Emmanuel Hermans

Structural, signalling and regulatory properties of the group I metabotropic glutamate receptors: prototypic family C G-protein-coupled receptors.

In 1991 a new type of G-protein-coupled receptor (GPCR) was cloned, the type 1a metabotropic glutamate (mGlu) receptor, which, despite possessing the defining seven-transmembrane topology of the GPCR superfamily, bore little resemblance to the growing number of other cloned GPCRs. Subsequent studies have shown that there are eight mammalian mGlu receptors that, together with the calcium-sensing receptor, the GABA(B) receptor (where GABA is gamma-aminobutyric acid) and a subset of pheromone, olfactory and taste receptors, make up GPCR family C. Currently available data suggest that family C GPCRs share a number of structural, biochemical and regulatory characteristics, which differ markedly from those of the other GPCR families, most notably the rhodopsin/family A GPCRs that have been most widely studied to date. This review will focus on the group I mGlu receptors (mGlu1 and mGlu5). This subgroup of receptors is widely and differentially expressed in neuronal and glial cells within the brain, and receptor activation has been implicated in the control of an array of key signalling events, including roles in the adaptative changes needed for long-term depression or potentiation of neuronal synaptic connectivity. In addition to playing critical physiological roles within the brain, the mGlu receptors are also currently the focus of considerable attention because of their potential as drug targets for the treatment of a variety of neurological and psychiatric disorders.

Biochemical and pharmacological control of the multiplicity of coupling at G-protein-coupled receptors.

For decades, it has been generally proposed that a given receptor always interacts with a particular GTP-binding protein (G-protein) or with multiple G-proteins within one family. However, for several G-protein-coupled receptors (GPCR), it now becomes generally accepted that simultaneous functional coupling with distinct unrelated G-proteins can be observed, leading to the activation of multiple intracellular effectors with distinct efficacies and/or potencies. Multiplicity in G-protein coupling is frequently observed in artificial expression systems where high densities of receptors are obtained, raising the question of whether such complex signalling reveals artefactual promiscuous coupling or is a genuine property of GPCRs. Multiple biochemical and pharmacological evidence in favour of an intrinsic property of GPCRs were obtained in recent studies. Thus, there are now many examples showing that the coupling to multiple signalling pathways is dependent on the agonist used (agonist trafficking of receptor signals). In addition, the different couplings were demonstrated to involve distinct molecular determinants of the receptor and to show distinct desensitisation kinetics. Such multiplicity of signalling at the level of G-protein coupling leads to a further complexity in the functional response to agonist stimulation of one of the most elaborate cellular transmission systems. Indeed, the physiological relevance of such versatility in signalling associated with a single receptor requires the existence of critical mechanisms of dynamic regulation of the expression, the compartmentalisation, and the activity of the signalling partners. This review aims at summarising the different studies that support the concept of multiplicity of G-protein coupling. The physiological and pharmacological relevance of this coupling promiscuity will be discussed.

Neuroinflammation and regulation of glial glutamate uptake in neurological disorders.

Oxidative stress, neuroinflammation, and excitotoxicity are frequently considered distinct but common hallmarks of several neurological disorders, including Parkinsons disease, amyotrophic lateral sclerosis, multiple sclerosis, and Alzheimers disease. Although neuron degeneration and death are the ultimate consequences of these pathological processes, it is now widely accepted that alterations in the function of surrounding glial cells are key features in the progression of these diseases. In response to alteration in their local environment, microglia, commonly considered the resident immune cells of the nervous parenchyma, become activated and release a variety of soluble factors. Among these, proinflammatory cytokines and free radicals actively participate in the degenerative insults. In addition, excitotoxic neuronal damage resulting from excessive glutamate is frequently associated with impaired handling of extracellular glutamate by gliotic astrocytes. Although several research projects have focused on the biochemical mechanisms of the regulation of glial glutamate transporters, a relationship between activation of microglia and modulation of astrocytic glutamate uptake is now suggested. The aim of this review is to summarize and discuss the data showing an influence of inflammatory mediators and related free radicals on the expression and activity of glial glutamate transporters.

Directed evolution of a G protein-coupled receptor for expression, stability, and binding selectivity

We outline a powerful method for the directed evolution of integral membrane proteins in the inner membrane of Escherichia coli. For a mammalian G protein-coupled receptor, we arrived at a sequence with an order-of-magnitude increase in functional expression that still retains the biochemical properties of wild type. This mutant also shows enhanced heterologous expression in eukaryotes (12-fold in Pichia pastoris and 3-fold in HEK293T cells) and greater stability when solubilized and purified, indicating that the biophysical properties of the protein had been under the pressure of selection. These improvements arise from multiple small contributions, which would be difficult to assemble by rational design. In a second screen, we rapidly pinpointed a single amino acid substitution in wild type that abolishes antagonist binding while retaining agonist-binding affinity. These approaches may alleviate existing bottlenecks in structural studies of these targets by providing sufficient quantities of stable variants in defined conformational states.

Functionally selective cannabinoid receptor signalling : therapeutic implications and opportunities

The CB(1) and CB(2) cannabinoid receptors are G protein-coupled receptors (GPCRs) recognized by a variety of endogenous ligands and activating multiple signalling pathways. This multiplicity of ligands and intracellular transduction mechanisms supports a complex control of physiological functions by the endocannabinoid system, but requires a finely tuned regulation of the signalling events triggered on receptor activation. Here we review the diverse signalling pathways activated by the cannabinoid receptors and discuss the mechanisms allowing for specificity in the associated functional responses triggered by endogenous or exogenous ligands. At variance with the classical concept that all agonists at a given GPCR induce a similar repertoire of downstream events in all tissues, we also summarize the experimental evidence supporting the existence of functional selectivity and protean agonism at cannabinoid receptors. By placing emphasis on the ligand- or constitutive activity-dependent specifications of receptor-G protein coupling, these concepts explain how distinct cannabinoid ligands may activate specific downstream mediators. Finally, although both the diversity and specificity in cannabinoid signalling are now established in vitro, few data are available from in vivo studies. Therefore, we conclude this review by examining the experimental evidence supporting the physiological relevance of this complexity in the cannabinoid system. The ability to selectively manipulate physiological functions, through activation of defined signalling cascades, will in all likelihood help in the development of efficacious and safe cannabinoid-based therapeutics for a variety of indications.

Analogues and homologues of N-palmitoylethanolamide, a putative endogenous CB(2) cannabinoid, as potential ligands for the cannabinoid receptors

The presence of CB(2) receptors was reported in the rat basophilic cell line RBL-2H3 and N-palmitoylethanolamide was proposed as an endogenous, potent agonist of this receptor. We synthesized a series of 10 N-palmitoylethanolamide homologues and analogues, varying by the elongation of the fatty acid chain from caproyl to stearoyl and by the nature of the amide substituent, respectively, and evaluated the affinity of these compounds to cannabinoid receptors in the rat spleen, RBL-2H3 cells and CHO-CB(1) and CHO-CB(2) receptor-transfected cells. In rat spleen slices, CB(2) receptors were the predominant form of the cannabinoid receptors. No binding of [(3)H]SR141716A was observed. [(3)H]CP-55,940 binding was displaced by WIN 55,212-2 and anandamide. No displacement of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 by palmitoylethanolamide derivatives was observed in rat spleen slices. In RBL-2H3 cells, no binding of [(3)H]CP-55,940 or [(3)H]WIN 55,212-2 could be observed and conversely, no inhibitory activity of N-palmitoylethanolamide derivatives and analogues was measurable. These compounds do not recognize the human CB(1) and CB(2) receptors expressed in CHO cells. In conclusion, N-palmitoylethanolamide was, in our preparations, a weak ligand while its synthesized homologues or analogues were essentially inactive. Therefore, it seems unlikely that N-palmitoylethanolamide is an endogenous agonist of the CB(2) receptors but it may be a compound with potential therapeutic applications since it may act via other mechanisms than cannabinoid CB(1)-CB(2) receptor interactions.

Acute up-regulation of glutamate uptake mediated by mGluR5a in reactive astrocytes.

Excitatory transmission in the CNS necessitates the existence of dynamic controls of the glutamate uptake achieved by astrocytes, both in physiological conditions and under pathological circumstances characterized by gliosis. In this context, this study was aimed at evaluating the involvement of group I metabotropic glutamate receptors (mGluR) in the regulation of glutamate transport in a model of rat astrocytes undergoing in vitro activation using a cocktail of growth factors (G5 supplement). The vast majority of the cells were found to take up aspartate, mainly through the glutamate/aspartate transporter (GLAST), and at least 60% expressed functional mGluR5a. When exposed for 15 s to the selective group I mGluR agonist (S)‐3,5‐dihydroxyphenylglycine, reactive astrocytes showed a significant increase in their capacity to take up aspartate. This effect was confirmed at the single‐cell level, since activation of mGluRs significantly increased the initial slope of aspartate‐dependent Na+ entry associated with the activity of glutamate transporters. This up‐regulation was inhibited by an antagonist of mGluR5 and, more importantly, was sensitive to a specific glutamate transporter 1 (GLT‐1) blocker. The acute influence of mGluR5 on aspartate uptake was phospholipase C‐ and protein kinase C‐dependent, and was mimicked by phorbol esters. We conclude that mGluR5a contributes to a dynamic control of GLT‐1 function in activated astrocytes, acting as a glial sensor of the extracellular glutamate concentration in order to acutely regulate the excitatory transmission.

Mechanisms of regulation of neurotensin receptors.

Since its discovery in 1973, the neuropeptide neurotensin has been demonstrated to be involved in the control of a broad variety of physiological activities in both the central nervous system and in the periphery. Pharmacological studies have shown that the biological effects elicited by neurotensin result from its specific binding to cell membrane neurotensin receptors that have been characterized in various tissue and in cell preparations. In addition, it is now well documented that most of these responses are subject to rapid desensitization. Such desensitization results in transient responses to sustained peptide applications, or to tachyphylaxis during successive stimulations in the same conditions. More recently, desensitization of neurotensin signalling was investigated at the cellular and molecular levels. In cultured cells, regulation at the second messenger level, receptor internalization, and receptor down-regulation processes have been reported. These are proposed to play a critical role in the control of cell responsiveness to neurotensin. This review aims to compile recent data on the different biochemical processes involved in the regulation of the neurotensin receptor and to discuss the physiological consequences of this regulation in vivo.

Modulation of the neuronal dopamine transporter activity by the metabotropic glutamate receptor mGluR5 in rat striatal synaptosomes through phosphorylation mediated processes

There is considerable evidence that the activity of the neuronal dopamine transporter (DAT) is dynamically regulated and a putative implication of its phosphorylation in this process has been proposed. However, there is little information available regarding the nature of physiological stimuli that contribute to the endogenous control of the DAT function. Based on the close relationship between glutamatergic and dopaminergic systems in the striatum, we investigated the modulation of the DAT activity by metabotropic glutamate receptors (mGluRs). Short‐term incubations of rat striatal synaptosomes with micromolar concentrations of the group I mGluR selective agonist (S)‐3,5‐dihydroxyphenylglycine were found to significantly decrease the DAT capacity and efficiency. This alteration was completely prevented by a highly selective mGluR5 antagonist, 2‐methyl‐6‐(phenylethynyl)pyridine hydrochloride (MPEP). The effect of (S)‐3,5‐dihydroxyphenylglycine was also inhibited by staurosporine and by selective inhibitors of protein kinase C and calcium calmodulin‐dependent protein kinase II. Co‐application of okadaic acid prolonged the transient effect of the agonist, supporting a critical role for phosphorylation in the modulation of the DAT activity by mGluRs. In conclusion, we propose that striatal mGluR5 contribute to the control of the DAT activity through concomitant activation of both protein kinase C and calcium calmodulin‐dependent protein kinase II.

Chimerization of astroglial population in the lumbar spinal cord after mesenchymal stem cell transplantation prolongs survival in a rat model of amyotrophic lateral sclerosis

Adult mesenchymal stem cells (MSCs) exhibit neuroprotective properties when introduced into the degenerating central nervous system through different putative mechanisms including secretion of growth factors and transdifferentiation. In the present study, we injected MSCs into the cerebrospinal fluid of symptomatic hSOD1G93A rats, a transgenic animal model of familial amyotrophic lateral sclerosis (ALS) expressing a mutated form of the human superoxide dismutase. MSCs were found to infiltrate the nervous parenchyma and migrate substantially into the ventral gray matter, where motor neurons degenerate. Even though overall astrogliosis was not modified, MSCs differentiated massively into astrocytes at the site of degeneration. The intrathecal delivery of MSCs and the subsequent generation of healthy astrocytes at symptomatic stage decreased motor neuron loss in the lumbar spinal cord, preserving motor functions and extending the survival of hSOD1G93A rats. This neuroprotection was correlated with decreased inflammation, as shown by the lower proliferation of microglial cells and the reduced expressiontion of COX‐2 and NOX‐2. Together, these data highlight the protective capacity of adult MSC‐derived astrocytes when grafted into the central nervous system and illustrate an attractive strategy to target excessive inflammation in ALS.