Antonio De Blasi

Molecular determinants of metabotropic glutamate receptor signaling.

Metabotropic glutamate (mglu) receptors are implicated in the regulation of many physiological and pathological processes in the CNS, including synaptic plasticity, learning and memory, motor coordination, pain transmission and neurodegeneration. Several recent studies have elucidated the molecular determinants of mglu receptor signaling and show that several mechanisms acting at different steps in signal propagation are involved. We attempt to offer an integrated view on how homologous and heterologous mechanisms regulate the initial steps of signal propagation, mainly at the level of mglu-receptor-G-protein coupling. Particular emphasis is placed on the role of phosphorylation mechanisms mediated by protein kinase C and G-protein-coupled receptor kinases, and on the emerging importance of some members of the regulators of G-protein signaling family, such as RGS2 and RGS4, which facilitate the GTPase activity that is intrinsic to the alpha-subunits of G(q) and G(i).

Metabotropic Glutamate Receptor Subtypes as Targets for Neuroprotective Drugs

Metabotropic glutamate (mGlu) receptors have been considered as potential targets for neuroprotective drugs, but the lack of specific drugs has limited the development of neuroprotective strategies in experimental models of acute or chronic central nervous system (CNS) disorders. The advent of potent and centrally available subtype-selective ligands has overcome this limitation, leading to an extensive investigation of the role of mGlu receptor subtypes in neurodegeneration during the last 2 years. Examples of these drugs are the noncompetitive mGlu1 receptor antagonists, CPCCOEt and BAY-36-7620; the noncompetitive mGlu5 receptor antagonists, 2-methyl-6-(phenylethynyl)pyridine, SIB-1893, and SIB-1757; and the potent mGlu2/3 receptor agonists, LY354740 and LY379268. Pharmacologic blockade of mGlu1 or mGlu5 receptors or pharmacologic activation of mGlu2/3 or mGlu4/7/8 receptors produces neuroprotection in a variety of in vitro or in vivo models. MGlu1 receptor antagonists are promising drugs for the treatment of brain ischemia or for the prophylaxis of neuronal damage induced by synaptic hyperactivity. MGlu5 receptor antagonists may limit neuronal damage induced by a hyperactivity of N-methyl-d-aspartate (NMDA) receptors, because mGlu5 and NMDA receptors are physically and functionally connected in neuronal membranes. A series of observations suggest a potential application of mGlu5 receptor antagonists in chronic neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer disease. MGlu2/3 receptor agonists inhibit glutamate release, but also promote the synthesis and release of neurotrophic factors in astrocytes. These drugs may therefore have a broad application as neuroprotective agents in a variety of CNS disorders. Finally, mGlu4/7/8 receptor agonists potently inhibit glutamate release and have a potential application in seizure disorders. The advantage of all these drugs with respect to NMDA or AMPA receptor agonists derives from the evidence that mGlu receptors do not “mediate,” but rather “modulate” excitatory synaptic transmission. Therefore, it can be expected that mGlu receptor ligands are devoid of the undesirable effects resulting from the inhibition of excitatory synaptic transmission, such as sedation or an impairment of learning and memory.

G protein-coupled receptors: heterologous regulation of homologous desensitization and its implications.

Two patterns of rapid desensitization have been characterized for G protein-coupled receptors: homologous desensitization, which mainly involves G protein-coupled receptor kinases and arrestins, and heterologous desensitization, which mainly involves protein kinases A (PKA) and C (PKC). In this review, Tsu Tshen Chuang and colleagues discuss evidence to show that PKA and PKC can modify the functional state of the G protein-coupled receptor kinases/arrestin homologous desensitization machinery, providing a novel level of cross-talk in signal transduction. Studies on regulation of G protein-coupled receptor kinases and arrestins confirm that the functional state of this machinery may have important consequences for cellular responsiveness and may represent new targets for therapeutic strategies.

Role of 5-HT2C receptors in the control of central dopamine function

Substantial evidence suggests that the functional status of the mesocorticolimbic dopamine (DA) system originating in the ventral tegmental area is under a phasic and tonic inhibitory control by the 5-HT system that acts by stimulating 5-HT(2C) receptor subtypes. Indeed, electrophysiological and biochemical data demonstrate that 5-HT(2C) receptor agonists decrease, whereas 5-HT(2C) receptor antagonists enhance, mesocorticolimbic DA function. However, 5-HT(2C) receptors do not appear to play a relevant role in the control of the nigrostriatal DA system originating in the substantia nigra pars compacta. In this article, the role of 5-HT(2C) receptors in the control of brain DA function will be reviewed, and the search for new therapies for neuropsychiatric disorders, such as depression, schizophrenia and drug addiction, based on these findings will be discussed.

Stress-Related Methylation of the Catechol-O-Methyltransferase Val158 Allele Predicts Human Prefrontal Cognition and Activity

DNA methylation at CpG dinucleotides is associated with gene silencing, stress, and memory. The catechol-O-methyltransferase (COMT) Val158 allele in rs4680 is associated with differential enzyme activity, stress responsivity, and prefrontal activity during working memory (WM), and it creates a CpG dinucleotide. We report that methylation of the Val158 allele measured from peripheral blood mononuclear cells (PBMCs) of Val/Val humans is associated negatively with lifetime stress and positively with WM performance; it interacts with stress to modulate prefrontal activity during WM, such that greater stress and lower methylation are related to reduced cortical efficiency; and it is inversely related to mRNA expression and protein levels, potentially explaining the in vivo effects. Finally, methylation of COMT in prefrontal cortex and that in PBMCs of rats are correlated. The relationship of methylation of the COMT Val158 allele with stress, gene expression, WM performance, and related brain activity suggests that stress-related methylation is associated with silencing of the gene, which partially compensates the physiological role of the high-activity Val allele in prefrontal cognition and activity. Moreover, these results demonstrate how stress-related DNA methylation of specific functional alleles impacts directly on human brain physiology beyond sequence variation.

Methamphetamine produces neuronal inclusions in the nigrostriatal system and in PC12 cells

Mice treated with the psychostimulant methamphetamine (MA) showed the appearance of intracellular inclusions in the nucleus of medium sized striatal neurones and cytoplasm of neurones of the substantia nigra pars compacta but not in the frontal cortex. All inclusions contained ubiquitin, the ubiquitin activating enzyme (E1), the ubiquitin protein ligase (E3‐like, parkin), low and high molecular weight heat shock proteins (HSP 40 and HSP 70). Inclusions found in nigral neurones stained for α‐synuclein, a proteic hallmark of Lewy bodies that are frequently observed in Parkinsons disease and other degenerative disorders. However, differing from classic Lewy bodies, MA‐induced neuronal inclusions appeared as multilamellar bodies resembling autophagic granules. Methamphetamine reproduced this effect in cultured PC12 cells, which offered the advantage of a simple cellular model for the study of the molecular determinants of neuronal inclusions. PC12 inclusions, similar to those observed in nigral neurones, were exclusively localized in the cytoplasm and stained for α‐synuclein. Time‐dependent experiments showed that inclusions underwent a progressive fusion of the external membranes and developed an electrodense core. Inhibition of dopamine synthesis by α‐methyl‐p‐tyrosine (αMpT), or administering the antioxidant S‐apomorphine largely attenuated the formation of inclusions in PC12 cells exposed to MA. Inclusions were again observed when αMpT‐treated cells were loaded with l‐DOPA, which restored intracellular dopamine levels.

Regulation of G-protein-coupled receptor kinase subtypes by calcium sensor proteins.

The process of G‐protein‐coupled receptor (GPCR) homologous desensitization is intrinsically related to the function of a class of S/T kinases named G‐protein‐coupled receptor kinases (GRK). GRK family is so far composed of six cloned members, named GRK1 to 6, which are classified into three subfamilies: GRK1 is alone in the first (rhodopsin kinase subfamily), GRK2 and 3 form the second [β‐adrenergic receptor kinase (βARK) subfamily], and GRK4, 5, and 6 constitute the third (GRK4 subfamily). Recent studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor‐specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1, and hippocalcin) are also able to inhibit GRK1 in a calcium‐dependent manner. The ubiquitous calcium binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC50=40–50 nM). A direct interaction between GRK5 and Ca2+/CaM was documented and this binding did not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogs in mediating the regulation of different GRK subtypes by Ca2+. This mechanism, however, is highly selective with respect to the GRK subtypes: GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, but GRK4, 5, and 6, which belong to the GRK4 subfamily are potently inhibited by CaM, which has little or no effect on members of other GRK subfamilies. Calcium‐dependent inhibition of rhodopsin kinase by recoverin represents one of the mechanisms that control adaptation to light. For the other GPCR, CSP‐GRK interaction provides a feedback mechanism that can modulate homologous desensitization of these receptors.— Iacovelli, L., Sallese, M., Mariggió, S., De Blasi, A. Regulation of G‐protein‐coupled receptor kinase subtypes by calcium sensor proteins. FASEB J. 13, 1–8 (1999)

Inhibition of G Protein-coupled Receptor Kinase Subtypes by Ca2+/Calmodulin

G protein-coupled receptor kinases (GRKs) are implicated in the homologous desensitization of G protein-coupled receptors. Six GRK subtypes have so far been identified, named GRK1 to GRK6. The functional state of the GRKs can be actively regulated in different ways. In particular, it was found that retinal rhodopsin kinase (GRK1), but not the ubiquitous βARK1 (GRK2), can be inhibited by the photoreceptor-specific Ca2+-binding protein recoverin through direct binding. The present study was aimed to investigate regulation of other GRKs by alternative Ca2+-binding proteins such as calmodulin (CaM). We found that Gβγ-activated GRK2 and GRK3 were inhibited by CaM to similar extents (IC50 ∼ 2 μM), while a 50-fold more potent inhibitory effect was observed on GRK5 (IC50 = 40 nM). Inhibition by CaM was strictly dependent on Ca2+ and was prevented by the CaM inhibitor CaMBd. Since Gβγ, which is a binding target of Ca2+/CaM, is critical for the activation of GRK2 and GRK3, it provides a possible site of interaction between these proteins. However, since GRK5 is Gβγ-independent, an alternative mechanism is conceivable. A direct interaction between GRK5 and Ca2+/CaM was revealed using CaM-conjugated Sepharose 4B. This binding does not influence the catalytic activity as demonstrated using the soluble GRK substrate casein. Instead, Ca2+/CaM significantly reduced GRK5 binding to the membrane. The mechanism of GRK5 inhibition appeared to be through direct binding to Ca2+/CaM, resulting in inhibition of membrane association and hence receptor phosphorylation. The present study provides the first evidence for a regulatory effect of Ca2+/CaM on some GRK subtypes, thus expanding the range of different mechanisms regulating the functional states of these kinases.

Role of G Protein-coupled Receptor Kinase 4 and β-Arrestin 1 in Agonist-stimulated Metabotropic Glutamate Receptor 1 Internalization and Activation of Mitogen-activated Protein Kinases

The metabotropic glutamate 1 (mGlu1) receptor in cerebellar Purkinje cells plays a key role in motor learning and motor coordination. Here we show that the G protein-coupled receptor kinases (GRK) 2 and 4, which are expressed in these cells, regulate the mGlu1 receptor by at least in part different mechanisms. Using kinase-dead mutants in HEK293 cells, we found that GRK4, but not GRK2, needs the intact kinase activity to desensitize the mGlu1 receptor, whereas GRK2, but not GRK4, can interact with and regulate directly the activated Gαq. In cells transfected with GRK4 and exposed to agonist, β-arrestin was first recruited to plasma membranes, where it was co-localized with the mGlu1 receptor, and then internalized in vesicles. The receptor was also internalized but in different vesicles. The expression of β-arrestin V53D dominant negative mutant, which did not affect the mGlu1 receptor internalization, reduced by 70–80% the stimulation of mitogen-activated protein (MAP) kinase activation by the mGlu1 receptor. The agonist-stimulated differential sorting of the mGlu1 receptor and β-arrestin as well as the activation of MAP kinases by mGlu1 agonist was confirmed in cultured cerebellar Purkinje cells. A major involvement of GRK4 and of β-arrestin in agonist-dependent receptor internalization and MAP kinase activation, respectively, was documented in cerebellar Purkinje cells using an antisense treatment to knock down GRK4 and expressing β-arrestin V53D dominant negative mutant by an adenovirus vector. We conclude that GRK2 and GRK4 regulate the mGlu1receptor by different mechanisms and that β-arrestin is directly involved in glutamate-stimulated MAP kinase activation by acting as a signaling molecule.

Regulation of G protein-coupled receptor kinase subtypes by calcium sensor proteins

G protein-coupled receptor homologous desensitization is intrinsically related to the function of a class of S/T kinases named G protein-coupled receptor kinases (GRK). The GRK family is composed of six cloned members, named GRK1 to 6. Studies from different laboratories have demonstrated that different calcium sensor proteins (CSP) can selectively regulate the activity of GRK subtypes. In the presence of calcium, rhodopsin kinase (GRK1) is inhibited by the photoreceptor-specific CSP recoverin through direct binding. Several other recoverin homologues (including NCS 1, VILIP 1 and hippocalcin) are also able to inhibit GRK1. The ubiquitous calcium-binding protein calmodulin (CaM) can inhibit GRK5 with a high affinity (IC(50)=40-50 nM). A direct interaction between GRK5 and Ca(2+)/CaM was documented and this binding does not influence the catalytic activity of the kinase, but rather reduced GRK5 binding to the membrane. These studies suggest that CSP act as functional analogues in mediating the regulation of different GRK subtypes by Ca(2+). This mechanism is, however, highly selective with respect to the GRK subtypes: while GRK1, but not GRK2 and GRK5, is regulated by recoverin and other NCS, GRK4, 5 and 6, that belong to the GRK4 subfamily, are potently inhibited by CaM, which had little or no effect on members of other GRK subfamilies.