Establishment and characterization of RNA-edited serotonin 2C receptor isoform cell models and alteration of amyloid precursor protein ectodomain secretion in HEK293 APPSwe cells

RNA editing is a mechanism for generating molecular diversity by altering the genetic code at the level of RNA. The 5-HT(2C) receptor is the only G protein-coupled receptor known to be edited. It has been reported that the non-edited 5-HT(2C) receptor stimulates secretion of the APP metabolite APP ectodomain (APPs). However, it remains unknown whether RNA-edited 5-HT(2C) receptors can also affect APPs secretion. In this study, cDNAs of five non-edited or partially/fully edited 5-HT(2C) receptor isoforms (INI, VNI, VNV, VSV and VGV) were stably transfected into HEK293APPSwe cells to detect the cell proliferation and APPs secretion. The results demonstrated that the overexpression of INI and VNI caused increased proliferation of host cells while VNV, VSV and VGV caused inverse effects (P?相似文献   

Serotonin 5-HT2C Receptor RNA Editing Alters Receptor Basal Activity

Rat and human serotonin 5-HT2C receptor isoforms were evaluated for agonist-independent activation of inositol phosphate production in COS-7 cells. The nonedited isoform (5-HT(2C-INI)) displayed the greatest basal activity, stimulating inositol phosphate production fourfold over the fully edited isoform (5-HT(2C--VGV)). All of the other isoforms tested displayed intermediate levels of basal activity. Decreasing receptor expression levels by 50% produced a parallel decrease in basal activity. 5-HT stimulated inositol phosphate production twofold over basal levels through the 5-HT(2C-INI) receptor and eightfold over basal levels through the 5-HT(2C-VGV) receptor but produced similar maximal levels of inositol phosphate. 5-HT competition for [3H]mesulergine binding to 5-HT(2C-INI) best fit a two-site analysis with K(H) = 7.6 nM and K(L) = 160 nM, whereas 5-HT(2C-VGV) best fit a one-site model with Ki = 163 nM. [3H]5-HT labeled 36% of the total population of 5-HT(2C-INI) receptors labeled by [3H]mesulergine but only 12% of 5-HT(2C-VGV) receptors. [H]5-HT K(D) values increased from 5.1 nM for 5-HT(2C-INI) to 20 nM for 5-HT(2C-VGV). [3H]Mesulergine K(D) values were the same for both isoforms. 5-HT EC50 values for inositol phosphate production increased from 6.1 nM for 5-HT(2C-INI) to 30 nM for 5-HT(2C-VGV). These results demonstrate that RNA editing decreases 5-HT2C receptor basal activity, agonist affinity, and potency, indicating that RNA editing may play a role in regulating serotonergic signal transduction and response to drug therapy.     

Altered G protein-coupling functions of RNA editing isoform and splicing variant serotonin2C receptors

Different isoforms of serotonin subtype 2C receptor (5-HT(2C)R) with altered G protein-coupling efficacy are generated by RNA editing, which converts genomically encoded adenosine residues into inosines. In combination, editing of five sites all located within the second intracellular loop region of 5-HT(2C)R mRNA changes the gene-encoded Ile, Asn, and Ile at positions 156, 158, and 160, respectively. We analyzed the G protein-coupling functions of previously unreported editing isoform receptors. An approximately 13-fold reduction in the agonist potency for G protein-coupling stimulation as well as a significantly reduced basal level activity was observed with the thalamus-specific isoform carrying Ile156, Gly158, and Val160 (5-HT(2C)R-IGV). In contrast, the agonist was four- to five-fold less potent with 5-HT(2C)R-MSV and -IDV, detected in the amygdala and choroid plexus, respectively, indicating a dominant role for the amino acid residue at position 158 in receptor functions. We also identified a splicing variant receptor with a truncated C terminus that displayed no ligand binding capacity or G protein-coupling activity. Examination of the alternatively spliced RNA encoding this truncated receptor suggests that editing of this variant RNA occurs after completion of splicing, resulting in complete editing at all five sites.     

Differences in conformational properties of the second intracellular loop (IL2) in 5HT(2C) receptors modified by RNA editing can account for G protein coupling efficiency

Adenosine-to-inosine RNA editing events that have been demonstrated for 5HT (2C) receptors resulted in alterations of the amino acid sequence at positions 156, 158 and 160 in the intracellular loop 2 (IL2) region. The edited receptor isoforms were shown to have reduced basal activity, but similar maximum responses to agonist binding. To identify the molecular mechanism of these pharmacological effects of editing we explored the conformational properties of the edited IL2 in comparison with the wild type. The results from conformational studies of the IL2 isoforms, using biased Monte Carlo simulations with an implicit solvent model based on a screened Coulomb potential, show that the compared loops differ in their preferred spatial orientations as a result of differences in the conformational space that is accessible to them by energy criteria. For the IL2 of the unedited (5HT (2C-INI) ) receptor, the preference for structures oriented towards the 7TM bundle is larger than for the 5HT (2C-VGV) edited receptor. This difference in preferred orientation can affect the association of IL2 with other intracellular loop domains involved in G protein coupling and hence the coupling efficiency. The results illustrate the high sensitivity of the system to small changes in the interaction surface presented to other intracellular loops, and/or the G protein.     

AGS proteins, GPR motifs and the signals processed by heterotrimeric G proteins

A long term objective of our research effort is to define factors that influence the specificity and efficiency of signal propagation by heterotrimeric G-proteins (G). G-proteins play a central role in cellular communication mediating the cell response to numerous hormones and neurotransmitters. A major determinant of signalling specificity for heterotrimeric G-proteins is the cell specific expression of the subtypes of the primary signalling entities, receptor, G and effector (E). Another major site for regulating signalling specificity lies at the R-G or G-E interface where these interactions are influenced by cell architecture, the stoichiometry of signalling components and accessory proteins that may segregate the receptor to microdomains of the cell, regulate the efficiency and/or specificity of signal transfer and/or influence the activation state of G-protein independent of a classical G-protein coupled receptor. One strategy to address these issues in our laboratory involves the identification of cellular proteins that regulate the transfer of signal from receptor to G or directly influence the activation state of G independent of a classical G-protein coupled receptor. We identified three proteins, AGS1, AGS2 and AGS3 (for Activators of G-protein Signaling), that activated heterotrimeric G-protein signalling pathways in the absence of a typical receptor. AGS1, 2 and 3 interact with different subunits and/or conformations of heterotrimeric G-proteins, selectively activate different G-proteins, provide unexpected mechanisms for regulation of the G-protein activation cycle and have opened up a new area of research related to the cellular role of G-proteins as signal transducers.     

RNA Editing of the Glutamate Receptor Subunits GluR2 and GluR6 in Human Brain Tissue

Abstract: Editing of mRNA in the coding region of the second transmembrane domain of glutamate receptor subunits GluR2, GluR5, and GluR6 involves a change of the base A in genomic DNA to the base G in mRNA as described in rat brain. To determine whether this reaction occurs in humans as well as rats, we studied RNA editing of GluR2 and GluR6 in human brain. We compared the extent of editing in controls and cases with Huntington's disease. To assay the extent of editing in brain RNA, first strand cDNA was amplified using the polymerase chain reaction yielding a product across the region of the second transmembrane spanning segment in which editing takes place in rats. The PCR product was incubated with the restriction enzyme BbvI, which recognizes the sequence GCAGC present in the nonedited sequence of the mRNA in subunits GluR2 and GluR6. Thus, BbvI cuts the nonedited version but leaves the edited version intact. As in the rat, the GluR2 subunit mRNA was completely edited in human brain. The GluR6 subunit was nearly completely edited in all gray matter structures investigated including cortex, striatum, thalamus, hippocampus, amygdala, and cerebellum with extent of editing ranging from 89% in the cerebellum to 95% in the cortex and striatum. No significant differences in the extent of RNA editing were apparent in control versus Huntington's disease brains. To compare the extent of editing in neurons and glia in the brain, editing in cerebral cortex (predominantly gray matter and thus neurons) was compared with editing in corpus callosum (white matter and thus nearly completely glial cells). In white matter, GluR2 was completely edited, whereas GluR6 was only ～10% edited compared with ～90% edited in gray matter. Thus, these studies indicate that RNA editing is seen in human brain as well as rat brain and that the extent of editing is similar in Huntington's disease compared with controls. The differences in editing in white matter for GluR6, but not for GluR2, suggest that different templates could be subject to different editing activities that undergo tissue-specific regulation.     

Basal activity of GIRK5 isoforms

G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.     

Extent of RNA editing of glutamate receptor subunit GluR5 in different brain regions of the rat

Summary 1. The structure and function of glutamate receptor subunits GluR2, GluR5, and GluR6 are changed by RNA editing. This reaction produces a base transition in the second transmembrane spanning region. The triplet CAG (coding for glutamine) is changed to CGG (coding for arginine). This transition has a pronounced effect on calcium fluxes through the respective ion channels, because calcium currents decrease with the rate of editing.2. In the present study the extent of RNA editing of the glutamate receptor subunit GluR5 was studied in different brain regions of control rats using a newly developed analysis system. This system is based on restriction analysis of the polymerase chain reaction (PCR) product, derived from reverse-transcribed mRNA as template, with the enzymeBbv1.Bbv1 recognizes the sequence of the nonedited receptor subunit around the edited base (sequence GCAGC) but not that of the edited subunit (sequence GCGGC; A edited to G).3. Total RNA was isolated from the cerebral cortex, striatum, hippocampus, thalamus, hypothalamus, cerebellum, pons/medulla oblongata, and white matter and reverse transcribed into cDNA. The region across the edited sequence was amplified by PCR using GluR5-specific primers and the cDNA as template. PCR products were cleaned by ethanol precipitation, incubated withBbv1, and electrophoresed on an agarose gel together with standards. Gels were photographed and the extent of GluR5 mRNA editing was quantified using an image analysis system. A calibration curve was obtained using PCR products amplified from plasmids with edited and nonedited GluR5 as inserts.4. In the brain of control rats the extent of RNA editing of the GluR5 subunit amounted to 62±6.0% of total (cortex), 43±5.3% (striatum), 52±5.3% (hippocampus), 91±6.3% (thalamus), 85±10.2% (hypothalamus), 82±6.5% (cerebellum), 88±6.8% (pons/medulla oblongata), and 41±2.7% (white matter).5. The extent of RNA editing varied, thus, considerably in different brain regions, being lowest in the white matter and striatum and highest in the thalamus and pons/medulla oblongate. RNA editing of glutamate receptor subunits may play an important role in the control of calcium fluxes through non-N-methyl-D-aspartate receptor channels in different physiological and/or pathological states of the brain.     

Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3

We have shown that progesterone (10 pM-10 nM) and progesterone covalently bound to bovine serum albumin (P-CMO BSA; 100 pM-1 microM) rapidly increased (within 5 s) the cytosolic free Ca(2+) concentration and inositol 1,4,5 trisphosphate (InsP(3)) formation in confluent female and male rat osteoblasts via a pertussis toxin-insensitive G-protein. The activation of G-proteins coupled to effectors such as phospholipase C (PLC) is an early event in the signal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta1 and PLC-beta 3 were involved in the Ca(2+) mobilization and InsP(3) formation induced by both progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamma 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta gamma subunits were not. The membrane effects were independent of the concentration of nuclear progesterone receptor, because the concentration of nuclear progesterone receptors was lower in male than in female osteoblasts. These data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of second messengers without involving the nuclear receptor.     

Receptor-independent activators of heterotrimeric G-protein signaling pathways

Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif. Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins. As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae. We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor. beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers. AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1. In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS. Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor. AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways. AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.     

RNA editing and alternative splicing of human serotonin 2C receptor in schizophrenia

Serotonin 2C receptor (5-HT2CR) heterogeneity in the brain occurs mostly from two different sources: (i) 5-HT2CR mRNA undergoes adenosine-to-inosine editing events at five positions, which leads to amino acid substitutions that produce receptor variants with different pharmacological properties; (ii) 5-HT2CR mRNA is alternatively spliced, resulting in a truncated mRNA isoform (5-HT2CR-tr) which encodes a non-functional serotonin receptor. 5-HT2CR mRNA editing efficiencies and the expression of the full-length and the truncated 5-HT2CR mRNA splice isoforms were analyzed in the prefrontal cortex of elderly subjects with schizophrenia vs. matched controls (ns = 15). No significant differences were found, indicating that there are no alterations in editing or alternative splicing of 5-HT2CRs that are associated with schizophrenia in persons treated with antipsychotic medications. Quantitation of 5-HT2CR and 5-HT2CR-tr mRNA variants revealed that the expression of 5-HT2CR-tr was approximately 50% of that observed for the full-length isoform.     

The roles of phospholipase C activation and alternative ADAR1 and ADAR2 pre-mRNA splicing in modulating serotonin 2C-receptor editing in vivo

The serotonin 2C receptor (5-HT2CR), a Gq-protein-coupled neurotransmitter receptor, exists in multiple isoforms that result from RNA editing of five exonic adenosines that are converted to inosines. In the adult brain, editing of 5-HT2C pre-mRNA exhibits remarkable plasticity in response to environmental and neurochemical stimuli. Here, we investigated two potential mechanisms underlying these plastic changes in adult 5-HT2CR editing phenotypes in vivo: activation of phospholipase C (PLC) and alternative splicing of pre-mRNA encoding the editing enzymes ADAR1 and ADAR2. Studies on two inbred strains of mice (C57Bl/6 and Balb/c) revealed that sustained stimulation of PLC—a downstream effector of activated Gαq protein—increased editing of forebrain neocortical 5-HT2C pre-mRNA at two sites known to be targeted by ADAR2. Moreover, changes in relative expression of the alternatively spliced “a” and “b” mRNA isoforms of ADAR1 and ADAR2 also correlate with changes in 5-HT2CR editing. The site-specific changes in 5-HT2CR editing detected in mice with different “a” over “b” ADAR mRNA isoform ratios only partially overlap with those evoked by sustained PLC activation and are best explained by the increased editing efficiency of ADAR1. Thus, activation of PLC and alternative splicing of ADAR pre-mRNA have both overlapping and specific roles in modulating 5-HT2CR editing phenotypes.     

Plant hormone perception and action: a role for G-protein signal transduction?

Plants perceive and respond to a profusion of environmental and endogenous signals that influence their growth and development. The G-protein signalling pathway is a mechanism for transducing extracellular signals that is highly conserved in a range of eukaryotes and prokaryotes. Evidence for the existence of G-protein signalling pathways in higher plants is reviewed, and their potential involvement in plant hormone signal transduction evaluated. A range of biochemical and molecular studies have identified potential components of G-protein signalling in plants, most notably a homologue of the G-protein coupled receptor superfamily (GCR1) and the G alpha and G beta subunits of heterotrimeric G-proteins. G-protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate heterotrimeric G-proteins in gibberellin and possibly auxin signalling. Antisense suppression of GCR1 in Arabidopsis leads to a phenotype which supports a role for this receptor in cytokinin signalling. These observations suggest that higher plants have at least some of the components of G-protein signalling pathways and that these might be involved in the action of certain plant hormones.