RNA Editing of the 5HT2C Receptor

RNA editing is a posttranscriptional 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. Editing of the 5-HT2C receptor was discovered by comparing sequences from genomic DNA and cDNAs from the rat striatum when four A-to-G discrepancies were identified in rat brain (Burns et al. 1997). It was proposed that these four adenosine residues in the 5-HT2C receptor pre-mRNA were converted into inosines in the mature mRNA by enzymes called adenosine deaminases that act on RNA (ADAR). The ADAR family consists of three members: ubiquitously expressed ADAR1 and ADAR 2, and ADAR3, which expression appears to be restricted to brain (Chen et al. 2000; Sanders-Bush et al. 2003). ADAR enzymes bind to double-stranded RNA and catalyze the conversion of adenosine to inosine by hydrolytic deamination. Because inosines have base-pairing properties similar to that of guanosines, they are recognized and translated as guanosine by the ribosomes. RNA transcripts encoding the human 5-HT2C receptor undergo adenosine-inosine (A-I) editing events at five positions, A, B, C, D, and E (Fig. 5.1), altering the amino acid sequence in the putative second intracellular loop of the protein and resulting in at least 21 discrete mRNA species encoding 14 different editing variants of the 5-HT2C receptor (Burns et al. 1997; Niswender et al. 1998). In human brain, the genomic (unedited) receptor expresses amino acids isoleucine, asparagine, and isoleucine (INI) at positions 156, 158, and 160, respectively (in the sequence IRNPI in the intracellular loop 2), whereas conver-

Fig. 5.1 RNA editing sites of 5-HT2C receptor mRNA. This figure depicts the human 5-HT2C receptor with seven membrane-spanning domains and an amino terminus facing the extracellular space. The genomic DNA encoding the second intracellular loop contains six mRNA editing sites (white oblongs). Five of those mRNA editing sites (A, B, E, C, and D) are modified by adenosine deaminase (ADAR1 and/or ADAR2), and one (cyt) is modified by cytidine deaminase (CDA). ADAR edits the mRNA sequences from adenosine to inosine (A ^ I), and CDA edits the mRNA sequences from cytidine to uridine (C ^ U), resulting in cDNA sequences that are modified from the genomic sequences. This results in the translation of amino acids that are distinct from those coded by genomic DNA. Amino acids are designated by their one-letter codes (I for isoleucine, R for arginine, etc.) followed by the superscripted amino acid number in the sequence of the 5-HT2C receptor protein (Adapted from Niswender et al. 1998; Tohda et al. 2006)

Fig. 5.1 RNA editing sites of 5-HT2C receptor mRNA. This figure depicts the human 5-HT2C receptor with seven membrane-spanning domains and an amino terminus facing the extracellular space. The genomic DNA encoding the second intracellular loop contains six mRNA editing sites (white oblongs). Five of those mRNA editing sites (A, B, E, C, and D) are modified by adenosine deaminase (ADAR1 and/or ADAR2), and one (cyt) is modified by cytidine deaminase (CDA). ADAR edits the mRNA sequences from adenosine to inosine (A ^ I), and CDA edits the mRNA sequences from cytidine to uridine (C ^ U), resulting in cDNA sequences that are modified from the genomic sequences. This results in the translation of amino acids that are distinct from those coded by genomic DNA. Amino acids are designated by their one-letter codes (I for isoleucine, R for arginine, etc.) followed by the superscripted amino acid number in the sequence of the 5-HT2C receptor protein (Adapted from Niswender et al. 1998; Tohda et al. 2006)

sion of all three amino acids results in valine, glycine, and valine (VGV), valine, serine, and valine (VSV), valine, asparagine, and valine (VNV), or valine, aspartic acid, and valine (VDV) (Niswender et al. 1998; Backstrom et al. 1999; Fitzgerald et al. 1999). These are referred to as 5-HT2C-INI, 5-HT2C-VNV, 5-HT2C-VSV, 5-HT2C-VGV, and 5-HT2C-vdv receptors. Other partially edited human receptor variants include 5-HT , 5-HT , 5-HT , 5-HT , 5-HT , 5-HT , 5-HT , 5-HT

2C-VNI 2C-VSI 2C-ISI 2C-INV 2C-ISV 2C-IGI 2C-VGI 2C-

IDI, and 5-HT2C-idv receptors. In addition to the five A-to-I RNA editing sites, a novel sixth site has recently been reported, which is deaminated from cytidine to uracil by citidine deaminase; this variant is thought to plays role in neuronal differentiation (Flomen et al. 2004; Tohda et al. 2006).

In the rat 5-HT2C receptor, only four (A-I) conversion sites (termed sites A-D) have been identified, resulting in 11 discrete mRNAs and seven different receptor protein isoforms (Fitzgerald et al. 1999; Niswender et al. 1999). Because the second intracellular loop of the protein is involved in the interaction of 5-HT2C receptors with G proteins, specific isoforms of 5-HT2C receptors show different coupling profiles, agonist potency, and ligand binding affinity (Niswender et al. 1999; Herrick-Davis et al. 1999). They also show varying degrees of constitutive activity, ranging from pronounced for the wild-type 5-HT2C-INI, to intermediate for partially edited isoforms such as 5-HT2C-VSV, to minimal for the fully edited 5-HT2C-VGV iso-forms (Niswender et al. 1999; Herrick-Davis et al. 1999). The variants also exhibit differential abilities to mobilize intracellular Ca2+ and to stimulate accumulation of inositol phosphates (Backstrom et al. 1999; Fitzgerald et al. 1999).

The 5-HT2C receptor is the only G-protein-coupled receptor known to be edited, and even mRNAs encoding closely related 5-HT2A and 5-HT2B receptors have not been shown to undergo RNA editing (Niswender et al. 1998). RNA editing of the 5-HT2C receptor is prominent in the central nervous system; indeed, most of the receptors in the brain exist as edited isoforms (Burns et al. 1997; Fitzgerald et al. 1999; Niswender et al. 2001). Because the 5-HT2C receptor is involved in the pathogenesis of various psychological disorders, RNA editing of the 5-HT2C receptor has been proposed to play role in the etiology of schizophrenia (Sodhi et al. 2001), depression (Tohda et al. 2006), and affective disorders (Niswender et al. 2001; Gurevich et al. 2002).

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