RÉSUMÉ
A disintegrin and metalloproteinase 10 (ADAM10) is the major α-secretase that catalyzes the amyloid precursor protein (APP) ectodomain shedding in the brain and prevents amyloid formation. Its activity depends on correct intracellular trafficking and on synaptic membrane insertion. Here, we describe that in hippocampal neurons the synapse-associated protein-97 (SAP97), an excitatory synapse scaffolding element, governs ADAM10 trafficking from dendritic Golgi outposts to synaptic membranes. This process is mediated by a previously uncharacterized protein kinase C phosphosite in SAP97 SRC homology 3 domain that modulates SAP97 association with ADAM10. Such mechanism is essential for ADAM10 trafficking from the Golgi outposts to the synapse, but does not affect ADAM10 transport from the endoplasmic reticulum. Notably, this process is altered in Alzheimer's disease brains. These results help in understanding the mechanism responsible for the modulation of ADAM10 intracellular path, and can constitute an innovative therapeutic strategy to finely tune ADAM10 shedding activity towards APP.
Sujet(s)
Protéines ADAM/métabolisme , Protéines adaptatrices de la transduction du signal/métabolisme , Amyloid precursor protein secretases/métabolisme , Appareil de Golgi/métabolisme , Protéines membranaires/métabolisme , Protéine kinase C/métabolisme , Protéines ADAM/composition chimique , Protéine ADAM10 , Protéines adaptatrices de la transduction du signal/composition chimique , Maladie d'Alzheimer/métabolisme , Maladie d'Alzheimer/anatomopathologie , Séquence d'acides aminés , Amyloid precursor protein secretases/composition chimique , Animaux , Cellules COS , Chlorocebus aethiops , Homologue-1 de la protéine Discs Large , Activation enzymatique , Cellules HEK293 , Humains , Protéines membranaires/composition chimique , Modèles moléculaires , Données de séquences moléculaires , Phosphorylation , Phosphothréonine/métabolisme , Densité post-synaptique/métabolisme , Liaison aux protéines , Rats , Synapses/métabolismeRÉSUMÉ
Serotonin 4 receptors (5-HT(4)Rs) were discovered 15 years ago. They are coded by a very complex gene (700Kb, 38 exons) which generates eight carboxy-terminal variants (a, b, c, d, e, f, g, n). Their sequences differ after position L(358). Another variant is characterized by a 14 residue insertion within the extracellular loop 2. Highly selective potent 5-HT(4) receptor antagonists and partial agonists which cross the blood-brain barrier have been synthesized, but a specific full agonist for brain studies is still missing. Based on physiological and behavioral experiments, 5-HT(4)Rs may be targets to treat cognitive deficits, abdominal pain and feeding disorders. One 5-HT(4)R-directed drug (SL65.0155) is already in phase II to treat patients suffering from memory deficits or dementia.
Sujet(s)
Maladies gastro-intestinales/traitement médicamenteux , Récepteurs de la sérotonine de type 5-HT4/classification , Récepteurs de la sérotonine de type 5-HT4/effets des médicaments et des substances chimiques , Antisérotonines/pharmacologie , Agonistes des récepteurs de la sérotonine/pharmacologie , Animaux , Encéphale/effets des médicaments et des substances chimiques , Encéphale/métabolisme , Maladies du système nerveux central/traitement médicamenteux , Maladies du système nerveux central/physiopathologie , Clonage moléculaire , Système digestif/effets des médicaments et des substances chimiques , Système digestif/métabolisme , Évaluation de médicament/méthodes , Protéines G/métabolisme , Maladies gastro-intestinales/physiopathologie , Humains , Immunohistochimie , Récepteurs de la sérotonine de type 5-HT4/génétique , Récepteurs de la sérotonine de type 5-HT4/métabolisme , Transduction du signal , Relation structure-activité , Distribution tissulaireRÉSUMÉ
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
We have recently shown that benzodiazepines with high affinity for peripheral-type receptors such as 4'-chlordiazepam inhibit insulin secretion in vitro. PK 11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxami de], a potent and selective ligand for peripheral benzodiazepine binding sites, was also shown to inhibit insulin release from rat pancreatic islets. Both substances have been reported to interact with mitochondrial binding sites. Hence, the present study was performed to investigate the effects of PK 11195 on insulin secretion induced by either a metabolic or a non-metabolic stimulus. In the rat isolated pancreas perfused at a constant pressure with a Krebs-bicarbonate buffer containing a slightly stimulating glucose concentration (8.3 mM), PK 11195 (10(-7)-10(-5) M) induced a progressive and concentration-dependent decrease in insulin secretion. Simultaneously, we recorded the effects on the pancreatic flow rate; in contrast to 4'-chlordiazepam, previously shown to induce vasodilation in the same preparation, PK 11195 was ineffective. The differential effects of these two substances on vascular resistance and insulin secretion may suggest the existence of different subtypes of peripheral benzodiazepine receptors on pancreatic beta-cells and vessels. A metabolic stimulation of insulin secretion was induced by a glucose increment from 4.2 mM to 8.4 mM or by 2 mM alpha-ketoisocaproic acid (KIC), which is directly metabolized in the mitochondria; these stimulations could be reduced by 10(-5) M PK 11195 (P<0.05). In contrast, the drug was ineffective on the insulin secretion induced by 5 mM or 10 mM KCl in the presence of a nonstimulating glucose concentration (4.2 mM). These results suggest that PK 11195 inhibits insulin secretion by interfering with mitochondrial oxidative metabolism.
Sujet(s)
Antagonistes du récepteur GABA-A , Glucose/antagonistes et inhibiteurs , Insuline/métabolisme , Isoquinoléines/pharmacologie , Pancréas/métabolisme , Système nerveux périphérique/effets des médicaments et des substances chimiques , Animaux , Glucose/pharmacologie , Techniques in vitro , Sécrétion d'insuline , Ilots pancréatiques/effets des médicaments et des substances chimiques , Ilots pancréatiques/métabolisme , Cétoacides/pharmacologie , Mâle , Pancréas/effets des médicaments et des substances chimiques , Chlorure de potassium/pharmacologie , Rats , Rat WistarRÉSUMÉ
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.
Sujet(s)
Épissage alternatif , Récepteurs sérotoninergiques/génétique , Séquence d'acides aminés , Substitution d'acide aminé , Animaux , Encéphale/métabolisme , Cellules cultivées , AMP cyclique/biosynthèse , Délétion de gène , Humains , Souris , Données de séquences moléculaires , Mutation , Conformation des protéines , Rats , Récepteurs bêta-adrénergiques/génétique , Récepteurs sérotoninergiques/composition chimique , Récepteurs de la sérotonine de type 5-HT4 , Similitude de séquences d'acides aminés , Sérotonine/métabolismeRÉSUMÉ
In this paper we discuss 1) the primary structures, pharmacology, and brain distribution of cloned 5-HT4 receptors; 2) the chromosomal localization of the h5-HT4 receptor; 3) whether benzamides are full or partial agonists because of a species or a coupling difference; 4) the intrinsic activity of 5-HT4 receptors and inverse agonism of GR125487 in COS-7 cells but not in colliculi neurons; 5) the modulation of 5-HT4 receptor binding and activity; and 6) the long-term blockade of K+ channels by 5-HT4 agonists and its effect on olfactory memory. We conclude that 1) the cloning of 5-HT4 receptors in different species using RT-PCR from different tissues reveals the presence of several splice variants for 5-HT4 receptors differing in the C-terminal part, downstream from the amino acid L358; 2) the pharmacological properties of 5-HT4 receptors are dependent on the cellular context in which they are expressed; and 3) 5-HT4 agonists can be added to the list of compounds having pro-cognitive properties.
Sujet(s)
Encéphale/métabolisme , Mémoire/physiologie , Perception/physiologie , Récepteurs sérotoninergiques/génétique , Récepteurs sérotoninergiques/physiologie , Odorat , Épissage alternatif , Séquence d'acides aminés , Animaux , Cellules COS , Cartographie chromosomique , Humains , Données de séquences moléculaires , Structure secondaire des protéines , Récepteurs sérotoninergiques/composition chimique , Récepteurs de la sérotonine de type 5-HT4 , Transduction du signal , TransfectionRÉSUMÉ
On the basis of differences in the potencies and intrinsic activity of 5-HT4 receptor agonists in different biological models it has been suggested that there is heterogeneity among 5-HT4 receptors. Here, we report the molecular cloning of several 5-HT4 receptor splice variants in mouse, rat, and human brain. Our data suggest that the differences in efficacy of 5-HT4 ligands on 5-HT4 receptor-mediated responses in several tissues is due to differences in coupling efficiency rather than to the presence of different 5-HT4 receptor isoforms.
Sujet(s)
Épissage alternatif , Variation génétique , Récepteurs sérotoninergiques/génétique , Récepteurs sérotoninergiques/physiologie , Séquence d'acides aminés , Animaux , Encéphale/métabolisme , Cartographie chromosomique , Chromosomes humains de la paire 5 , Clonage moléculaire , Humains , Souris , Données de séquences moléculaires , Rats , Récepteurs sérotoninergiques/composition chimique , Récepteurs de la sérotonine de type 5-HT4 , Protéines recombinantes/biosynthèse , Protéines recombinantes/composition chimique , Protéines recombinantes/métabolisme , Alignement de séquences , Similitude de séquences d'acides aminésRÉSUMÉ
We have isolated a cDNA encoding the 5-HT4S receptor by RT-PCR on poly (A)+ RNA from both human heart and brain. The sequence homology with the rat and mouse 5-HT4 receptors was high: 93.8% of identity in the amino acid sequence. None of the 24 amino acid substitutions observed between rat and human receptors are at positions likely to modify their pharmacology. Comparing the pharmacological properties of six agonists and five antagonists on rat and human 5-HT4S receptors revealed no significant differences. We have analyzed the behavior of renzapride, a full and a partial agonist on mouse colliculi neurons and human heart biological responses respectively. The coupling efficiency of renzapride was two-fold lower than that of 5-HT for the stimulation of 5-HT4S receptors transfected in two different cell lines (LLC-PK1 and COS-7), but increasing the receptor density suppressed the partial agonist effect of renzapride.
Sujet(s)
Adenylate Cyclase/métabolisme , Récepteurs sérotoninergiques/biosynthèse , Séquence d'acides aminés , Animaux , Benzamides/pharmacologie , Chimie du cerveau/effets des médicaments et des substances chimiques , Chimie du cerveau/physiologie , Clonage moléculaire , AMP cyclique/métabolisme , Humains , Cellules LLC-PK1 , Membranes/métabolisme , Souris , Données de séquences moléculaires , Myocarde/métabolisme , Réaction de polymérisation en chaîne , Dosage par compétition , Rats , Récepteurs sérotoninergiques/effets des médicaments et des substances chimiques , Récepteurs sérotoninergiques/génétique , Spécificité d'espèce , SuidaeRÉSUMÉ
Since most of our knowledge on pharmacological properties of brain 5-HT4 receptors have been discussed for mouse colliculi neurons, we cloned the corresponding receptor using the RT-PCR approach. As expected, the homology with the already cloned rat 5-HT(4L) receptor was high, revealing only 16 differences at the amino-acid level. One of the differences, proline75 in mouse, alanine75 in the already published rat sequences was not confirmed. Therefore this proline is part of the consensus sequence present in all 5-HT receptor transmembrane domain II (LVMP). Comparing the affinities of 11 agonists and five antagonists for the cloned mouse receptor (5-HT(4L))expressed in LLCPK1 and the corresponding receptor in mouse colliculi shows an excellent correlation. The transfected mouse 5-HT(4L) receptor stimulated cAMP production. When expressed at high density, it exhibited intrinsic activity. In contrast to the previously described distribution, we found that mRNA encoding for both the short (5-HT(4S))and the long form (5-HT(4L)) of 5-HT4 receptors are expressed in all mouse and rat brain areas.