A Conservative, Single-Amino Acid Substitution in the Second Cytoplasmic Domain of the Human Serotonin2C Receptor Alters Both Ligand-Dependent and -Independent Receptor Signaling

Abstract

The post-transcriptional process of mRNA editing changes up to three amino acids in the second intracellular domain (i2) of the serotonin 2C (5-HT 2C ) receptor and alters some signaling characteristics of the receptor. Here, we report that the substitution of valine for isoleucine (I156V; 5-HT 2C-VNI ), which occurs naturally as a result of mRNA editing, alters both ligand-dependent and -independent signaling. Agonist functional selectivity at the 5-HT 2C-VNI receptor differed from the nonedited 5-HT 2C-INI receptor. Ligands with selectivity for phospholipase C (PLC) signaling in 5-HT 2C-INI cells retained this selectivity in 5-HT 2C-VNI -expressing cells. However, ligands with selectivity for phospholipase A2 (PLA2) signaling in 5-HT 2C-INI cells lost the capacity for preferential PLA2 activation in 5-HT 2C-VNI cells. Maximal PLC responses elicited by 5-HT (full agonist) and lysergic acid diethylamide and 2,5-dimethoxy-4-iodophenylisopropylamine (partial agonists) at edited receptors (5-HT 2C-VNI , 5-HT 2C-VSV , and 5-HT 2C-VGV ) were not different from 5-HT 2C-INI receptors, suggesting that the capacity of the agonist-occupied receptor to couple to G q/11 proteins was not different. Ligand-independent (i.e., constitutive) receptor activity toward PLC for the 5-HT 2C-VNI receptor was markedly reduced to a level similar to that for the fully edited 5-HT 2C-VSV isoform. However, there was no difference in the thermal stability of the edited receptors, suggesting that mRNA editing does not alter the capacity of receptors to adopt active conformations. These results indicate that a conservative change in one amino acid (I156V) located in i2 of the 5-HT 2C receptor produces profound changes in receptor function that differ depending upon whether the receptor is unoccupied or occupied by agonist.The serotonin 2C (5-HT 2C ) receptor is a member of the 5-HT2 seven transmembrane-spanning (7-TMS) receptor family, also known as G protein-coupled receptors. The 5-HT 2C receptor is widely expressed in numerous brain regions and plays significant roles in many physiological functions and behaviors, such as sleep, affective state, feeding behavior, and temperature regulation. In addition, the 5-HT 2C receptor is a focus for the development of drugs to treat schizophrenia, depression, Parkinson's disease, and obesity and is also a likely target for hallucinogenic drugs of abuse (Di Giovanni et al., 2006).It is noteworthy that the 5-HT 2C receptor is the only 7-TMS receptor whose mRNA undergoes adenosine-inosine editing events that change the coding for amino acids located within the putative second intracellular domain (i2) of the receptor. In human brain, the nonedited receptor contains the amino acids isoleucine, asparagine, and isoleucine (INI) at positions 156, 158, and 160, respectively, whereas the principle fully edited isoforms express valine, serine, and valine (VSV) or valine, glycine, and valine (VGV). Partially edited receptor isoforms also exist where alteration of one or two amino acids within i2 occurs....

Developmental Changes in Serotonin 2C Receptor mRNA Editing in the Rat Cerebral Cortex and Primary Cultured Cortical Neurons

“…Occurrence of adenosine-to-inosine editing at site A of 5-HT2C mRNA is known to generate a 5-HT2CR isoform with substitution of valine for isoleucine at a position 156 (5-HT2CR I156V ) within the putative second intracellular domain of the receptor. Recently, Berg et al 17) have demonstrated that a conservative change in one amino acid (I156V) of the 5-HT2CR produces profound changes in receptor function that differ depending on whether the receptor is unoccupied or occupied by agonist. They also suggest that phospholipase A2 (PL-A2) signaling of 5-HT2CR is influenced by this substitution (I156V) without affecting the phospholipase C signaling.…”

Constitutive Activity of Serotonin2C Receptors at G Protein-Independent Signaling: Modulation by RNA Editing and Antidepressants

“…RNA editing decreases binding affinity of agonists and alters some coupling, ligand-functional selectivity, and signaling characteristics of the receptor Niswender et al, 1999;Berg et al, 2008a;Werry et al, 2008a). Differentially edited receptors exhibit varying degrees of constitutive activity at G protein-dependent signaling, ranging from the highest for the nonedited 5-HT 2C-INI receptor to intermediate for partially edited isoforms and negligible for the fully edited 5-HT 2C-VGV receptor Niswender et al, 1999).…”

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

“…The mammalian cells (NIH3T3, CHO, COS-7, HEK293 or CV1), which stably or transiently transfected with nonedited or edited serotonin 5-HT 2C receptor isoforms, have been used as the cellular models for the studies of related signaling pathways and the modification of receptor function by RNA editing [14,15,18,24,25]. However, only a few serotonin receptor cell models of AD were utilized [11] and no RNA edited 5-HT 2C receptor isoforms cell model exists to provide appropriate cellular system to study the association between the RNA edited serotonin 5-HT 2C receptor isoforms and APP metabolism.…”

“…The partially edited receptor isoforms express valine, asparagine, and isoleucine (VNI), valine, asparagine, and valine (VNV), or valine, serine, and valine (VSV), whereas the fully edited isoforms express valine, glycine, and valine (VGV) (Fig. 1) [15][16][17][18][19][20].…”