Real-time G-protein-coupled receptor imaging to understand and quantify receptor dynamics.

Understanding the trafficking of G-protein-coupled receptors (GPCRs) and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 µm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

Hydrogen peroxide impairs GRK2 translation via a calpain-dependent and cdk1-mediated pathway.

Oxidative mechanisms of injury are involved in many neurodegenerative diseases such as stroke, ischemia-reperfusion injury and multiple sclerosis. G protein-coupled receptor kinase 2 (GRK2) plays a key role in G protein-coupled receptor (GPCR) signaling modulation, and its expression levels are decreased after brain hypoxia/ischemia and reperfusion as well as in several inflammatory conditions. We report here that hydrogen peroxide downregulates GRK2 expression in C6 rat glioma cells. The hydrogen peroxide-induced decrease in GRK2 is prevented by a calpain protease inhibitor, but does not involve increased GRK2 degradation or changes in GRK2 mRNA level. Instead we show that hydrogen peroxide treatment impairs GRK2 translation in a process that requires Cdk1 activation and involves the mTOR pathway. This novel mechanism for the control of GRK2 expression in glial cells upon oxidative stress challenge may contribute to the modulation of GPCR signaling in different pathological conditions.

Rapid desensitization of neonatal rat liver beta-adrenergic receptors. A role for beta-adrenergic receptor kinase.

Exposure of beta-adrenergic receptors (BAR) to agonists often leads to a rapid loss of receptor responsiveness. The proposed mechanisms of such rapid receptor desensitization include receptor phosphorylation by either cAMP-dependent protein kinase or the specific beta-adrenergic receptor kinase (BARK), leading to functional uncoupling from adenylyl cyclase and sequestration of the receptors away from the cell surface. To evaluate the physiological role of such mechanisms, we have investigated whether rapid regulation of BAR occurs in the neonatal rat liver immediately after birth, a physiological situation characterized by a dramatic but transient increase in plasma catecholamines. We have detected a rapid, transient uncoupling of liver plasma membrane BARs from adenylyl cyclase (corresponding to a desensitization of approximately 45%) within the first minutes of extrauterine life, followed by a transient sequestration of approximately 40% of the BARs away from the plasma membrane. In agreement with such pattern of desensitization, we have detected (by enzymatic and immunological assays) rapid changes in BARK specific activity in different neonatal rat liver subcellular fractions that take place within the same time frame of BAR uncoupling and sequestration. Our results provide new evidence on the potential role of BAR desensitization mechanisms in vivo and suggest that they are involved in modulating catecholamines actions at the moment of birth. Furthermore, our data indicate that in addition to its suggested role as a rapid modulator of adrenergic receptor function at synapse, rapid BARK-mediated receptor regulation may have functional relevance in other tissues in response to high circulating or local levels of agonists.

Tyrosine transport into isolated rat brain synaptosomes. Ionic dependence and kinetic studies.

The ionic dependence and kinetics of the uptake of L-tyrosine into isolated rat brain synaptosomes has been investigated. L-Tyrosine has been found to enter the synaptosomes through three different transport systems showing distinct ionic requirements and kinetic characteristics. The one with the lowest affinity for tyrosine (Km 0.6 mM) showed a strong Na+ dependence. This system seems to provide the nerve cell with a safety mechanism that ensures the supply of tyrosine even in the presence of high levels of competing amino acids. The second one (Km 50 microM) does not appear to exhibit any strong ionic requirements and features most of the characteristics of the L-system for large neutral amino acids. Finally, the third shows the most interesting ionic dependence. Its activity increases at very low Na+ external concentrations, but this increase is prevented by the removal of divalent cations, Ca2+ and Mg2+. This ionic behaviour, along with the affinity constant of this system (Km 6 microM) (within the range of tyrosine extraneural concentrations), suggests that it is an initial regulatory step in the synthesis of catecholamines.

Tyrosine transport by membrane vesicles isolated from rat brain.

Tyrosine uptake by membrane vesicles derived from rat brain has been investigated. The uptake is dependent on an Na+ gradient ([Na+]outside greater than [Na+]inside). The uptake is transport into an osmotically active space and not a binding artifact as indicated by the effect of increasing the medium osmolarity. The process is stimulated by a membrane potential (negative inside) as demonstrated by the effect of the ionophores valinomycin and carbonyl cyanide m-chlorophenylhydrazone and anions with different permeabilities. Kinetic data show that tyrosine is accumulated by two systems with different affinities. Tyrosine uptake is inhibited by the presence of phenylalanine and tryptophan.

Analysis of the human G protein-coupled receptor kinase 2 (GRK2) gene promoter: regulation by signal transduction systems in aortic smooth muscle cells.

BACKGROUND: Desensitization of G protein-coupled receptors (GPCR) is emerging as an important feature of several cardiovascular diseases. G protein-coupled receptor kinase 2 (GRK2) plays a key role in the regulation of a variety of these receptors, and its cardiac expression levels are altered in pathological situations such as chronic heart failure. However, very little is known about the signals and mechanisms that modulate GRK2 expression in cardiovascular cells. METHODS AND RESULTS: We have studied the transcriptional activity of the 1.6-kb-long proximal genomic region of the human GRK2 gene. In an aortic smooth muscle cell line, agents that lead to physiological vasoconstriction and hypertrophy, such as phorbol esters, increased GRK2 promoter activity. Activation of signaling pathways by cotransfected G(alphaq) subunits or alpha(1)-adrenergic receptors also markedly enhanced the expression of the GRK2 promoter constructs. Conversely, proinflammatory cytokines, such as interleukin-1beta, tumor necrosis factor-alpha, or interferon-gamma, led to the opposite effect, decreasing the activity of the GRK2 promoter. CONCLUSIONS: Our results suggest that the expression of GRK2 in vascular cells is tightly controlled at the transcriptional level by the interplay between several extracellular messengers, which may trigger alterations of normal GRK2 levels in some physiopathological circumstances, thus promoting changes in the efficacy of the GPCR signal transduction.

Expression of the G protein-coupled receptor kinase 2 during early mouse embryogenesis.

Whilst several G protein-coupled receptors (GPCRs) have been shown to play important roles during development, little study has been carried out on the G protein-coupled receptor kinases (GRKs) that modulate their activity in embryos. Here, we have analyzed the expression of GRK2, the predominant GRK expressed during embryogenesis. We show that at early stages, the expression of GRK2 is restricted to populations of cells that are undifferentiated, multipotent and in many cases, migratory. As such, GRK2 transcripts were found in the early mesoderm and neural crest as they migrate from the primitive streak and the neural tube, respectively. In the limb bud, GRK2 transcripts were observed in cells of the progress zone and in the interdigital areas. At later stages, the expression in the heart is compatible with the phenotype observed in the GRK2 deficient mice.

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.

Effect of hypothyroidism on G protein-coupled receptor kinase 2 expression levels in rat liver, lung, and heart.

GRK2 is a member of the G protein-coupled receptor kinase family that phosphorylates the activated form of beta-adrenergic and other G protein-coupled receptors and plays an important role in their desensitization and modulation. Alterations in thyroid hormone levels have been reported to lead to important changes in adrenergic receptor responsiveness and signaling in a variety of tissues. In this context, we have explored the effects of experimental hypothyroidism on GRK2 protein levels in rat heart, lung, and liver using a specific antibody. Hypothyroid animals show significant up-regulation ( approximately 50% increase compared with controls) in GRK2 levels in heart and lung at 60 days after birth, whereas a 50% reduction is detected in the liver at this stage. These alterations are selective, as beta-adrenergic receptors or other G protein-coupled receptor regulatory proteins, such as G protein-coupled receptor kinase 5 or beta-arrestin-1, display a different pattern of expression changes in the hypothyroid animals. The reported changes in GRK2 levels and in the receptor/kinase ratio predict alterations in adrenergic receptor desensitization and signal transduction efficacy consistent with those observed in thyroid disorders, thus suggesting a relevant role for the modulation of GRK2 expression in this physiopathological condition.

Production of the Alzheimer's beta-amyloid peptide by C6 glioma cells.

The beta-amyloid peptide (A beta) is a 4 kDa proteolytic fragment derived from the beta-amyloid precursor protein (beta APP) which is deposited as amyloid fibrils in the brains of patients with Alzheimer's disease. beta APP processing was investigated in C6 glioma cells using several affinity-purified anti-peptide antibodies raised against different domains of the protein. Both direct immunoblot analysis of C6 glioma conditioned medium and metabolic labeling of cells followed by immunoprecipitation of extracellular medium with specific antibodies revealed that these glial cells normally produce and release a soluble 4 kDa peptide which co-migrates with synthetic A beta (1-40) and is specifically recognized by antibodies raised against N- or C-terminal domains of the beta-amyloid peptide. Our results further suggest that glial cells may prove a major source of beta-amyloid production in the nervous tissue.

Rapid agonist-induced beta-adrenergic receptor kinase translocation in C6 glioma cells.

Exposure of C6 glioma cells to 1 microM isoproterenol leads to fast desensitization of the beta-adrenergic receptor/adenylyl cyclase system and transient receptor sequestration. It also triggers a very rapid and transient translocation to the plasma membrane of beta-adrenergic receptor kinase (beta ARK), a specific cytoplasmic kinase that phosphorylates only the agonist-occupied form of several G protein-coupled receptors. beta ARK-mediated receptor phosphorylation appears to be a suitable mechanism for the rapid regulation of adrenergic receptor function in the nervous tissue.

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.

Alanine and lactate as gluconeogenic substrates during late gestation.

Rates of alanine incorporation into glucose by isolated liver cells of fed rats are 5-fold higher than those observed when lactate was used as substrate. The rates of gluconeogenesis from alanine and lactate in isolated liver cells of fed pregnant rats increase 50 and 200-400%, respectively, over virgins during the last 3 days of gestation. The results support the existence of an increase in the alanine-glucose cycle in the late pregnancy as an important homeostatic pathway in the supply of glucose to the growing fetus.

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.

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.

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.

The subcellular and cellular distribution of G protein-coupled receptor kinase 2 in rat brain.

G protein-coupled receptor kinase 2 has been found to phosphorylate and thus regulate the activity of several G protein-coupled receptors implicated in neuronal signalling pathways. Although this kinase was initially described as a soluble protein, our laboratory has recently found that a significant amount of G protein-coupled receptor kinase 2 is associated with microsomal membranes in liver and different types of cultured cells. In the present report we show that high G protein-coupled receptor kinase 2 specific activity and protein levels are present in microsomal fractions of rat brain homogenates. On the other hand, immunochemical detection using a new antibody raised against the N-terminus of the kinase revealed a specific and widely distributed staining in different areas of the central nervous system, and the association of G protein-coupled receptor kinase 2 with intracellular structures in nervous cells. Our results further suggest that this receptor kinase may be involved in the modulation of G protein-coupled receptor-mediated neurotransmission and that association with microsomal membranes may play a role in G protein-coupled receptor kinase 2 functions in the brain.

Post-transcriptional induction of beta 1-adrenergic receptor by retinoic acid, but not triiodothyronine, in C6 glioma cells expressing thyroid hormone receptors.

Thyroid hormone (triiodothyronine; T3) has been shown to control the expression of beta 1-adrenergic receptors (beta 1-AR) in cardiac myocytes, but not in C6 glioma cells. This cell specificity has been attributed to low expression of T3 receptors and high expression of the c-erbA alpha 2 splice variant that interferes with the action of T3. To check this hypothesis we have expressed the c-erbA/thyroid hormone receptor (TR) alpha 1 gene in C6 glioma cells and investigated their response to thyroid hormone. Cells expressing TR alpha 1, but not wild-type cells, were responsive to T3 as shown by increased expression of mitochrondrial hydroxymethylglutaryl CoA synthase after T3 exposure. However, T3 had no effect on beta 1-AR gene expression in either set of cells. The beta 1-AR mRNA concentrations were, however, altered by retinoic acid (RA) treatment. Retinoic acid caused a rapid up-regulation of beta 1-AR mRNA levels that was blocked by cycloheximide. Retinoic acid did not increase the beta 1-AR gene transcription rate in run-on experiments. These results indicate an indirect post-transcriptional effect of RA. Control of beta 1-AR expression in C6 cells is also exerted at the translational level, because there was no correlation between mRNA and protein induction, as determined by radioligand binding studies. We conclude that lack of responsiveness of the beta 1-AR gene in C6 cells to T3 is not due to high expression of c-erbA alpha 2 but to undefined cell-specific factors.

Interaction of bilirubin with gangliosides.

Gangliosides seem to play an important role in the interaction of the neurotoxic pigment bilirubin with the synaptosomal plasma membrane (Vázquez et al. [1988] J. Biol. Chem. 263, 1255-1265). In this report, a further characterization of the bilirubin-ganglioside interaction is presented. The interaction is fast, and it is observed at any pH in the range 7.0-9.0. The characteristics of the interaction are different from those observed with other membrane lipids, including sphingomyelin. A model of binding to a single population of sites is able to adequately fit the experimental data. This model predicts a decrease in the tendency of bilirubin to interact with gangliosides and an increase in the binding capacity as the pH is decreased from 8.0 to 7.0. Our data would suggest a role for gangliosides in explaining the preferential accumulation of bilirubin in some areas of the brain and the toxic effect of this pigment in neuronal membrane-related functions.

Sensitive procedures for measuring chloride fluxes mediated by the purified glycine receptor incorporated into phospholipid vesicles.

Two novel methods have been developed to directly measure chloride influx into purified glycine receptor-containing phospholipid vesicles. Using a method based on the fluorescence quenching of a chloride-sensitive probe entrapped into the vesicles, a chloride influx was detected which could be enhanced by glycine and completely abolished by the antagonist strychnine. In addition, by tracing the 36Cl- influx into the proteoliposomes, a stimulatory effect of both glycine and beta-alanine could be seen, which can be inhibited by strychnine and other glycine antagonists. These data, together with a previous report demonstrating ligand-mediated iodide fluxes in the same preparation (Biochemistry, 28 (1989) 6405-6409), clearly demonstrate the utility of the reconstituted receptor preparation to investigate some ion channel and pharmacological properties of the glycine receptor.