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The recent completion of the human genome predicted the presence of only 30,000 genes, stressing the importance of mechanisms that increase molecular diversity at the post-transcriptional level. One such posttranscriptional event is RNA editing, which generates multiple protein isoforms from a single gene, often with profound functional consequences. The human serotonin 5-HT 2C receptor undergoes RNA editing that creates multiple receptor isoforms. One consequence of RNA editing of cell surface receptors may be to alter the pattern of activation of heterotrimeric G-proteins and thereby shift preferred intracellular signaling pathways. We examined the ability of the nonedited 5-HT 2C receptor isoform (INI) and two extensively edited isoforms, VSV and VGV, to interact with various G-protein ␣ subunits. Two functional assays were utilized: the cellbased functional assay, Receptor Selection/Amplification Technology TM , in which the pharmacological consequences of co-expression of 5HT 2C receptor isoforms with G-protein ␣ subunits in fibroblasts were studied, and 5HT 2C receptor-mediated rearrangements of the actin cytoskeleton in stable cell lines. These studies revealed that the nonedited 5-HT 2C receptor functionally couples to G q and G 13 . In contrast, coupling to G 13 was not detected for the extensively edited 5-HT 2C receptors. Thus, RNA editing represents a novel mechanism for regulating the pattern of activation of heterotrimeric G-proteins, molecular switches that control an enormous variety of biological processes.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 (1). The 2C subtype of serotonin receptor (5-HT 2C R) is a member of the G-protein-coupled receptor superfamily and interacts with G q to stimulate phospholipase C, resulting in the production of inositol phosphates and diacylglycerol (2). RNA transcripts encoding the human 5-HT 2C R undergo adenosineto-inosine RNA editing events at five positions, termed A, B, C, D, and E (Fig. 1A), altering the amino acid coding potential within the putative second intracellular loop of the protein (3, 4). We and others have demonstrated a decrease in agonist potency at the human edited VSV and VGV isoforms (named for the amino acids at positions 156, 158, and 160) compared with the nonedited isoform, which codes for INI at these positions. This decrease in agonist potency is reflected as a rightward shift in the dose-response curve for inositol phosphate accumulation (5, 6) and calcium release (7). The decreased agonist potency was proposed to result from a reduced G qprotein coupling efficiency induced by the introduction of these novel amino acids into the second intracellular loop, a region known to be important for G-protein coupling (8 -16). Another consequence of RNA editing may be to alter the specificity of activation of heterotrimeric G-proteins and thereby shift intracellular signaling pathways. To test this hypothesi...

1 Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons within the peripheral and central nervous systems. 2 Neurophilin ligands, including FK506, potentiate NGF-induced neurite outgrowth in several experimental models, although the mechanism of this potentiation is unclear. 3 Therefore, we tested which signaling pathways were involved in FK506-potentiated neurite outgrowth in SH-SY5Y neuroblastoma cells using specific pharmacological inhibitors of various signaling molecules. 4 Inhibitors of Ras (lovastatin), Raf (GW5074), or MAP kinase (PD98059 and U0126) blocked FK506 activity, as did inhibitors of phospholipase C (U73122) and phosphatidylinositol 3 0 kinase (LY294002). 5 Protein kinase C inhibitors (Go6983 and Ro31-8220) slightly but significantly inhibited neurite outgrowth, whereas inhibitors of p38 MAPK (SB203580) or c-Jun N-terminal kinase (SP600125) had no effect. 6 These data suggest that FK506 potentiates neurite outgrowth through the Ras/Raf/MAP kinase signaling pathway downstream of phospholipase C and phosphatidylinositol 3 0 kinase.

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Phosphorylation-deficient serotonin 5-HT 2C receptors were generated to determine whether phosphorylation promotes desensitization of receptor responses. Phosphorylation of mutant 5-HT 2C receptors that lack the carboxyl-terminal PDZ recognition motif (Ser 458 -SerVal-COOH; ⌬PDZ) was not detectable based on a bandshift phosphorylation assay and incorporation of 32 Serotonin 5-HT 2C (formerly 5-HT 1C ) receptors exists as several isoforms throughout the brain, due to RNA editing and alternative splicing, and function to stimulate phospholipase C through activation of the G protein G q (1). Whereas RNA editing generates isoforms in the second intracellular loop that modify receptor signaling (2-4), alternative splicing creates truncated nonfunctional receptors (5, 6). In addition, agonists promote phosphorylation of nonedited 5-HT 2C receptors (7), raising the possibility that 5-HT 2C receptors are also regulated dynamically.Phosphorylation of G protein-coupled receptors regulates signaling through multiple mechanisms including receptor desensitization, which attenuates second messenger responses. In the case of phospholipase C-linked receptors, phosphorylation has been demonstrated to promote desensitization based on observations that receptors lacking the corresponding phosphorylation sites display sustained phosphoinositide hydrolysis responses relative to wild-type receptors (8 -13). Furthermore, a second application of agonist produces amplified phosphoinositide hydrolysis (14) and calcium release (13, 15, 16) by phosphorylation-deficient mutants relative to wild-type receptors, illustrating that mutation of phosphorylation sites involved in desensitization enhances both initial and secondary responses.Although desensitization of 5-HT 2C receptor-mediated responses has been observed in assays that examine phosphoinositide hydrolysis (7, 17), release of intracellular calcium (18,19), and Ca 2ϩ -activated currents (20), it is unknown whether phosphorylation of the 5-HT 2C receptor is involved in desensitization. To address this issue, we identified a 5-HT 2C receptor domain that is required for receptor phosphorylation and performed functional assays with phosphorylation-deficient receptor mutants. First, we determined that phosphorylation of the 5-HT 2C receptor requires the carboxyl-terminal PDZ (PSD-95 discs-large ZO-1) recognition motif, a domain present in nonedited and edited isoforms of the 5-HT 2C receptor. Next, we found that phosphorylation-deficient receptors display identical initial responses as the wild-type 5-HT 2C receptor in phosphoinositide hydrolysis and calcium release assays. However, phosphorylation-deficient receptors exhibit diminished secondary responses and a delayed recovery relative to wild-type 5-HT 2C receptors. Cells stably expressing 5-HT 2C receptors with a single serine-to-alanine mutation also display decreased receptor phosphorylation and diminished secondary calcium responses. We therefore propose that 5-HT 2C receptor phosphorylation promotes resensitization of 5-HT 2C re...