The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) elicits a wide array of physiological effects by binding to several receptor subtypes. The 5-HT2 family of receptors belongs to a large group of seven-transmembrane-spanning G-protein-coupled receptors and includes three receptor subtypes (5-HT2A, 5-HT(2B) and 5-HT(2C)) which are linked to phospholipase C, promoting the hydrolysis of membrane phospholipids and a subsequent increase in the intracellular levels of inositol phosphates and diacylglycerol. Here we show that transcripts encoding the 2C subtype of serotonin receptor (5-HT(2C)R) undergo RNA editing events in which genomically encoded adenosine residues are converted to inosines by the action of double-stranded RNA adenosine deaminase(s). Sequence analysis of complementary DNA isolates from dissected brain regions have indicated the tissue-specific expression of seven major 5-HT(2C) receptor isoforms encoded by eleven distinct RNA species. Editing of 5-HT(2C)R messenger RNAs alters the amino-acid coding potential of the predicted second intracellular loop of the receptor and can lead to a 10-15-fold reduction in the efficacy of the interaction between receptors and their G proteins. These observations indicate that RNA editing is a new mechanism for regulating serotonergic signal transduction and suggest that this post-transcriptional modification may be critical for modulating the different cellular functions that are mediated by other members of the G-protein-coupled receptor superfamily.

RNA editing is a post-transcriptional modification resulting in an alteration of the primary nucleotide sequence of RNA transcripts by mechanisms other than splicing. The enzymatic conversion of adenosine to inosine by RNA editing has been identified within an increasing number of RNA transcripts, indicating that this modification represents an important mechanism for the generation of molecular diversity. Several of these editing events have been shown to have significant consequences for cellular function. Transcripts encoding the Bsubunit (GluR-B) of the ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype of glutamate receptor undergo RNA editing events that modulate both the ion permeation and electrophysiological characteristics of this glutamate-gated ion channel (1-3). Mice that are deficient in their ability to edit GluR-B transcripts die at 3 weeks of age due to epileptic seizures, suggesting that editing of GluR-B RNA is important in the modulation of normal glutamatergic neurotransmission (4). These results suggest that the consequences of editing events within other, diverse RNA molecules might also have important ramifications for cellular function.The monoamine 5-hydroxytryptamine (serotonin; 5-HT) 1 interacts with a large family of receptors to induce signal transduction events important in the modulation of neurotransmission (5). The 2C subtype of serotonin receptor (5-HT 2C R) is a member of the G protein-coupled receptor superfamily and stimulates phospholipase C, resulting in the production of inositol phosphates and diacylglycerol (6). We have recently shown that RNA transcripts encoding the rat, mouse, and human 5-HT 2C R undergo adenosine-to-inosine RNA editing events at five positions, termed A, B, C, D, and E (previously termed CЈ) (7,8), resulting in an alteration of amino acid coding potential within the putative second intracellular loop of the encoded protein. Editing at the A site, or at the A and B sites concurrently, converts an isoleucine to a valine at amino acid 156 of the human receptor, while editing at the B position alone generates a methionine codon at this site (Fig. 1A). Editing at C converts asparagine 158 to a serine; editing at E generates an aspartate at this site, and conversion at both C and E generates a glycine triplet. Editing at D results in the substitution of a valine for an isoleucine at position 160.We have previously demonstrated a decrease in 5-HT potency when interacting with the rat 5-HT 2C R isoform 5-HT 2C-VSV , which is simultaneously edited at the A, B, C, and D positions encoding valine, serine, and valine at positions 157, 159, 161, respectively. This decrease in potency was reflected as a rightward shift in the dose-response curve for [ 3 H]inositol monophosphate accumulation (7). We proposed that the decreased potency resulted from a reduced G protein coupling efficiency induced by the introduction of novel amino acids into the second intracellular loop, a region known to be important for G protein coupling (9 -16). In the present study,...

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Fifty years ago, increased whole-blood serotonin levels, or hyperserotonemia, first linked disrupted 5-HT homeostasis to Autism Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene (SLC6A4) has been associated with whole blood 5-HT levels and ASD susceptibility. Previously, we identified multiple gain-of-function SERT coding variants in children with ASD. Here we establish that transgenic mice expressing the most common of these variants, SERT Ala56, exhibit elevated, p38 MAPK-dependent transporter phosphorylation, enhanced 5-HT clearance rates and hyperserotonemia. These effects are accompanied by altered basal firing of raphe 5-HT neurons, as well as 5HT 1A and 5HT 2A receptor hypersensitivity. Strikingly, SERT Ala56 mice display alterations in social function, communication, and repetitive behavior. Our efforts provide strong support for the hypothesis that altered 5-HT homeostasis can impact risk for ASD traits and provide a model with construct and face validity that can support further analysis of ASD mechanisms and potentially novel treatments. development | monoamine | neurotransmitter A utism spectrum disorder (ASD) is a male-predominant disorder that is characterized by deficits in social interactions and communication, as well as repetitive behavior (1). Hyperserotonemia, or increased whole-blood serotonin [i.e., 5-hydroxytryptamine (5-HT)], is a well replicated biomarker that is present in approximately 30% of subjects with ASD (2, 3). Some data suggest an association of hyperserotonemia with stereotyped or self-injurious behavior, but results have been inconsistent (4, 5). Despite the high heritability of whole-blood 5-HT levels (6), a mechanistic connection between hyperserotonemia and specific components of the pathophysiology of ASD remains enigmatic. In blood, 5-HT is contained almost exclusively in platelets (7) that lack 5-HT biosynthetic capacity but accumulate the monoamine via the antidepressant-sensitive serotonin transporter (SERT; 5-HTT). A genome-wide study of whole-blood 5-HT as a quantitative trait found association with the SERT-encoding gene SLC6A4, as well as with ITGB3, which encodes the SERT-interacting protein integrin β3. In both cases, the strongest evidence for association was found in males (8-10). Linkage studies in ASD also implicate the 17q11.2 region containing SLC6A4, again with stronger evidence in males (11, 12).As common SLC6A4 variants are only modestly associated with ASD (13), we and our colleagues previously screened SLC6A4 for rare variants in multiplex families that demonstrate strong linkage to 17q11.2. In this effort, we identified five rare SERT coding variants, each of which confers increased 5-HT transport in transfected cells as well as in lymphoblasts derived from SERT variant-expressing probands (11,14,15). We found the most common of these variants, Ala56 (allele frequency in subjects of European ancestry of 0.5-1%), to be overtransmitted to autism probands, and to be associated with both rigid-compulsive behavior and sensory aversion (11,16). N...

Nonspecific binding is a frequently encountered problem with fluorescent labeling of tissue cultures when labeled with quantum dots. In these studies various cell lines were examined for nonspecific binding. Evidence suggests that nonspecific binding is related to cell type and may be significantly reduced by functionalizing quantum dots with poly(ethylene glycol) ligands (PEG). The length of PEG required to give a significant reduction in nonspecific binding may be as short as 12-14 ethylene glycol units.

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Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the ␤ 2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37°C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N, Ndimethyl-N-(iodoacetyl)-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the ␤ 2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.In classical receptor theory, binding of agonist has been considered essential for receptor activation and transmission of the biological signal across the plasma membrane. However, expression of many members of the seven-transmembrane, G protein-coupled receptor family in transfected cell lines has revealed that activation of intracellular messenger systems can occur in the absence of agonists (1-6). Moreover, it has been found that mutations in several different regions of G proteincoupled receptors can dramatically enhance agonist-independent receptor activity and in some cases confer oncogenic properties to the receptor (1-3, 7-14). Nevertheless, the molecular and structural changes in the receptor that are responsible for constitutive, agonist-independent activation are poorly understood. According to the prevailing two-state model for activation of G protein-coupled receptors, constitutive activation has been explained as a disturbance of the normal equilibrium between the inactive (R) 1 state and the active (R*) state leading to a higher proportion of receptor molecules in the active R* state (3, 15). However, the conformational state of a constitutively active receptor protein has so far only been deduced from its effects on intracellular second messenger systems; therefore, this hypothesis has not been substantiated by any direct structural analysis.Recently, we have described the use of fluorescence spectroscopy to directly analyze ligand-induced conformational changes in the purified wild type ␤ 2 adrenergic receptor (16). The approach is based on the sensitivity of many fluorescent molecules to the polarity of their molecular environment (17). In this study, we use the same techniques to directly study conformational changes associated with constitutive activation. Our...