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Laurent Fagni

Publications and source records attributed to Laurent Fagni.

13 recordsLinked to original sources
PubMed2006

MacMARCKS interacts with the metabotropic glutamate receptor type 7 and modulates G protein-mediated constitutive inhibition of calcium channels.

We have previously shown that the interaction of Ca2+/calmodulin with the metabotropic glutamate receptor type 7 (mGluR7) promotes the G-protein-mediated inhibition of voltage-sensitive Ca2+ channels (VSCCs) seen upon agonist activation. Here, we performed a yeast two-hybrid screen of a new-born rat brain cDNA library using the cytoplasmic C-terminal tail of mGluR7 as bait and identified macrophage myristoylated alanine-rich c-kinase substrate (MacMARCKS) as a binding protein. The interaction was confirmed in vitro and in vivo by pull-down assays, immunoprecipitation, and colocalization of mGluR7 and MacMARCKS in transfected HEK293 cells and cultured cerebellar granule cells. Binding of MacMARCKS to mGluR7 was antagonized by Ca2+/calmodulin. In neurons, cotransfection of MacMARCKS with mGluR7, but not mGluR7 mutants unable to bind MacMARCKS, reduced the G-protein-mediated tonic inhibition of VSCCs in the absence of mGluR7 agonist. These results suggest that competitive interactions of Ca2+/calmodulin and MacMARCKS with mGluR7 control the tonic inhibition of VSCCs by G-proteins.

Amino Acid Sequence↗
PubMed2005

Key role of the postsynaptic density scaffold proteins Shank and Homer in the functional architecture of Ca2+ homeostasis at dendritic spines in hippocampal neurons.

A key aspect of postsynaptic function, also important for plasticity, is the segregation within dendritic spines of Ca2+ rises attributable to release from intracellular stores. Previous studies have shown that overexpression in hippocampal neurons of two postsynaptic density (PSD) scaffold proteins, Shank1B and Homer1b, induces spine maturation, including translocation of the intracellular Ca2+ channel inositol trisphosphate receptor (IP3R). The structural and functional significance of these processes remained undefined. Here, we show that in its relocation, IP3R is accompanied by other endoplasmic reticulum (ER) proteins: the Ca2+ pump sarcoendoplasmic reticulum calcium ATPase, the lumenal Ca2+-binding protein calreticulin, the ER lumen-addressed green fluorescent protein, and, to a lesser extent, the membrane chaperone calbindin. The specificity of these translocations was demonstrated by their inhibition by both a Shank1 fragment and the dominant-negative Homer1a. Activation in Shank1B-transfected neurons of the metabotropic glutamatergic receptors 1/5 (mGluRs1/5), which induce IP3 generation with ensuing Ca2+ release from the stores, triggered considerable increases in Ca2+-dependent responses: activation of the big K+ channel, which was revealed by patch clamping, and extracellular signal-regulated protein kinase (ERK) phosphorylation. The interaction of Shank1B and Homer1b appears as the molecular mechanism linking mGluRs1/5, strategically located in the spines, to IP3R with the integration of entire ER cisternas in the PSD and with consequences on both local Ca2+ homeostasis and overall neuronal signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Shank expression is sufficient to induce functional dendritic spine synapses in aspiny neurons.

Shank proteins assemble glutamate receptors with their intracellular signaling apparatus and cytoskeleton at the postsynaptic density. Whether Shank plays a role in spinogenesis and synaptogenesis remained unclear. Here, we report that knock-down of Shank3/prolinerich synapse-associated protein-2 by RNA interference reduces spine density in hippocampal neurons. Moreover, transgene expression of Shank 3 is sufficient to induce functional dendritic spines in aspiny cerebellar neurons. Transfected Shank protein recruits functional glutamate receptors, increases the number and size of synaptic contacts, and increases amplitude, frequency, and the AMPA component of miniature EPSCs, similar to what is observed during synapse developmental maturation. Mutation/deletion approaches indicate that these effects require interactions of Shank3 with the glutamate receptor complex. Consistent with this observation, chronic treatment with glutamate receptor antagonists alters maturation of the Shank3-induced spines. These results strongly suggest that Shank proteins and the associated glutamate receptors participate in a concerted manner to form spines and functional synapses.

Animals↗
PubMed2004

GPCR interacting proteins (GIP).

G protein-coupled receptors (GPCR) interact not only with heterotrimeric G proteins but also with accessory proteins called GPCR interacting proteins (GIP). These proteins have important functions. They are implicated in GPCR targeting to specific cellular compartments, in their assembling into large functional complexes called "receptosomes," in their trafficking to and from the plasma membrane, and in the fine-tuning of their signaling properties. There are several types of GIPs. Some are transmembrane proteins such as another GPCR (homodimerization and heterodimerization), ionic channels, ionotropic receptors, and single transmembrane proteins. The latter is implicated in the fine-tuning of receptor pharmacology or signaling. Other GIPs are soluble proteins interacting mainly with the "magic" C-terminal tail. Among them, PDZ domain-containing proteins are the most abundant. They generally, but not always, interact with the extreme C-terminal domain of GPCRs. Some GIPs interact with specific sequences of the C-terminal such as the Homer binding sequence (-PPxxFR-), the dopamine receptor interacting protein (DRIP) binding sequence (-FxxxFxxxF-), etc. Finally, only few GIPs have been found thus far to interact with the third intracellular loop of GPCRs. The future will tell us if this situation is only due to technical reasons.

Animals↗
PubMed2004

Identification and functional roles of metabotropic glutamate receptor-interacting proteins.

In the mammalian brain, a majority of excitatory synapses use glutamate as a neurotransmitter. Glutamate activates ligand-gated channels (ionotropic receptors) and G protein-coupled (metabotropic) receptors. During the past decade, a number of intracellular proteins have been described to interact with these receptors. These proteins not only scaffold the glutamate receptors at the pre- and post-synaptic membranes, but also regulate their subcellular targeting and intracellular signaling. Thus, identification of these proteins has been essential for further understanding the functions of glutamate receptors. Here we will focus on those proteins that interact with the subgroup of metabotropic glutamate (mGlu) receptors, and review the methods used for their identification, as well as their functional roles in neurons.

Animals↗
PubMed2004

GPCR-GIP networks: a first step in the discovery of new therapeutic drugs?

G protein-coupled receptors (GPCRs) are transmembrane molecules that, on interaction with G proteins upon ligand binding, can associate with a large variety of transmembrane or soluble proteins, termed 'GPCR-interacting proteins' (GIPs). Some special transmembrane GIPs are themselves GPCRs that form homo- or heterodimers, while other transmembrane GIPs are ionic channels, ionotropic receptors and single transmembrane proteins that control GPCR pharmacology and trafficking. Most soluble GIPs interact with the C-termini of GPCRs and often physically link GPCRs to large protein networks, called 'receptosomes', that include ionic channels, protein kinases, small G proteins, cytoskeletal proteins and adhesion molecules. Here, we review the nature and functions of some of these networks, such as the glutamate and serotonin receptosomes, and focus on the fine-tuning of GPCR signaling by GIPs. Finally, we discuss the possibilities for developing new therapeutic drugs capable of modulating GPCR signaling by disrupting or reinforcing specific GPCR-GIP interactions.

Animals↗
PubMed2003

The 'magic tail' of G protein-coupled receptors: an anchorage for functional protein networks.

All cell types express a great variety of G protein-coupled receptors (GPCRs) that are coupled to only a limited set of G proteins. This disposition favors cross-talk between transduction pathways. However, GPCRs are organized into functional units. They promote specificity and thus avoid unsuitable cross-talk. New methodologies (mostly yeast two-hybrid screens and proteomics) have been used to discover more than 50 GPCR-associated proteins that are involved in building these units. In addition, these protein networks participate in the trafficking, targeting, signaling, fine-tuning and allosteric regulation of GPCRs. To date, proteins that interact with the GPCR C-terminus are the most abundant and are the focus of this review.

Allosteric Regulation↗
PubMed2002

Inhibition of voltage-gated Ca2+ channels by antazoline.

We showed recently that imidazolines exert neuroprotection against hypoxia and NMDA toxicity in cerebellar and striatal neuronal cultures, through a voltage-dependent blockade of glutamatergic NMDA receptors. Here, we report that in striatal neuronal cultures from mouse embryos the imidazoline compound, antazoline, inhibits voltage-gated Ca2+ channels by acting at a phencyclidine-like site. This effect was fast, fully reversible, voltage-dependent and predominant on P/Q- and N-type Ca2+ channels. Taken together, these results suggest that imidazolines may elicit neuroprotective effects also by decreasing the release of glutamate through inhibition of presynaptic Ca2+ channels.

Animals↗
PubMed2002

Homer as both a scaffold and transduction molecule.

Increasing evidence shows that scaffold proteins not only control membrane assembly of receptors and channels, but also modulate intracellular signaling by assembled receptors. The Homer family of proteins act as scaffolds to bind clusters of proteins and glutamate receptors at postsynaptic sites. We review results of cloning and gene expression of this protein family, and summarize roles in glutamate receptor function and intracellular signaling in neurons. Homer proteins trigger the localization of metabotropic glutamate receptor subtype 5 (mGlu5 receptor) to the postsynaptic plasma membrane. They can also alter the kinetics and peak amplitude of the intracellular Ca2+ responses of mGlu1 and mGlu5 receptors. Homer proteins can either prevent or promote spontaneous activation of these receptors, depending on the type of Homer protein isoform expressed.

Animals↗
PubMed2002

Homer-dependent cell surface expression of metabotropic glutamate receptor type 5 in neurons.

The metabotropic glutamate (mGlu) receptors are a family of receptors involved in the tuning of fast excitatory synaptic transmission in the brain. Experiments performed in heterologous expression systems suggest that cell surface expression of group I mGlu receptors is controlled by their auxiliary protein, Homer. However, whether or not this also applies to neurons remains controversial. Here we show that in cultured cerebellar granule cells, the group I mGlu receptor subtype, mGlu5, transfected alone is functionally expressed at the surface of these neurons. Transfected Homer1b caused intracellular retention and clustering of this receptor at synaptic sites. Recombinant Homer1a alone did not affect cell surface expression of the receptor, but in neurons transfected with Homer1b, excitation-induced expression of native Homer1a reversed the intracellular retention of mGlu5 receptors, resulting in the receptor trafficking to synaptic membranes. Transfected Homer1a also increased the latency and amplitude of the mGlu5 receptor Ca2+ response. These results indicate that Homer1 proteins regulate synaptic cycling and Ca2+ signaling of mGlu5 receptors, in response to neuronal activity.

Animals↗
PubMed2001

A single subunit (GB2) is required for G-protein activation by the heterodimeric GABA(B) receptor.

Although G-protein-coupled receptors (GPCRs) have been shown to assemble into functional homo or heteromers, the role of each protomer in G-protein activation is not known. Among the GPCRs, the gamma-aminobutyric acid (GABA) type B receptor (GABA(B)R) is the only one known so far that needs two subunits, GB1 and GB2, to function. The GB1 subunit contains the GABA binding site but is unable to activate G-proteins alone. In contrast the GB2 subunit, which does not bind GABA, has an heptahelical domain able to activate G-proteins when assembled into homodimers (Galvez, T., Duthey, B., Kniazeff, J., Blahos, J., Rovelli, G., Bettler, B., Prézeau, L., and Pin, J.-P. (2001) EMBO J. 20, 2152-2159). In the present study, we have examined the role of each subunit within the GB1-GB2 heteromer, in G-protein coupling. To that end, point mutations in the highly conserved third intracellular loop known to prevent G-protein activation of the related Ca-sensing or metabotropic glutamate receptors were introduced into GB1 and GB2. One mutation, L686P introduced in GB2 prevents the formation of a functional receptor, even though the heteromer reaches the cell surface, and even though the mutated subunit still associates with GB1 and increases GABA affinity on GB1. This was observed either in HEK293 cells where the activation of the G-protein was assessed by measurement of inositol phosphate accumulation, or in cultured neurons where the inhibition of the Ca(2+) channel current was measured. In contrast, the same mutation when introduced into GB1 does not modify the G-protein coupling properties of the heteromeric GABA(B) receptor either in HEK293 cells or in neurons. Accordingly, whereas in all GPCRs the same protein is responsible for both agonist binding and G-protein activation, these two functions are assumed by two distinct subunits in the GABA(B) heteromer: one subunit, GB1, binds the agonists whereas the other, GB2, activates the G-protein. This illustrates the importance of a single subunit for G-protein activation within a dimeric receptor.

Animals↗
PubMed2001

Permissive effect of voltage on mGlu 7 receptor subtype signaling in neurons.

G protein-coupled receptors mobilize neuronal signaling cascades which until now have not been shown to depend on the state of membrane depolarization. Thus we have previously shown that the metabotropic glutamate receptor type 7 (mGlu7 receptor) blocks P/Q-type Ca(2+) channels via activation of a G(o) protein and PKC, in cerebellar granule cells. We show here that the transient depolarizations used to evoke the studied Ca(2+) current were indeed permissive to activate this pathway by a mGlu7 receptor agonist. Indeed, sustained depolarization to 0 mV was sufficient to inhibit P/Q-type Ca(2+) channels. This effect involved a conformational change in voltage-gated sodium channel independently of Na(+) flux, activation of a pertussis toxin-sensitive G-protein, inositol trisphosphate formation, intracellular Ca(2+) release, and PKC activity. Subliminal sustained membrane depolarization became efficient in inducing inositol trisphosphate formation, release of intracellular Ca(2+) and in blocking Ca(2+) channels, when applied concomitantly with the mGlu7a receptor agonist, d,l-aminophosphonobutyrate. This synergistic effect of membrane depolarization and mGlu7 receptor activation provides a mechanism by which neuronal excitation could control action of the mGlu7 receptor in neurons.

Animals↗