Federico Mayor

The adenosine A2A receptor interacts with the actin-binding protein α-actinin

Recently, evidence has emerged that heptaspanning membrane or G protein-coupled receptors may be linked to intracellular proteins identified as regulators of receptor anchoring and signaling. Using a yeast two-hybrid screen, we identified α-actinin, a major F-actin-cross-linking protein, as a binding partner for the C-terminal domain of the adenosine A2A receptor (A2AR). Colocalization, co-immunoprecipitation, and pull-down experiments showed a close and specific interaction between A2AR and α-actinin in transfected HEK-293 cells and also in rat striatal tissue. A2AR activation by agonist induced the internalization of the receptor by a process that involved rapid β-arrestin translocation from the cytoplasm to the cell surface. In the subsequent receptor traffic from the cell surface, the role of actin organization was shown to be crucial in transiently transfected HEK-293 cells, as actin depolymerization by cytochalasin D prevented its agonist-induced internalization. A2AΔCTR, a mutant version of A2AR that lacks the C-terminal domain and does not interact with α-actinin, was not able to internalize when activated by agonist. Interestingly, A2AΔCTR did not show aggregation or clustering after agonist stimulation, a process readily occurring with the wild-type receptor. These findings suggest an α-actinin-dependent association between the actin cytoskeleton and A2AR trafficking.

Glycine transport into plasma-membrane vesicles derived from rat brain synaptosomes.

Transport of β-alanine has been demonstrated in membrane vesicles isolated from rat brain, using artificially imposed ion gradients as the sole energy source. The uptake of β-alanine is strictly dependent on the presence of Na+ and Cl− in the medium, and the process can be driven either by an Na+ gradient (out > in) or by a Cl− gradient (out > in) when the other essential ion is present. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of ionophore valinomycin and anions with different permeabilities. β-Alanine uptake is inhibited by the presence of GABA.

G Protein–Coupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity

OBJECTIVE Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein–coupled receptor kinase 2 (GRK2), first identified as a G protein–coupled receptor regulator, could have as a modulator of insulin responses. RESEARCH DESIGN AND METHODS We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed. RESULTS Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity. CONCLUSIONS Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders.

Hormonal and metabolic changes in the perinatal period.

A review of some hormonal and metabolic changes occurring during the four stages of the perinatal period is presented. Glucocorticoids and insulin are the hormones that mediate liver glycogen accumulation during late fetal stage. In the presuckling period, muscle glycogenolysis supplies the lactate moieties that are oxidized by the neonatal tissues, representing the alternative substrate until glucose and ketone bodies become available. The postnatal increase in plasma catecholamine concentrations and the decrease in the insulin/glucagon ratio triggers liver glycogenolysis and gluconeogenesis, and hence postnatal hypoglycemia is reversed. In the suckling period, the oxidation of fatty acids, ketone bodies utilization and active gluconeogenesis supply the bulk of the energy and carbon components required to support the rapid growth rate of this period. The increase in the insulin/glucagon ratio that occurs with the change to a carbohydrate-rich diet starts the induction of lipogenesis at weaning.

G Protein-coupled Receptor Kinase 2-mediated Phosphorylation of Downstream Regulatory Element Antagonist Modulator Regulates Membrane Trafficking of Kv4.2 Potassium Channel

Downstream regulatory element antagonist modulator (DREAM)/potassium channel interacting protein (KChIP3) is a multifunctional protein of the neuronal calcium sensor subfamily of Ca2+-binding proteins with specific roles in different cell compartments. In the nucleus, DREAM acts as a Ca2+-dependent transcriptional repressor, and outside the nucleus DREAM interacts with Kv4 potassium channels, regulating their trafficking to the cell membrane and their gating properties. In this study we characterized the interaction of DREAM with GRK6 and GRK2, members of the G protein-coupled receptor kinase family of proteins, and their phosphorylation of DREAM. Ser-95 was identified as the site phosphorylated by GRK2. This phosphorylation did not modify the repressor activity of DREAM. Mutation of Ser-95 to aspartic acid, however, blocked DREAM-mediated membrane expression of the Kv4.2 potassium channel without affecting channel tetramerization. Treatment with the calcineurin inhibitors FK506 and cyclosporin A also blocked DREAM-mediated Kv4.2 channel trafficking and calcineurin de-phosphorylated GRK2-phosphorylated DREAM in vitro. Our results indicate that these two Ca2+-dependent posttranslational events regulate the activity of DREAM on Kv4.2 channel function.

Stoichiometry of sodium- and chloride-coupled glycine transport in synaptic plasma membrane vesicles derived from rat brain.

The stoichiometric properties of the glycine transporter were studied in synaptic plasma membrane vesicles from rat brain. The present results, together with previous data from our laboratory, allow us to suggest a Stoichiometry of 2 Na+ and 1 Cl− per glycine zwitterion for the translocation cycle catalyzed by the glycine carrier. We propose a kinetic model with an ordered mechanism for the binding/debinding of solutes.

Effect of Bilirubin on the Membrane Potential of Rat Brain Synaptosomes

Abstract: The effect of the neurotoxic pigment bilirubin on the membrane potential of rat brain synaptosomes was studied by using the tetraphenylphosphonium ion (TTP+) technique. Bilirubin induces a rapid depolarization of synaptosomes, as reflected by an efflux of previously accumulated [3H]TTP+. This phenomenon persisted when the membrane potential across either the plasma membrane of the synaptosome or the inner membrane of the entrapped mitochondria was selectively depressed, thus indicating that both components of the synaptosomal membrane potential were affected by bilirubin. Bovine serum albumin, used at a albumin/bilirubin molar ratio of 1:1, had the capacity to completely prevent and reverse the effect of bilirubin. This fact demonstrates that the bilirubin‐induced TPP+ release from synaptosomes is a reversible process that requires the presence of bilirubin interacting with the synaptosomal membranes. These results, together with the inhibition by bilirubin of [3H]TPP+ and [2‐14C]acetate uptake by synaptosomal plasma membrane vesicles isolated from rat brain, suggest that bilirubin depresses the membrane potential across the synaptosomal plasma membrane by a mechanism involving alterations in ion permeability. This effect could be of relevance in the pathogenesis of bilirubin encephalopathy.

G protein–coupled receptor kinase 2 (GRK2) modulation and cell cycle progression

Cell cycle progression requires changes in the activity or levels of a variety of key signaling proteins. G protein–coupled receptor kinase 2 (GRK2) plays a central role in G protein–coupled receptor regulation. Recent research is uncovering its involvement in additional cellular functions, but the potential role of GRK2 in the cell cycle has not been addressed. We report that GRK2 protein levels are transiently down-regulated during the G2/M transition by a mechanism involving CDK2-mediated phosphorylation of GRK2 at Serine670, which triggers binding to the prolyl-isomerase Pin1 and subsequent degradation. Prevention of GRK2 phosphorylation at S670 impedes normal GRK2 down-regulation and markedly delays cell cycle progression. Interestingly, we find that endogenous GRK2 down-regulation is prevented on activation of the G2/M checkpoint by doxorubicin and that stabilized GRK2 levels in such conditions inversely correlate with the p53 response and the induction of apoptosis, suggesting that GRK2 participates in the regulatory network controlling cell cycle arrest and survival in such conditions.

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

Abstract: 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°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‐Msoxazolo[5,4‐c]azepin‐3‐ol, and the ligands bicuculline, norharmane, and PK‐8165 decreased at higher temperatures. The binding of these ligands was enthalpydriven. In contrast, the affinity of the agonists glycine, β‐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.

Interfering with MAP kinase docking interactions: implications and perspective for the p38 route.

Docking interactions are key to understand the dynamic assembly of signal transductioncomplexes in the cell. In particular, the docking domain (D domain)-dependentinteractions described so far for several MAPK routes are essential to specify theupstream regulators, downstream mediators and also inactivators that complex with thep38, JNK and ERK proteins. In addition to contributing to the maintenance of thelinearity and specificity of these pathways, novel data have revealed that dockingcontacts also regulate the activity, subcellular distribution and substrate selection ofeach MAPK. Moreover, phosphorylation inside or around a docking domain isemerging as a novel mechanism of regulation of MAPK association with cellularpartners, suggesting new potential strategies for the design of selective MAPKinhibitors. Here, we discuss these novel data and the biochemical and cellularimplications they may have with specific emphasis on the p38 route.