RNA editing of the serotonin 5HT2C receptor and its effects on cell signalling, pharmacology and brain function
Introduction
The revision, or editing, of written work is a concept familiar to most research scientists (and the bane of many). The aim of editing in this instance is to simplify or clarify the content for unambiguous interpretation. Similarly, certain cellular components are also subject to editing but, in the molecular setting, these changes actually promote diversity and ambiguity in transcribed genomic information that can translate into a protein product with often dramatically altered structure and function. This review focuses on one particular edited protein, the serotonin (5HT) 2C receptor (5HT2CR).
The 5HT2CR is a member of the superfamily of seven transmembrane domain receptors (7TMRs) that signal to the internal cellular environment via heterotrimeric guanine nucleotide-binding proteins (G proteins) in response to stimulation of the extracellular surface of the receptor by hormones, neurotransmitters and pharmacological ligands. The overall homology of the 5HT2CR with other members of the 5HT 7TMR family is broad, ranging from a 28% amino acid similarity with 5HT7 receptors to a 57% amino acid similarity with 5HT2A receptors (Hoyer et al., 2002). While the 5HT2CR was one of the first of the 5HT receptor family to be cloned, knowledge of its distribution and physiological functions has not progressed with equivalent pace to some of its cousins. This is, in part, because efforts to add depth to our understanding of this receptor have been hampered by a lack of truly selective 5HT2CR ligands, and the burgeoning number of functional forms of the receptor produced by alternative splicing and RNA editing (see below). Despite this, however, through dedication and ingenuity, many of these problems have been circumvented and a large body of evidence now exists in support of roles for the 5HT2CR in many physiological and patho-physiological roles.
The examination of the anatomical localisation of the 5HT2CR has enabled us to speculate on the role of the receptor in complex behaviours. Various techniques have been used to identify and quantify 5HT2CR expression in tissues, including measuring mRNA expression, 3H-mesulergine autoradiography, and immunohistochemistry. These techniques have shown the 5HT2CR to be almost entirely localised to the central nervous system (CNS), with little evidence to suggest that the receptor is expressed in high abundance in the periphery. Within the CNS, the distribution of 5HT2CR is arguably more extensive than that of the 5HT2A (Cornea-Hébert et al., 1999, Pompeiano et al., 1994) and 5HT2B (Duxon et al., 1997) receptors, with particularly high levels within the epithelial cells of the choroid plexus (Sanders-Bush & Breeding, 1988). Lower levels of expression are observed within limbic areas (prefrontal cortex, anterior olfactory nucleus and the lateral habenular nucleus), hippocampal and associated regions (the pyramidal cells of the CA3 region of the hippocampus, the subiculum and entorhinal cortex, and lateral septal nucleus), amygdala, portions of the basal ganglia (caudate and substantia nigra pars compacta), portions of the mesocortical/mesolimbic pathways (nucleus accumbens and ventral tegmental area), and in the hypothalamus (arcuate, periventricular and ventromedial nuclei) (Clemett et al., 2000, Eberle-Wang et al., 1997, López-Giménez et al., 2001, Marazziti et al., 1999, Mengod et al., 1996, Pasqualetti et al., 1999, Pompeiano et al., 1994, Wright et al., 1995). Little expression has been noted in the cerebellum.
The distribution pattern of the 5HT2CR in the brain is suggestive of specific roles in normal physiology and also, when dysregulated, in the development of certain disease states such as obesity, anxiety, epilepsy, sleep disorders and motor dysfunction. Many of these predictions are supported by data acquired through the use of knock-out mouse models that lack the 5HT2CR, and are summarised in Table 1. A significant body of pharmacological data has also been accumulated that confirms findings from the knock-out models and also reveals additional roles for the receptor that were not phenotypically evident in the genetically modified mice; this is summarised in Table 2. Examples of ligands from this group suggest that selectively targeting the 5HT2CR is viable for certain diseases. For instance, APD356 (lorcaserin) is a 5HT2CR agonist in phase IIb clinical trials for the treatment of obesity (Halford et al., 2007). Selective 5HT2CR antagonists alone have been shown to produce pronounced inhibition of anxiety-like behaviours (Harada et al., 2006, Kennett et al., 1997). Ro 60-0175 (an agonist at 5HT2CR) has been shown to reduce cocaine-induced locomotor activity and self-administration (Fletcher et al., 2004) and also to block some of the addiction-related behaviours associated with Δ9-THC and nicotine (Ji et al., 2006, Zaniewska et al., 2007). This is perhaps through its ability to inhibit the firing rate of dopaminergic neurons in the ventral tegmental area (VTA) (Di Giovanni et al., 2000, Pozzi et al., 2002, Prisco et al., 1994). It is to be hoped that more 5HT2CR-targeting ligands will be discovered that will improve the available pharmacotherapy of these disorders, and a better knowledge of the receptor and its idiosyncrasies may assist in this search.
On a cellular level, the 5HT2CR stimulates intracellular responses via Gαq/11, Gα12/13 and Gαi G proteins, and by doing so can regulate the levels of second messengers such as inositol trisphosphate (Ins(1,4,5)P3), calcium (Ca2+), cyclic AMP, arachadonic acid (Berg et al., 1994, Berg et al., 1998), cyclic GMP (Kaufman et al., 1995) and the activity of extracellular signal-regulated kinases 1 and 2 (ERK1/2) (Werry et al., 2005) and protein kinase C (PKC). Activation of one or more of these pathways will doubtless account for many of the patho-physiological actions attributable to the 5HT2CR discussed above. Given the multitude of roles for the 5HT2CR in cell signalling and the regulation of patho-physiological processes, this receptor is of clear significance as a drug target. However, the 5HT2CR has a number of interesting pharmacological properties, including ligand-independent (i.e. constitutive) activity, pleiotropic signalling and agonist-directed trafficking of receptor stimulus (ADTRS) (see below for further details and references), which make it both a fascinating case study of 7TMR behaviour and a highly complex and challenging drug discovery target. The 5HT2CR is also currently unique among 7TMRs as the only member thus far shown to undergo a process termed RNA editing (Burns et al., 1997), and thus be dramatically diversified in terms of sequence and function.
Section snippets
Mechanistic basis of RNA editing
Several processes are required for genomic DNA sequence to be extracted and used as a template for protein synthesis. The template is messenger RNA (mRNA), and is created by RNA polymerases that read the relevant genomic sequence and create a copy of the information (transcription) which is used to direct protein synthesis (translation). The immediate product of this transcription is often subject to modifications before it becomes available for translation into protein sequence. We herein term
Patterns of editing in the 5HT2CR transcript
The initial demonstration of RNA editing in rat 5HT2CR pre-mRNA identified four specific adenosine residues within the RNA at which the transcript underwent A-to-I conversion (Burns et al., 1997), and these sites were confirmed some time later in humans, along with the discovery of a fifth edited residue (Fitzgerald et al., 1999). These sites are known as A, B, C′ (previously called E), C and D (Fig. 2), and are all contained in the exonic sequence of the mRNA. A sixth site (Site F), that lies
Sub-cellular compartmentalisation of ADAR activity
The spatial localization of RNA editing within the cell has not yet been adequately resolved, although pieces of circumstantial evidence do allow speculation. ADAR1 and ADAR2 both localize (to different degrees) in the nucleolus, with ADAR2 residing almost exclusively there while ADAR1 shuttles rapidly between the nucleolus and the nucleoplasm (Desterro et al., 2003, Sansam et al., 2003). This does not, however, infer that RNA editing is a nucleolar event per se since it is at odds with the
Cellular signalling effects of RNA editing
Experiments in cell lines recombinantly expressing the unedited isoform of the 5HT2CR (5HT2CR-INI) have shown that this receptor transduces extracellular signals into a number of different intracellular responses. The best characterised of these is its ability to stimulate phospholipase Cβ (PLCβ) via Gαq/11 proteins, causing production of inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P3) (Berg et al., 1994, Lutz et al., 1993), the elevation of intracellular Ca2+concentration (Akiyoshi et al., 1995
Modulation of editing by extracellular signals
The traditional view of a receptor signalling system is that it has only two positions: ‘off’ and ‘on’. The 5HT2CR RNA editing system appears to depart from this paradigm, better approximating a rheostat in that there are many variations on activity levels between simply ‘on’ and ‘off’. It is also reasonable to postulate that the expression pattern of 5HT2CR isoforms in a cell may be dynamic, and responsive to changes in the cellular environment. Any dynamic mechanism with such flexibility
Editing changes in disease— hope for future therapies?
An interesting example of how cell lines can help to explain effects seen in whole organisms was provided by the work of Yang et al. (2004), who employed a glioblastoma primary cell line, HTB14, to investigate the effects of interferon-α (IFN-α) on 5HT2CR editing. Recombinant IFN-α is currently widely used in the treatment of chronic viral hepatitis, multiple sclerosis and certain types of malignancy, but its use is complicated by the development of depressive symptoms in patients receiving the
Summary
With only ~ 30,000 distinct genes in the human genome, RNA editing provides a mechanism by which the body can make multiple variants of a particular gene and thus make more of the comparatively few resources it has available to produce such a complex organism. Our understanding of this system is, however, still rather removed from our ability to predict it, influence it and use it to our therapeutic advantage. The ultimate aim of such endeavours is to identify what changes correlate with the
Acknowledgments
Work in the authors' laboratory is funded by project grant nos. 400133 and 436779 of the National Health and Medical Research Council (NHMRC) of Australia. AC is a Senior Research Fellow, and PMS a Principal Research Fellow, of the NHMRC.
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2020, NeuropharmacologyCitation Excerpt :Taken together these results highlight an important and bidirectional influence of serotonin signalling through the 5-HT2C receptor on behaviours which span the impulsive-compulsive spectrum. Since the 5-HT2C receptor protein may be expressed as multiple structural variants with differing functional properties, due to RNA editing and splice variants (Werry et al., 2008; Palacios et al., 2017; Stamm et al., 2017), the deregulation of this system can have profound phenotypic and behavioural consequences. For example, alterations in 5HT2C receptor editing have been observed in individuals with Prader-Willi syndrome, a condition associated with compulsive behaviour, and psychiatric conditions including those associated with impulsive-compulsive spectrum disorder (Dimitropoulos et al., 2000; Kishore and Stamm, 2006; Werry et al., 2008; O'Neill and Emeson, 2012; Di Giovanni and De Deurwaerdère, 2016; Stamm et al., 2017).
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Current address: Psychiatry Discovery Technology Group, GlaxoSmithKline, New Frontiers Science Park, Harlow, UK.