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A Ruiz-Gómez

Publications and source records attributed to A Ruiz-Gómez.

16 recordsLinked to original sources
PubMed2000

Phosphorylation of phosducin and phosducin-like protein by G protein-coupled receptor kinase 2.

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G protein-coupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind Gbetagamma subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 +/- 0.1 and 0.83 +/- 0.09 mol of P(i)/mol of protein, respectively). The phosphorylation reactions exhibit apparent K(m) values in the range of 40-100 nm, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G beta gamma subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G beta gamma binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.

Animals↗
PubMed1998

Degradation of the G protein-coupled receptor kinase 2 by the proteasome pathway.

GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family and has been shown to play a key role in determining the desensitization and resensitization patterns of a variety of G protein-coupled receptors. In this report, we show that GRK2 is actively degraded by the proteasome proteolytic pathway, unveiling a new mechanism for the rapid regulation of its expression levels. Interestingly, activation of beta2-adrenergic receptors (beta2AR) markedly increases GRK2 ubiquitination and degradation through the proteasome pathway. In addition, blocking GRK2 degradation notably alters beta2AR signaling and internalization, consistent with a relevant physiological role for GRK2 proteasomal degradation. Activity-dependent modulation of GRK2 cellular levels emerges as an important mechanism for modulating the cellular response to agonists acting through G protein-coupled receptors.

Cell-Free System↗
PubMed1998

G protein-coupled receptor kinase 2 (GRK2): mechanisms of regulation and physiological functions.

G protein-coupled receptor kinase 2 (GRK2) plays a key role in determining the rate and extent of G protein-coupled receptor (GPCR) desensitization and resensitization. Recent data indicate that GRK2 activity, subcellular distribution and expression are tightly regulated. The important physiological function of GRK2 as a modulator of the efficacy of GPCR signal transduction systems is exemplified by its relevance in cardiovascular physiopathology as well as by its emerging role in the regulation of chemokine receptors.

Animals↗
PubMed1998

Role of G protein-coupled receptor kinase 2 and arrestins in beta-adrenergic receptor internalization.

G protein-coupled receptors (GPCRs) mediate the action of messengers that are key modulators of the function, growth, and differentiation of cardiac and vascular cells. A general feature of GPCRs is the existence of complex regulatory mechanisms that modulate receptor responsiveness and underlie important physiologic phenomena such as signal integration and desensitization. The molecular mechanisms of desensitization have been investigated with the beta2-adrenergic receptor (beta2AR) used as the main model system. Rapid regulation of betaAR and other GPCRs appears to involve agonist-promoted receptor phosphorylation by G protein-coupled receptor kinases (GRKs). This is followed by binding of uncoupling proteins termed arrestins and transient receptor internalization, which plays a key role in resensitizing GPCR by allowing its dephosphorylation and recycling. Recent data indicate that, besides the uncoupling function, GRK2 and beta-arrestin also directly participate in beta2AR sequestration, thus providing the trigger for its resensitization. A detailed knowledge of the role of GRKs and arrestins in betaAR internalization would make their physiologic role in the modulation of cellular responses to messengers better understood.

Journal Article↗
PubMed1997

The basal subcellular distribution of beta-adrenergic receptor kinase is independent of G-protein beta gamma subunits.

beta-Adrenergic receptor kinase (beta ARK-1 or GRK2) is a key regulatory protein involved in the regulation of G-protein-coupled receptors which associates with microsomal and plasma membranes. beta gamma Subunits of G-proteins have been suggested to mediate agonist-dependent membrane translocation of beta ARK, but their possible role in maintaining the complex subcellular distribution of the kinase is not known. In this study we show that lovastatin-mediated inhibition of G gamma subunits isoprenylation in HEK-293 cells stably transfected with beta ARK1 leads to a significant release of G beta subunits to the cytosol without causing changes in total particulate beta ARK or in the association of this kinase to plasma or microsomal membrane fractions. In addition, transient overexpression of mutant forms of G gamma unable to become isoprenylated resulted in a marked sequestration of G beta to the soluble compartment, but caused no rearrangement in the distribution of cotransfected beta ARK. These results indicate that anchoring of beta ARK to cellular membranes under basal conditions is independent of the availability of heterotrimeric G-protein subunits.

Cell Line↗
PubMed1997

Beta-adrenergic receptor kinase (GRK2) colocalizes with beta-adrenergic receptors during agonist-induced receptor internalization.

Rapid regulation of G protein-coupled receptors appears to involve agonist-promoted receptor phosphorylation by G protein-coupled receptor kinases (GRKs). This is followed by binding of uncoupling proteins termed arrestins and transient receptor internalization. In this report we show that the beta-adrenergic receptor kinase (betaARK-1 or GRK2) follows a similar pattern of internalization upon agonist activation of beta2-adrenergic receptors (beta2AR) and that betaARK expression levels modulate receptor sequestration. Stable cotransfected cells expressing an epitope-tagged beta2AR and betaARK-1 show an increased rate and extent of beta2AR internalization compared with cells expressing receptor alone. Moreover, subcellular gradient fractionation studies suggest that betaARK colocalizes with the internalized receptors. In fact, double immunofluorescence analysis using confocal microscopy shows extensive colocalization of beta2AR and betaARK in intracellular vesicles upon receptor stimulation. Our results confirm a functional relationship between receptor phosphorylation and sequestration and indicate that betaARK does not only translocates from the cytoplasm to the plasma membrane in response to receptor occupancy, but shares endocytic mechanisms with the beta2AR. These data suggest a direct role for betaARK in the sequestration process and/or the involvement of receptor internalization in the intracellular trafficking of the kinase.

Adrenergic beta-Agonists↗
PubMed1996

High affinity binding of beta-adrenergic receptor kinase to microsomal membranes. Modulation of the activity of bound kinase by heterotrimeric G protein activation.

The beta-adrenergic receptor kinase (beta ARK) modulates beta-adrenergic and other G protein-coupled receptors by rapidly phosphorylating agonist-occupied receptors at the plasma membrane. We have recently shown that beta ARK also associates with intracellular microsomal membranes both "in vitro" and "in situ" (García-Higuera, I., Penela, P., Murga, C., Egea, G., Bonay, P., Benovic, J. L., and Mayor, F., Jr. (1994) J. Biol. Chem. 269, 1348-1355), thus suggesting a complex modulation of the subcellular distribution of beta ARK. In this report, we used recombinant [35S]methionine-labeled beta ARK to show that this kinase interacts rapidly with a high affinity binding site (Kd of 20 +/- 1 nM) present in salt-stripped rat liver microsomal membranes. Although beta ARK binding is not modulated by membrane preincubation with G protein activators, the activity of bound beta ARK toward rhodopsin or a synthetic peptide substrate was markedly enhanced upon stimulation of the endogenous heterotrimeric G proteins present in the microsomal membranes by AIF4- or mastoparan/guanosine 5'-(3-O-thio)triphosphate, thus strongly suggesting a functional link between these proteins and membrane-associated beta ARK. Interestingly, beta ARK association with microsomal membranes is not significantly affected by a fusion protein derived from the carboxyl terminus of beta ARK1 (the proposed location of the beta gamma subunit binding site), whereas it is markedly inhibited by fusion proteins corresponding to the amino-terminal region of the kinase. The main determinants of binding appear to be localized to an approximately 60-amino acid residue stretch (residues 88 to 145). Our results further indicate a functional relationship between beta ARK and heterotrimeric G proteins in different intracellular organelles, and suggest that additional proteins may be involved in modulating the cellular localization of the kinase through a new targeting domain of beta ARK.

Animals↗
PubMed1996

Carbamazepine poisoning in adolescent suicide attempters. Effectiveness of multiple-dose activated charcoal in enhancing carbamazepine elimination.

The aim of the study was to evaluate continuous administration of multiple-dose activated charcoal (MDAC) in enhancing elimination of carbamazepine (CBZ) in eight consecutive adolescent suicide attempters. Diluted charcoal was administered through a nasogastric tube at a dose of 1 g/kg every 4 h, and a saline cathartic at the same dosage was administered every 12 h. Plasma CBZ concentrations were measured at 0.0, 12, 24 and 36 h by means of a modified EMIT technique. As a measure of CBZ disappearance, half life of elimination (t1/2 beta) and exogenous total body clearance (CLB) were calculated. Clinical improvement occurred after 12 to 24 h, except in one patient who was the most severely intoxicated and who required advanced life-support therapy. Pharmacokinetic data reported a mean t1/2 beta of 9.5 h, shorter than the reference value of 18-54 h (p < 0.05), and a mean CLB of 103.13 ml/ min/kg, higher than the reference of 75.01 ml/min/ kg (p < 0.05). Initial mean CBZ levels of 27.9 decreased to 0.82 microgram/ml (97% of elimination, p < 0.05). MDAC was free from adverse side effects. In conclusion, MDAC is an effective procedure in enhancing CBZ elimination in overdosed patients as well as being relatively free from serious side effects, widely available, inexpensive and non-invasive.

Adolescent↗
PubMed1994

Modulation of inhibitory glycine receptors by phosphorylation by protein kinase C and cAMP-dependent protein kinase.

Recent evidence has suggested a role for phosphorylation in the regulation of ligand-gated ion channels. We have recently shown (Ruiz-Gómez, A., Vaello, M., Valdivieso, F., and Mayor, F., Jr. (1991) J. Biol. Chem. 266, 559-566) that the inhibitory glycine receptor (GlyR) alpha subunit is phosphorylated in vitro by protein kinase C (PKC). In this report we further show that alpha subunits of the GlyR can also be phosphorylated by cAMP-dependent protein kinase (PKA) in an in vitro assay. Moreover, incubation of intact rat spinal cord neurons with specific PKC or PKA activators leads to increased phosphorylation of the GlyR alpha subunits, strongly suggesting a physiological role in its functional modulation. The role of protein phosphorylation in modulating GlyR channels was explored in Xenopus oocytes injected with poly (A)+ mRNA isolated from nervous tissue. The treatment of oocytes with phorbol esters or dibutyryl cAMP resulted in a decrease or an enhancement, respectively, of glycine-evoked currents. Our results show that the GlyR can be phosphorylated in vivo in response to activation of either PKC or PKA with opposite functional consequences, suggesting that neurotransmitters affecting the activity of such kinases could profoundly alter glycine-mediated neuronal signaling and modulate synaptic efficacy.

Animals↗
PubMed1992

Glycinergic ligands modulate the rate of phosphorylation of the glycine receptor by protein kinase C.

The alpha subunit of the glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C (Ruiz-Gómez et al., (1991) J. Biol. Chem. 266, 559-566). We report here that the rate of phosphorylation of the glycine receptor by this kinase is higher in the presence of agonists (glycine, beta-alanine) than in the presence of antagonists (strychnine, RU-5135). These results suggest that activated glycine receptors would be a preferential target for functional regulation through phosphorylation mechanisms.

Alanine↗
PubMed1991

Phosphorylation of the 48-kDa subunit of the glycine receptor by protein kinase C.

The postsynaptic glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C. The target for phosphorylation is the strychnine-binding subunit of the receptor (molecular mass of approximately 48 kDa), which is phosphorylated on serine residues to a final stoichiometry of approximately 0.8 mol of phosphate/mol of subunit. The 48-kDa phosphoprotein was analyzed by proteolytic cleavage and peptide mapping in order to localize the site of phosphorylation within the receptor molecule. Examination of the 32P-labeled receptor fragments generated by digestion with N-chlorosuccinimide, cyanogen bromide, and endoproteinase lysine C and of the deduced amino acid sequence of the 48-kDa protein (Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E. D., and Betz, H. (1987) Nature 328, 215-220) indicates that the phosphorylation site is located in a region corresponding to the major intracellular loop of the predicted structure of the glycine receptor subunit and suggests serine 391 as the phosphorylated residue. In fact, a synthetic peptide corresponding to residues 384-392 of the 48-kDa subunit was specifically phosphorylated by protein kinase C. Moreover, tryptic digests of this phosphopeptide and of the phosphorylated 48-kDa subunit of the glycine receptor migrated to the same position in two-dimensional peptide mapping. Furthermore, antibodies elicited against peptide 384-392 were shown to inhibit the protein kinase C-dependent phosphorylation of the 48-kDa polypeptide. Interestingly, the relative position of the phosphorylated domain is similar to those known or proposed to be phosphorylated in other ligand-gated ion channel receptor subunits, thus suggesting further the existence of a homologous regulatory region in these receptor proteins.

Amino Acid Sequence↗
PubMed1991

Sulfhydryl groups modulate the allosteric interaction between glycine binding sites at the inhibitory glycine receptor.

We have investigated the effect of chemical reagents that modify sulfhydryl groups on the ligand binding properties of the glycine receptor (GlyR). The Hill coefficient (nH) for the displacement of [3H]strychnine binding by glycine was increased from approximately 0.8 to values significantly above 1 (approximately 1.2-1.4) in membranes pretreated with the disulfide-reducing agent dithiothreitol or glutathione. However, the affinity of strychnine or glycine for the GlyR was not affected by these treatments. This indicates that several glycine binding sites interact cooperatively for displacing bound strychnine under such experimental circumstances. A similar increase in the nH for glycine has been observed when the temperature of the binding assay was increased to 37 degrees C. Combination of dithiothreitol pretreatment and increased binding temperature led to nH variations similar to those observed with either of these treatments alone, a finding suggesting that their mechanisms of action are not independent. Conversely, modification of rat spinal cord membranes or of purified and reconstituted GlyR preparations with the sulfhydryl-alkylating agent N-ethylmaleimide or fluorescein-maleimide decreased nH values to approximately 0.5, without affecting glycine or strychnine affinities. This effect may be caused by an increased heterogeneity of GlyR populations. It is interesting that occupancy of the receptor by glycine or beta-alanine (but not by antagonists) specifically protects from the effects of the different sulfhydryl reagents. Moreover, the presence of some of the Eccles' anions, i.e., anions that permeate through the channels associated with GlyRs and gamma-aminobutyric acidA receptors, seems to be required for the action of both dithiothreitol and N-ethylmaleimide.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1990

Agonist binding to purified glycine receptor reconstituted into giant liposomes elicits two types of chloride currents.

Using 'inside-out' membrane patches obtained from reconstituted giant liposomes containing purified glycine receptor from rat spinal cord, we have detected chloride currents elicited in response to the presence of the agonists glycine or beta-alanine. Regardless of the agonist employed, two different patterns of single channel currents could be detected, which differ in their main conductance, complexity of substates and opening frequency. In agreement with the expectations of glycine receptor heterogeneity suggested recently at the mRNA and cDNA level, our results indicate the existence of functionally different glycine receptors in the adult rat spinal cord.

Alanine↗
PubMed1990

Localization of the strychnine binding site on the 48-kilodalton subunit of the glycine receptor.

Amino acid residues that participate in antagonist binding to the strychnine-sensitive glycine receptor (GlyR) have been identified by selectively modifying functional groups with chemical reagents. Moreover, a region directly involved with strychnine binding has been localized in the 48-kDa subunit of this receptor by covalent labeling and proteolytic mapping. Modification of tyrosyl or arginyl residues promotes a marked decrease of specific [3H]strychnine binding either to rat spinal cord plasma membranes or to the purified GlyR incorporated into phospholipid vesicles. Occupancy of the receptor by strychnine, but not by glycine, completely protects from the inhibition caused by chemical reagents. Furthermore, these tyrosine- or arginine-specific reagents decrease the number of binding sites (Bmax) for [3H]strychnine binding without affecting the affinity for the ligand (Kd). These observations strongly suggest that such residues are present at, or very close to, the antagonist binding site. In order to localize the strychnine binding domain within the GlyR, purified and reconstituted receptor preparations were photoaffinity labeled with [3H]strychnine. The radiolabeled 48-kDa subunit was then digested with specific chemical proteolytic reagents, and the peptides containing the covalently bound radioligand were identified by fluorography after gel electrophoresis. N-Chlorosuccinimide treatment of [3H]strychnine-labeled 48K polypeptide yielded a single labeled peptide of Mr approximately 7300, and cyanogen bromide gave a labeled peptide of Mr 6200.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Thermodynamics of agonist and antagonist interaction with the strychnine-sensitive glycine receptor.

The thermodynamic parameters associated with the interactions of agonists and antagonists with glycine receptors in rat spinal cord membranes were determined. The binding of the antagonist [3H]strychnine and the inhibition of strychnine binding by 11 different glycinergic ligands were examined at temperatures between 0.5 and 37 degrees C. The density of receptors was not affected by the temperature at which the incubation was performed, but the ability of glycine receptor agonists and antagonists to compete with [3H]strychnine binding varied markedly. The affinity of the receptor for the antagonists strychnine, 2-aminostrychnine, RU-5135, 5,6,7,8-tetrahydro-4H-isoxazolo[5,4-c]azepin-3-ol, and the ligands bicuculline, norharmane, and PK-8165 decreased at higher temperatures. The binding of these ligands was enthalpy-driven. In contrast, the affinity of the agonists glycine, beta-alanine, and taurine and of the antihelmintic ivermectin increased at higher temperatures, and their binding was characterized by substantial increases in entropy. In addition, temperature affected the allosteric interaction between the glycine and strychnine sites of the receptor, as indicated by changes in the Hill number of the competition curves for glycine. Our results clearly indicate that the binding of agonists and antagonists to the glycine receptor is differentially affected by temperature, probably as a consequence of the different changes induced in the receptor conformation.

Animals↗