B)-Adrenergic receptors mediate many of the actions of the natural catecholamines, adrenaline and noradrenaline. They belong to the seven transmembrane domains G protein-coupled receptor superfamily and exert their actions mainly through activation of Gq proteins and phosphoinositide turnover/calcium signaling. Many hormones and neurotransmitter...

Adrenergic receptors are a heterogeneous family of the G protein-coupled receptors that mediate the actions of adrenaline and noradrenaline. Adrenergic receptors comprise three subfamilies (α(1), α(2), and β, with three members each) and the α(1D)-adrenergic receptor is one of the members of the α(1) subfamily with some interesting traits. The α(1D...

Confocal images of HEK 293 cells expressing non-tagged eGFP. Cells were treated for 5 min with vehicle (BASAL), 10 μM noradrenaline (NA) or 1 μM TPA; at the end of incubation FM4-64 (membrane marker, red) was added and fluorescence recorded. Right panels: merge images are presented. No colocalization was observed

Schematic representation of: a Bovine α1A-Adrenergic Receptor; b Hamster α1B-Adrenergic Receptor; c Chimeric α1AB-Adrenergic Receptor; d Chimeric α1BA-Adrenergic Receptor; e Carboxyl terminus-truncated bovine α1A-Adrenergic Receptor. Putative phosphorylation sites are indicated in red

Receptor tyrosine kinases and G protein-coupled receptors modulate physiological processes and are also involved in the pathogenesis of some diseases. These receptors have intense bidirectional crosstalks leading to interactions in their signaling pathways and also modulation of the receptors themselves. In some cases, the receptor tyrosine kinases...

Noradrenaline- and tetradecanoyl phorbol acetate (TPA)-induced phosphorylation and functional desensitization of the following receptors were studied: (1) wild-type bovine α(1A)- and hamster α(1B)-adrenergic receptors (ARs), (2) chimeric ARs in which the carboxyl terminus tails were exchanged (α(1AB)- and α(1BA)-ARs), and (3) carboxyl terminus-trun...

alpha(1)-Adrenoceptors are differentially regulated by protein kinase C-mediated phosphorylation. The most sensitive member of this family is the alpha(1D)-subtype, which is also characterized by a constitutive activity and a reduced expression at the plasma membrane controlled by the amino terminus. Information on the structural domains that deter...

Human alpha(1D)-adrenoceptors (truncated at the amino terminus (Delta1-79) to increase their membrane expression) were stably expressed in Rat-1 fibroblasts (1-1.5 pmol/mg protein). The receptors were functional as evidenced by a robust increase in intracellular calcium in response to noradrenaline. Using this cell line, the possibility that activa...

Receptor tyrosine kinases (RTKs) can induce G protein-coupled receptor (GPCR) desensitization and phosphorylation. In some cases phosphorylation by RTKs on GPCRs tyrosine residues is involved, while in others indirect action through diverse protein kinases takes place. GPCRs stimulation can lead to RTK activation through the action of metalloprotei...

The effect of insulin-like growth factor-I (IGF-I) on human alpha(1B)-adrenoceptor function, phosphorylation state and cellular location was studied. Rat-1 fibroblasts were transfected with a plasmid construction containing enhanced green fluorescent protein joined to the carboxyl terminus of the human alpha(1B)-adrenoceptor. Receptors were identif...

IGF-I induces alpha(1B)-adrenoceptor (alpha(1B)-AR) phosphorylation. The effect of IGF-I was rapid and transient, reaching near-maximal values at 10 min and decreasing after 30 min; it was observed at low IGF-I concentrations (EC(50) approximately 10 ng/ml) and was associated to receptor desensitization as evidenced by a decreased alpha(1B)-adrener...

The ability of insulin to induce alpha1B-adrenoceptor phosphorylation and desensitization was tested in two model systems: rat-1 cells that stably express alpha1B-adrenoceptors, through transfection, and endogenously express insulin receptors and DDT1 MF2 cells that endogenously express both receptors. Insulin induced concentration-dependent increa...

Rat-1 fibroblast were transfected with a plasmid containing the cDNA of the human alpha(1D)-adrenoceptor. A cell line was isolated that stably expressed the receptor as evidenced by BMY 7378-sensitive noradrenaline-induced increases in intracellular calcium concentration. The effect of noradrenaline was blocked by active phorbol esters; such blocka...

Transforming growth factor-beta (TGF-beta) induced alpha(1B)-adrenergic receptor phosphorylation in Rat-1 fibroblasts stably expressing these adrenoceptors. This effect of TGF-beta was rapid, reaching a maximum within 30 min and decreasing thereafter, and concentration-dependent (EC(50) 0.3 pM). The phosphoinositide 3-kinase inhibitors wortmannin a...

In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]pr...

In the present paper, the cloning and expression of the guinea pig α1A-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of ≈51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [3H]prazosin and [3H]tamsul...

In rat-1 fibroblasts stably expressing rat alpha(1d)-adrenoceptors, noradrenaline and PMA markedly decreased alpha(1d)-adrenoceptor function (noradrenaline-elicited increases in calcium in whole cells and [(35)S]guanosine 5'-[gamma-thio]triphosphate binding in membranes), suggesting homologous and heterologous desensitizations. Photoaffinity labell...

Maximal adrenergic responses in Rat-1 fibroblasts expressing α1a-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of α1b-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on α1a-adrenoreceptor phosphorylation and co...

Maximal adrenergic responses in Rat-1 fibroblasts expressing α1a-adrenergic receptors are not blocked by activation of protein kinase C. In contrast, activation of protein kinase C induces the phosphorylation of α1b-adrenoreceptors and blocks their actions. The effect of norepinephrine and phorbol esters on α1a-adrenoreceptor phosphorylation and co...

Human α1b-adrenoceptors stably expressed (Bmax∼800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca2+ and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the α1b-adrenergic-mediated actions but did not block the ability of the cells to respond to lysophosph...

Human alpha(1b)-adrenoceptors stably expressed (B(max) approximately 800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca(2+) and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the alpha(1b)-adrenergic-mediated actions but did not block the ability of the...

It is now well documented that changes in gene expression take place during cell isolation and culture. Here, we report the change in the expression of the mRNAs for alpha(1)-adrenoceptor subtypes, during dissociation of guinea pig liver cells with collagenase. Using Reverse Transcription-Polymerase Chain Reaction (RT-PCR) assays, it was observed t...

In guinea pig hepatocytes angiotensin II induced phosphorylase a activation. This effect was mimicked by other angiotensins with the potency order: angiotensin II (EC 50 ≈1 nm)>angiotensin III (EC 50 ≈30 nm)>angiotensin I (EC 50 ≈300 nm). The effect of 10 nm angiotensin II was blocked by the angiotensin II receptor AT 1 -selective antagonists irbes...

Although P2 receptors mediate a myriad of physiological effects of extracellular adenine nucleotides, study of this broad class of receptors has been compromised by a lack of P2 receptor-selective antagonist molecules. The adenine nucleotide-promoted inositol lipid hydrolysis response of turkey erythrocyte membranes, which has been used extensively...

The alpha 1-adrenoceptors present in the liver of rhesus monkeys was characterized using [3H]prazosin. This radioligand binds to monkey liver membranes with high affinity (KD 0.33 nM) to a moderately abundant number of sites (97 fmol/mg of protein). These sites were characterized pharmacologically, by binding competition, observing two affinities f...

The beta-adrenoceptors present in dog liver were characterized by radioligand binding techniques. [3H]Dihydroalprenolol bound with high affinity (KD 0.9 nM) to a relatively low number of sites (approximately 68 fmol/mg protein) in partially purified dog liver plasma membranes. These sites were characterized by binding competition; the orders of aff...

A series of chain‐extended 2‐thioether derivatives of adenosine monophosphate were synthesized and tested as agonists for activation of the phospholipase C‐linked P 2Y ‐purinoceptor of turkey erythrocyte membranes, the adenylyl cyclase‐linked P 2Y ‐purinoceptor of C6 rat glioma cells, and the cloned human P 2U ‐receptor stably expressed in 1321N1 h...

The α1-adrenoceptors present in the liver of rhesus monkeys was characterized using [3H]prazosin. This radioligand binds to monkey liver membranes with high affinity (KD 0.33 nM) to a moderately abundant number of sites (97 fmol/mg of protein). These sites were characterized pharmacologically, by binding competition, observing two affinities for mo...

The β-adrenoceptors present in dog liver were characterized by radioligand binding techniques. [3H]Dihydroalprenolol bound with high affinity (KD 0.9 nM) to a relatively low number of sites (≈68 fmol/mg protein) in partially purified dog liver plasma membranes. These sites were characterized by binding competition; the orders of affinity were: (a)...

The subtype selectivity of the alpha 1-adrenoceptor antagonist, tamsulosin, was tested using hepatocytes and liver membranes from guinea pigs and rabbits (expressing alpha 1-adrenoceptors with alpha 1A pharmacology) and rats (alpha 1B-adrenoceptors). Tamsulosin blocked the alpha 1-adrenergic activation of phosphorylase with higher affinity in hepat...

The alpha 1-adrenoceptor subtype present in human liver membranes was studied using radioligand binding techniques. [3H]Prazosin binding was rapid, saturable and reversible. A kinetically derived Kd of 0.22 nM was obtained. Rosenthal analysis of saturation isotherms indicated a single class of binding sites with a Kd of 0.47 nM and a Bmax of 70 fmo...

The α1-adrenoceptor subtype present in human liver membranes was studied using radioligand binding techniques. [3H]Prazosin binding was rapid, saturable and reversible. A kinetically derived Kd of 0.22 nM was obtained. Rosenthal analysis of saturation isotherms indicated a single class of binding sites with a Kd of 0.47 nM and a Bmax of 70 fmol/mg...

The subtype selectivity of the α1-adrenoceptor antagonist, tasulsion, was tested using hepatocytes and liver membranes from guinea pigs and rabbits (expressing α1-adrenoceptors with α1A pharmacology) and rats (α1B-adrenoceptors). Tamsulosin blocked the α1-adrenergic activation of phosphorylase with higher affinity in epatocytes from guinea pigs and...

Using dog liver membranes we observed that [125I]HEAT ((+/-)-beta-([125I]iodo-4-hydroxyphenyl)-ethyl-aminomethyl-tetralone) binds with high affinity (KD 97 pM) to a discrete number of sites (Bmax 40 fmol/mg protein) with the pharmacological characteristics expected for alpha 1-adrenoceptors. Such sites were inactivated by pretreatment with chloroet...

The alpha 1-adrenoceptors present in liver membranes from rats, hamsters and mice were characterized using [3H]prazosin. In the liver membranes from the three species a relatively large number of receptors was observed (500-900 fmol/mg of protein) and the affinities for [3H]prazosin were very similar (0.2-0.3 nM). Membrane preincubation with 10 mic...

Binding of 5-[3H]methylurapidil to guinea pig liver membranes was rapid, saturable, and reversible. Scatchard analysis of saturation isotherms indicated a single class of binding sites with a Kd of 0.86 nM and a Bmax of 36 fmol/mg of protein. Preincubation of the membranes with chlorethylclonidine did not alter significantly the binding parameters...

In guinea pig hepatocytes, histamine increased phosphorylase activity and inositol phosphate production. Similar effects were obtained with 2-(2-aminoethyl)-thiazole, a histamine H1 receptor agonist, but not with dimaprit or impromidine, H2 receptor agonists. These effects of histamine were dose-dependently inhibited by the H1 antihistamines, (+)-c...

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 t...

Activation of guinea pig hepatocyte α1-adrenoceptors increases phosphatidylinositol (PI) labeling, [3H]inositol phosphate production and phosphorylasc 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 la...

In guinea pig hepatocytes, histamine increased phosphorylase activity and inositol phosphate production. Similar effects were obtained with 2-(2-aminoethyl)-thiazole, a histamine H1 receptor agonist, but not with dimaprit or impromidine, H2 receptor agonists. These effects of histamine were dose-dependently inhibited by the H1 antihistamines, (+)-c...

In isolated rat white adipocytes, epinephrine (in the presence of 10 microM propranolol) increased the uptake of [32P]Pi into phosphatidylinositol in a dose-dependent fashion. When the cells were pretreated with the irreversible antagonist chlorethylclonidine, this alpha 1-adrenergic effect was markedly diminished. The effect of epinephrine was dos...

alpha 1-Adrenergic activation stimulated phosphorylase and phosphoinositide turnover in hepatocytes from guinea pigs, rats and rabbits. Chlorethylclonidine inhibited these effects in rat and rabbit cells but not in guinea pig hepatocytes; low concentrations of 5-methyl urapidil blocked the alpha 1 actions in guinea pig and rabbit liver cells, but n...

An acellular pertussis vaccine was prepared from pertussis vaccine concentrates by eliminating the cells by centrifugation and subsequent acid precipitation. SDS-PAGE analysis indicated the presence of 17 clearly defined bands including the subunits of pertussis toxin. The acellular preparation was detoxified with glutaraldehyde and lost its undesi...

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A which seems to be useful for identifying biological processes that are controlled by reversible phosphorylation of proteins. We report here that okadaic acid inhibits in isolated hepatocytes the stimulations of phosphoinositide turnover induced by epinephrine, angioten...