Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning and functional expression of an Aplysia 5-HT receptor negatively coupled to adenylate cyclase.

ABSTRACT: Serotonin (5-HT) is involved in the control of various behaviors in Aplysia californica, including reproduction, feeding, locomotion, circadian rhythm, synaptic plasticity, and synaptic growth. The large variety of functions of 5-HT is mediated by different receptor subtypes that are coupled to different second-messenger systems. Here, we report the cloning of a cDNA coding for an Aplysia G-protein-coupled 5-HT receptor (5-HTap1). Its deduced amino acid sequence resembles those of the 5-HT1 receptor subfamily. When expressed in stable cell lines, 5-HTap1 exhibits high-affinity binding for the serotonergic radioligand [N-methyl-3H]lysergic acid diethylamide. This binding is competed by several 5-HT agonists and antagonists, and the pharmacological profile of inhibition has some similarities with those of 5-HT1 and 5-HT7 receptors. Application of 5-HT or its agonists 5-carboxamidotryptamine maleate and (+/-)-8-hydroxy-2-(di-n-propyl-amino) tetralin hydrobromide on cells transformed with 5-HTap1 produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation. 5-HTap1 is thus negatively coupled to adenylate cyclase. The production of antiserum against the 5-HTap1 receptor allowed us to examine its expression in animal tissues. The receptor protein is detected in every tissue examined, although it seems only weakly expressed in some samples. The receptor is also found in every ganglia of the nervous system, both in the sheath and in the neurons. 5-HTap1 mRNA is absent from the sheath, indicating that the protein observed there is probably located on the nerve terminals.

SUBMITTER: Angers A

PROVIDER: S-EPMC6793050 | biostudies-literature | 1998 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor.

Project description:Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the vasoactive intestinal peptide (VIP)/secretion/glucagon family of peptides, interacts with a distinct high-affinity receptor (type I receptor) on a number of tissues. These PACAP type I receptors have a high affinity for PACAP and a low affinity for VIP and are present in the hypothalamus and anterior pituitary, where they regulate the release of adrenocorticotropin, luteinizing hormone, growth hormone, and prolactin, and in the adrenal medulla, where they regulate the release of epinephrine. Type I PACAP receptors are also present in high concentrations in testicular germ cells, where they may regulate spermatogenesis, and some transformed cell lines, such as the rat pancreatic acinar carcinoma cell AR4-2J. Here we report the molecular cloning and functional expression of the PACAP type I receptor isolated from an AR4-2J cell cDNA library by cross-hybridization screening with a rat VIP receptor cDNA. The cDNA sequence encodes a unique 495-amino acid protein with seven transmembrane domains characteristic of guanine nucleotide-binding regulatory protein-coupled receptors. A high degree of sequence homology with the VIP, secretin, glucagon-like peptide 1, parathyroid, and calcitonin receptors suggests its membership in this subfamily of Gs-coupled receptors. Results of binding studies and stimulation of cellular cAMP accumulation in COS-7 cells transfected with this cDNA are characteristic of a PACAP type I receptor. Cloning of the PACAP type I receptor will enhance our understanding of its distribution, structure, and functional properties and ultimately increase our understanding of its physiological role.

| S-EPMC46925 | biostudies-other

Molecular cloning of an orphan G-protein-coupled receptor that constitutively activates adenylate cyclase.

Project description:A human gene encoding an orphan G-protein-coupled receptor named ACCA (adenylate cyclase constitutive activator) was isolated from a genomic library using as a probe a DNA fragment obtained by low-stringency PCR. Human ACCA (hACCA) is a protein of 330 amino acids that exhibits all the structural hallmarks of the main family of G-protein-coupled receptors. Expression of hACCA resulted in a dramatic stimulation of adenylate cyclase, similar in amplitude to that obtained with other Gs-coupled receptors fully activated by their respective ligands. This stimulation was obtained in a large variety of stable cell lines derived from various organs, and originating from different mammalian species. hACCA was found to be the human homologue of a recently reported mouse orphan receptor (GPCR21). The mouse ACCA (mACCA) was therefore recloned by PCR, and expression of mACCA in Cos-7 cells demonstrated that the mouse receptor behaved similarly as a constitutive activator of adenylate cyclase. It is not known presently whether the stimulation of adenylate cyclase is the result of a true constitutive activity of the receptor or, alternatively, is the consequence of a permanent stimulation by a ubiquitous ligand. The tissue distribution of mACCA was determined by RNase protection assay. Abundant transcripts were found in the brain, whereas lower amounts were detected in testis, ovary and eye. Various hypotheses concerning the constitutive activity of ACCA and their potential biological significance are discussed.

| S-EPMC1135708 | biostudies-other

Identification of a serotonin receptor coupled to adenylyl cyclase involved in learning-related heterosynaptic facilitation in Aplysia.

Project description:Serotonin (5-HT) plays a critical role in modulating synaptic plasticity in the marine mollusc Aplysia and in the mammalian nervous system. In Aplysia sensory neurons, 5-HT can activate several signal cascades, including PKA and PKC, presumably via distinct types of G protein-coupled receptors. However, the molecular identities of these receptors have not yet been identified. We here report the cloning and functional characterization of a 5-HT receptor that is positively coupled to adenylyl cyclase in Aplysia neurons. The cloned receptor, 5-HT(apAC1), stimulates the production of cAMP in HEK293T cells and in Xenopus oocytes. Moreover, the knockdown of 5-HT(apAC1) expression by RNA interference blocked 5-HT-induced cAMP production in Aplysia sensory neurons and blocked synaptic facilitation in nondepressed or partially depressed sensory-to-motor neuron synapses. These data implicate 5-HT(apAC1) as a major modulator of learning related synaptic facilitation in the direct sensory to motor neuron pathway of the gill withdrawal reflex.

| S-EPMC2732834 | biostudies-literature

Cloning of another human serotonin receptor (5-HT1F): a fifth 5-HT1 receptor subtype coupled to the inhibition of adenylate cyclase.

Project description:An intronless gene encoding an additional human serotonin (5-HT) 5-HT1-like receptor subtype was isolated from a human genomic library with probes obtained from degenerate PCR primers used to amplify 5-HT-receptor-specific sequences. The highest degree of homology was found with the 5-HT1E subtype (70%) and the 5-HT1D alpha (63%) and 5-HT1D beta (60%) receptors. RNA for this gene was detected in the human brain but was not detected in kidney, liver, spleen, heart, pancreas, and testes. High-affinity (Kd = 9.2 nM) 3H-labeled 5-HT binding was detected. Competition studies revealed the following rank order of potencies for serotonergic ligands: 5-HT > sumatriptan >> 5-carboxyamidotryptamine > 8-hydroxy-2(di-1-propylamino)tetralin > spiperone. 5-HT produced a dose-dependent inhibition of forskolin-stimulated cAMP accumulation (EC50 = 7.9 nM) in transfected cells. These properties distinguish this receptor from any previously characterized and establish a fifth 5-HT1-like receptor subtype (5-HT1F) coupled to the inhibition of adenylate cyclase.

| S-EPMC45671 | biostudies-other

Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

Project description:G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A2A receptor and dopamine D2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

| S-EPMC5871782 | biostudies-literature

Expression data from human adipocytes after adenylate cyclase activation

Project description:Progenitors in human vasculature expanded in-vitro were differentiated with adipogenic cocktail for 12 days, following which they were stimulated with forskolin for 7 days Microarrays were used to detail the program of differentiation of thermogenic human adipose cells

2018-01-22 | GSE65190 | GEO

Functional dissection of adenylate cyclase R, an inducer of spore encapsulation.

Project description:Cyclic AMP acting on protein kinase A controls sporulation and encystation in social and solitary amoebas. In Dictyostelium discoideum, adenylate cyclase R (ACR), is essential for spore encapsulation. In addition to its cyclase (AC) domain, ACR harbors seven transmembrane helices, a histidine kinase domain, and two receiver domains. We investigated the role of these domains in the regulation of AC activity. Expression of an ACR-YFP fusion protein in acr(-) cells rescued their sporulation defective phenotype and revealed that ACR is associated with the nuclear envelope and endoplasmic reticulum. Loss of the transmembrane helices (?TM) caused a 60% reduction of AC activity, but ?TM-ACR still rescued the acr(-) phenotype. The isolated AC domain was properly expressed but inactive. Mutation of three essential ATP-binding residues in the histidine kinase domain did not affect the AC activity or phenotypic rescue. Mutation of the essential phosphoryl-accepting aspartate in receivers 1, 2, or both had only modest effects on AC activity and did not affect phenotypic rescue, indicating that AC activity is not critically regulated by phosphorelay. Remarkably, the dimerizing histidine phosphoacceptor subdomain, which in ACR lacks the canonical histidine for autophosphorylation, was essential for AC activity. Transformation of wild-type cells with an ACR allele (?CRA) that is truncated after this domain inhibited AC activity of endogenous ACR and replicated the acr(-) phenotype. Combined with the observation that the isolated AC domain was inactive, the dominant-negative effect of ?CRA strongly suggests that the defunct phosphoacceptor domain acquired a novel role in enforcing dimerization of the AC domain.

| S-EPMC3009899 | biostudies-literature

Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in aplysia sensory neurons.

Project description:Short-term behavioral sensitization of the gill-withdrawal reflex after tail stimuli in Aplysia leads to an enhancement of the connections between sensory and motor neurons of this reflex. Both behavioral sensitization and enhancement of the connection between sensory and motor neurons are importantly mediated by serotonin. Serotonin activates two types of receptors in the sensory neurons, one of which is coupled to the cAMP/protein kinase A (PKA) pathway and the other to the inositol triphosphate/protein kinase C (PKC) pathway. Here we describe a genetic approach to assessing the isolated contribution of the PKA pathway to short-term facilitation. We have cloned from Aplysia an octopamine receptor gene, Ap oa(1), that couples selectively to the cAMP/PKA pathway. We have ectopically expressed this receptor in Aplysia sensory neurons of the pleural ganglia, where it is not normally expressed. Activation of this receptor by octopamine stimulates all four presynaptic events involved in short-term synaptic facilitation that are normally produced by serotonin: (i) membrane depolarization; (ii) increased membrane excitability; (iii) increased spike duration; and (iv) presynaptic facilitation. These results indicate that the cAMP/PKA pathway alone is sufficient to produce all the features of presynaptic facilitation.

| S-EPMC26521 | biostudies-literature

Bordetella pertussis Adenylate Cyclase Toxin Disrupts Functional Integrity of Bronchial Epithelial Layers.

Project description:The airway epithelium restricts the penetration of inhaled pathogens into the underlying tissue and plays a crucial role in the innate immune defense against respiratory infections. The whooping cough agent, Bordetella pertussis, adheres to ciliated cells of the human airway epithelium and subverts its defense functions through the action of secreted toxins and other virulence factors. We examined the impact of B. pertussis infection and of adenylate cyclase toxin-hemolysin (CyaA) action on the functional integrity of human bronchial epithelial cells cultured at the air-liquid interface (ALI). B. pertussis adhesion to the apical surface of polarized pseudostratified VA10 cell layers provoked a disruption of tight junctions and caused a drop in transepithelial electrical resistance (TEER). The reduction of TEER depended on the capacity of the secreted CyaA toxin to elicit cAMP signaling in epithelial cells through its adenylyl cyclase enzyme activity. Both purified CyaA and cAMP-signaling drugs triggered a decrease in the TEER of VA10 cell layers. Toxin-produced cAMP signaling caused actin cytoskeleton rearrangement and induced mucin 5AC production and interleukin-6 (IL-6) secretion, while it inhibited the IL-17A-induced secretion of the IL-8 chemokine and of the antimicrobial peptide beta-defensin 2. These results indicate that CyaA toxin activity compromises the barrier and innate immune functions of Bordetella-infected airway epithelia.

| S-EPMC5820963 | biostudies-literature

Cloning and characterization of a Drosophila serotonin receptor that activates adenylate cyclase.

Project description:Using a strategy based on nucleotide sequence homology between genes encoding receptors that interact with guanine nucleotide-binding proteins, we have isolated Drosophila genomic and cDNA clones encoding a functional serotonin receptor (5HT-dro receptor). This protein is expressed predominantly in Drosophila heads and exhibits highest homology with the human 5HT1A receptor. The predicted structure of the 5HT-dro receptor reveals two unusual features: (i) eight putative transmembrane domains instead of the expected seven and (ii) a Gly-Ser repeat that is a potential glycosaminoglycan attachment site. When stably introduced into mouse NIH 3T3 cells, the 5HT-dro receptor activates adenylate cyclase in response to serotonin and is inhibited by serotonin receptor antagonists such as dihydroergocryptine. The 5HT-dro receptor or closely related receptors might be responsible for the serotonin-sensitive cyclase that has been suggested to play a role in learning and modulation of circadian rhythm in a number of invertebrate systems.

| S-EPMC55076 | biostudies-other

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data