RÉSUMÉ
Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) approaches to transfect melanopsin into cortical pyramidal cells maintained in organotypic slice culture. Whole cell recordings from transfected neurons indicated that application of blue light effectively activated the transfected melanopsin to elicit the canonical biphasic modulation of membrane excitability previously associated with the activation of GPCRs coupling to Gαq-11 Remarkably, full mimicry of exogenous agonist concentration could be obtained with pulses as short as a few milliseconds, suggesting that their triggering required a single melanopsin activation-deactivation cycle. The resulting temporal control over melanopsin activation allowed us to compare the activation kinetics of different components of the electrophysiological response. We also replaced the intracellular loops of melanopsin with those of the 5-HT2A receptor to create a light-activated GPCR capable of interacting with the 5-HT2A receptor interacting proteins. The resulting chimera expressed weak activity but validated the potential usefulness of melanopsin as a tool for the study of G protein signaling in CNS neurons.
Sujet(s)
Cortex cérébral/cytologie , Phototransduction/physiologie , Neurones/physiologie , Récepteurs couplés aux protéines G/métabolisme , Opsines des bâtonnets/métabolisme , Acétylcholine/pharmacologie , Analyse de variance , Animaux , Animaux nouveau-nés , Techniques in vitro , Lumière , Phototransduction/effets des médicaments et des substances chimiques , Phototransduction/génétique , Neurones/effets des médicaments et des substances chimiques , Agents neuromédiateurs/pharmacologie , Techniques de culture d'organes , Techniques de patch-clamp , Stimulation lumineuse , Quinoxalines/pharmacologie , Rats , Rat Sprague-Dawley , Récepteurs couplés aux protéines G/génétique , Opsines des bâtonnets/génétique , Tétrodotoxine/pharmacologie , TransfectionRÉSUMÉ
The simplistic idea that seven transmembrane receptors are single monomeric proteins that interact with heterotrimeric G-proteins after agonist binding is definitively out of date. Indeed, GPCRs (G-protein-coupled receptors) are part of multiprotein networks organized around scaffolding proteins. These GIPs (GPCR-interacting proteins) are either transmembrane or cytosolic proteins. Proteomic approaches can be used to get global pictures of these 'receptosomes'. This approach allowed us to identify direct but also indirect binding partners of serotonin receptors. GIPs are involved in a wide range of functions including control of the targeting, trafficking and signalling of GPCRs. One of them, Shank, which is a secondary and tertiary partner of metabotropic and ionotropic glutamate receptors, respectively, can induce the formation of a whole functional glutamate 'receptosome' and the structure to which it is associated, the dendritic spine.
Sujet(s)
Protéines de transport/composition chimique , Protéines adaptatrices de la transduction du signal/composition chimique , Animaux , Protéines de transport/métabolisme , Cytosol/métabolisme , Humains , Modèles biologiques , Protéines de tissu nerveux , Liaison aux protéines , Conformation des protéines , Structure tertiaire des protéines , Protéomique , Récepteur de l'AMPA/composition chimique , Récepteurs sérotoninergiques/composition chimique , Transduction du signal , Synapses/métabolismeRÉSUMÉ
Somatic mutations leading to constitutively active G-protein coupled receptors (GPCRs) are responsible for certain human diseases. A consistent structural description of the molecular change underlying the conversion of GPCRs from an inactive R state to an active R* state is lacking. Here, we show that a series of constitutively active 5-HT4 receptors (mutated or truncated in the C-terminal and the third intracellular loop) were characterized by an increase in their denaturation rate at 55 degrees C. The thermal denaturation kinetics were monophasic, suggesting that we were measuring mainly the denaturation rate of R*. Analysis of these kinetics revealed that constitutively active C-terminal domain mutants, were due to a change in the J constant governing the R/R* equilibrium. However, the constitutive activity of the receptor mutated within the third intracellular loop was the result of both a change in the allosteric J constant and a change in the R* conformation.
Sujet(s)
Récepteurs sérotoninergiques/génétique , Récepteurs sérotoninergiques/métabolisme , Animaux , Acide aspartique/composition chimique , Technique de Western , Cellules COS , AMP cyclique/métabolisme , ADN complémentaire/métabolisme , Immunotransfert , Immunohistochimie , Cinétique , Modèles chimiques , Mutation , Liaison aux protéines , Conformation des protéines , Dénaturation des protéines , Structure secondaire des protéines , Structure tertiaire des protéines , Récepteurs sérotoninergiques/composition chimique , Récepteurs de la sérotonine de type 5-HT4 , Sérotonine/pharmacologie , Température , Facteurs temps , TransfectionRÉSUMÉ
By using the yeast two-hybrid system, we previously isolated a cDNA clone encoding a novel member of the multivalent PDZ protein family called MUPP1 containing 13 PDZ domains. Here we report that the C terminus of the 5-hydroxytryptamine type 2C (5-HT(2C)) receptor selectively interacts with the 10th PDZ domain of MUPP1. Mutations in the extreme C-terminal SSV sequence of the 5-HT(2C) receptor confirmed that the SXV motif is critical for the interaction. Co-immunoprecipitations of MUPP1 and 5-HT(2C) receptors from transfected COS-7 cells and from rat choroid plexus verified this interaction in vivo. Immunocytochemistry revealed an SXV motif-dependent co-clustering of both proteins in transfected COS-7 cells as well as a colocalization in rat choroid plexus. A 5-HT(2C) receptor-dependent unmasking of a C-terminal vesicular stomatitis virus epitope of MUPP1 suggests that the interaction triggers a conformational change within the MUPP1 protein. Moreover, 5-HT(2A) and 5-HT(2B), sharing the C-terminal EX(V/I)SXV sequence with 5-HT(2C) receptors, also bind MUPP1 PDZ domains in vitro. The highest MUPP1 mRNA levels were found in all cerebral cortical layers, the hippocampus, the granular layer of the dentate gyrus, as well as the choroid plexus, where 5-HT(2C) receptors are highly enriched. We propose that MUPP1 may serve as a multivalent scaffold protein that selectively assembles and targets signaling complexes.
Sujet(s)
Protéines de transport/composition chimique , Protéines de transport/métabolisme , Récepteurs sérotoninergiques/composition chimique , Récepteurs sérotoninergiques/métabolisme , Séquence d'acides aminés , Animaux , Encéphale/métabolisme , Cellules COS , Membrane cellulaire/métabolisme , Chlorocebus aethiops , Plexus choroïde/métabolisme , Séquence consensus , Épitopes/composition chimique , Protéines et peptides de signalisation intracellulaire , Données de séquences moléculaires , Rats , Récepteur de la sérotonine de type 5-HT2A , Récepteur de la sérotonine de type 5-HT2B , Récepteur de la sérotonine de type 5-HT2C , Récepteurs sérotoninergiques/génétique , Protéines recombinantes/composition chimique , Protéines recombinantes/métabolisme , Alignement de séquences , Similitude de séquences d'acides aminés , Transcription génétique , Transfection , Virus de la stomatite vésiculeuse de type Indiana/composition chimiqueRÉSUMÉ
We studied the pharmacological properties of twenty-four 5-hydroxytryptamine (5-HT)(4) receptor ligands known to act as antagonists on 5-HT(4) receptors positively coupled to adenylyl cyclase endogenously expressed in mouse colliculi neurons. In COS-7 cells expressing human or mouse 5-HT(4(a)) receptors (100-8000 fmol/mg of protein), we found neutral antagonists, partial agonists, and inverse agonists. The majority of neutral antagonists belong to the benzodioxanyl ketone class, whereas partial agonists belong to different chemical classes. We found only two inverse agonists, GR 125487 and SB 207266, which are both indoles. Analysis of pharmacological characteristics of the constitutively active wild-type and constitutively active mutated receptors revealed that 1) the ratio between the efficiencies of the full agonist 5-HT and the partial agonist RS 23597 was invariable when the receptor density increased, but was dependent on receptor structure; 2) similarly, the efficacy of the inverse agonist SB 207266 was not dependent on receptor density but was dependent on receptor structure; 3) when the receptor concentration increased, the EC(50) values of the full agonist 5-HT were not modified and the increase in basal constitutive activity, as well as its stimulation by 5-HT, followed a parallel evolution; and 4) the stimulation of basal constitutive activity by 5-HT was not modified by the overexpression of Galphas. All these results indicate that in COS-7 cells, the coupling of the 5-HT(4) receptor to adenylyl cyclase was linear with no indication of spare receptors even at high receptor density (8 pmol/mg). These results are also in accordance with a precoupling between the activated receptor (f(R*)) and adenylyl cyclase. Such observations allowed us to use the two-state model to calculate the constant J, i.e., the equilibrium allosteric constant denoting the ratio of the receptor in the inactive versus active state (J = [R]/[R*]). We found that J was a receptor structural characteristic, independent of receptor density.
Sujet(s)
AMP cyclique/métabolisme , Récepteurs sérotoninergiques/métabolisme , Antisérotonines/pharmacologie , Adenylate Cyclase/métabolisme , Régulation allostérique , Aminobenzoates/pharmacologie , Animaux , Cellules COS , Relation dose-effet des médicaments , Sous-unités alpha Gs des protéines G/métabolisme , Humains , Indoles/pharmacologie , Souris , Modèles biologiques , Mutation , Pipéridines/pharmacologie , Récepteurs sérotoninergiques/effets des médicaments et des substances chimiques , Récepteurs sérotoninergiques/génétique , Récepteurs de la sérotonine de type 5-HT4 , Sulfonamides/pharmacologie , Transfection , para-AminobenzoatesRÉSUMÉ
We have cloned new 5-Hydroxytryptamine 4 (5-HT4) receptor splice variants from mouse (m5-HT4(e)R and m5-HT4(f)R), rat (r5-HT4(e)R), and human brain tissue (h5-HT4(e)R) which differ, as do the previously described 5-HT4 receptor variants, in the length and composition of their intracellular C termini after the common splicing site (L358). These new variants have a unique C-terminal sequence made of two PV repeats and are only expressed in brain tissue. All of the 5-HT4 receptor splice variants have a high constitutive activity when expressed at low and physiological densities (<500 fmol/mg protein). At similar density, they showed a much higher constitutive activity than the native and the mutated beta2-adrenergic receptors. The constitutive activity of the new splice variants with short C-terminal sequences (m5-HT4(e)R and m5-HT4(f)R) was higher than that of the long C-terminal sequence variants (m5-HT4(a)R and m5-HT4(b)R). This may indicate that the short variants have a higher capacity for isomerization from the inactive to the active conformation. Moreover, we further identified a sequence within the C-terminal tail upstream of L358, rich in serine and threonine residues, that played a crucial role in maintaining 5-HT4R under its inactive conformation.