RNA editing of the serotonin 5HT2C receptor and its effects on cell signalling, pharmacology and brain function.

The process of RNA editing involves the modification of mRNA at specific sites by adenosine deaminases that act on RNA (ADAR) enzymes. By catalyzing the conversion of adenosine to inosine, these enzymes alter the way in which the mRNA is translated, and consequently alter the primary structure of the resultant proteins. The serotonin (5HT) 2C receptor (5HT2CR) is currently the only known member of the superfamily of seven transmembrane domain receptors (7TMRs) to undergo this modification, and provides a fascinating case study in the effects of such changes. Here we review the current state of knowledge surrounding the editing of the 5HT2CR, the stark differences in signalling arising due to this process, and the potential for (and difficulties in) exploiting the phenomenon for improved therapeutic intervention in various neurological disorders.

Receptor activity-modifying proteins differentially modulate the G protein-coupling efficiency of amylin receptors.

Receptor activity-modifying proteins (RAMPs) 1, 2, and 3 are prototypic G protein-coupled receptor accessory proteins that can alter not only receptor trafficking but also receptor phenotype. Specific RAMP interaction with the calcitonin receptor (CTR) generates novel and distinct receptors for the peptide amylin; however, the role of RAMPs in receptor signaling is not understood. The current study demonstrates that RAMP interaction with the CTRa in COS-7 or HEK-293 cells leads to selective modulation of signaling pathways activated by the receptor complex. There was a 20- to 30-fold induction in amylin potency at CTR/RAMP1 (AMY1) and CTR/RAMP3 (AMY3) receptors, compared with CTR alone, for formation of the second-messenger cAMP that parallels an increase in amylin binding affinity. In contrast, only 2- to 5-fold induction of amylin potency was seen for mobilization of intracellular Ca++ or activation of ERK1/2. In addition, in COS-7 cells, the increase in amylin potency for Ca++ mobilization was 2-fold greater for AMY3 receptors, compared with AMY1 receptors and this paralleled the relative capacity of overexpression of Galphaq proteins to augment induction of high affinity 125I-amylin binding. These data demonstrate that RAMP-complexed receptors have a different signaling profile to CTRs expressed in the absence of RAMPs, and this is likely due to direct effects of the RAMP on G protein-coupling efficiency.

Mechanisms of ERK1/2 regulation by seven-transmembrane-domain receptors.

Control of cell growth and differentiation has long been a focus of intense research interest, particularly in the context of cancer therapeutics. The evolutionarily-conserved extracellular signal-regulated kinases 1 and 2 (ERK1/2) are serine-threonine kinases that respond to a wide range of mitogens and growth factors to initiate changes in cellular proliferation and differentiation, and are the most important members of the mitogen-activated protein kinase (MAPK) family in terms of seven transmembrane-domain receptor (7TMR)-mediated regulation of mitogenic processes. Regulation of the ERK1/2 signaling cascade by 7TMRs is highly complex and cell type-specific. Recent advances in our knowledge of this effector pathway have revealed that its regulation is at least partly independent of traditional G protein-mediated actions arising from the stimulation of 7TMRs. This review summarizes the current position of our knowledge of ERK1/2 regulation, and illustrates the wealth of potential targets available for the development of new strategies for the treatment of proliferative and other ERK-related disorders.

"Ins and outs" of seven-transmembrane receptor signalling to ERK.

Extracellular-signal-regulated kinases 1 and 2 (ERK1/2) are important members of the mitogen-activated protein kinase (MAPK) family and have emerged as key effector targets of activation by seven-transmembrane-spanning (G-protein-coupled) receptors (7TMRs). Regulation of ERK by 7TMRs is highly complex and dependent on cell type. Numerous studies have linked specific G protein pathways to ERK activation, but recent evidence suggests that some 7TMR-linked ERK signalling pathways might not be exclusively mediated by G proteins. In addition, the emergence of an "inside-out" model for receptor tyrosine kinase (RTK) "transactivation" by 7TMRs has enhanced our understanding of the ERK signalling system and further underscores the complexity of mitogenic regulation by 7TMRs.

Mechanisms of cross-talk between G-protein-coupled receptors resulting in enhanced release of intracellular Ca2+.

Alteration in [Ca(2+)](i) (the intracellular concentration of Ca(2+)) is a key regulator of many cellular processes. To allow precise regulation of [Ca(2+)](i) and a diversity of signalling by this ion, cells possess many mechanisms by which they are able to control [Ca(2+)](i) both globally and at the subcellular level. Among these are many members of the superfamily of GPCRs (G-protein-coupled receptors), which are characterized by the presence of seven transmembrane domains. Typically, those receptors able to activate PLC (phospholipase C) enzymes cause release of Ca(2+) from intracellular stores and influence Ca(2+) entry across the plasma membrane. It has been well documented that Ca(2+) signalling by one type of GPCR can be influenced by stimulation of a different type of GPCR. Indeed, many studies have demonstrated heterologous desensitization between two different PLC-coupled GPCRs. This is not surprising, given our current understanding of negative-feedback regulation and the likely shared components of the signalling pathway. However, there are also many documented examples of interactions between GPCRs, often coupling preferentially to different signalling pathways, which result in a potentiation of Ca(2+) signalling. Such interactions have important implications for both the control of cell function and the interpretation of in vitro cell-based assays. However, there is currently no single mechanism that adequately accounts for all examples of this type of cross-talk. Indeed, many studies either have not addressed this issue or have been unable to determine the mechanism(s) involved. This review seeks to explore a range of possible mechanisms to convey their potential diversity and to provide a basis for further experimental investigation.

Ca(2+) signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells.

1. Human embryonic kidney (HEK)-293 cells expressing recombinant G alpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca(2+)] ([Ca(2+)](i)) in response to interleukin-8 (IL-8) following pre-stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. 2. Pre-stimulation of cells with adenosine 5'-triphosphate (ATP) revealed a substantial Ca(2+) signalling component mediated by IL-8 (E(max)=83 +/- 8% of maximal ATP response, pEC(50) of IL-8 response=9.7 +/- 0.1). 3. 1 microM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 microM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca(2+)](i) elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca(2+)](i) rapidly returned to basal levels. 4. Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca(2+)](i) elevation but the effects of UTP (E(max) of IL-8 response increased to 50 +/- 1% of the maximal response to ATP, pEC(50) increased to 9.8 +/- 0.1) were greater than those of 2MeSADP (E(max) increased to 36 +/- 2%, pEC(50) increased to 8.7 +/- 0.2). 5. 5. The potentiation of IL-8-mediated Ca(2+) signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca(2+) signalling was apparent in the absence of extracellular Ca(2+), demonstrating the release of Ca(2+) from intracellular stores. 6. Activation of P2Y1 and P2Y2 receptors also revealed Ca(2+) signalling by an endogenously expressed, G alpha(s)-coupled beta-adrenoceptor. 7. In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca(2+) signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.

Performance of mouse neural stem cells as a screening reagent: characterization of PAC1 activity in medium-throughput functional assays.

The self-renewal and phenotypic properties of neural stem cells make them an abundant and more physiologically relevant alternative to recombinant cell lines for drug screens to identify ligands acting at neural targets. Here, the authors use high-throughput phenotypic and signaling assays to test the ability of neural stem cells isolated from postnatal mouse hippocampus (mNSCs) to deliver high-content and physiologically relevant data on native peptide receptor activity. The authors find that mNSCs express PAC1 but not the related VPAC1 and VPAC2 receptors. PAC1 promotes both the proliferation of mNSCs and their differentiation into neuronal-like cells. In addition, the authors show that PAC1 stimulates markedly different extracellular signal-regulated kinase signals in mNSCs than in recombinant CHO-PAC1 cells and is able to couple to Ca(2+) elevation only in CHO-PAC1 cells. These data suggest that G-protein coupling in CHO-PAC1 cells is nonphysiological, which may affect the ligand binding properties of the receptor and thus distort the results of a screen by increasing numbers of false positives/negatives. This work reinforces the emerging pharmacological theory that recombinant cell lines are often inappropriate models of natively expressing primary cells, and the authors conclude that mNSCs are a viable and relevant physiological alternative for use in high-throughput drug screens.

N-desmethylclozapine (NDMC) is an antagonist at the human native muscarinic M(1) receptor.

N-desmethylclozapine (NDMC) has been reported to display partial agonism at the human recombinant and rat native M(1) mAChR, a property suggested to contribute to the clinical efficacy of clozapine. However, the profile of action of NDMC at the human native M(1) mAChR has not been reported. The effect of NDMC on M(1) mAChR function was investigated in human native tissues by assessing its effect on (1) M(1) mAChR-mediated stimulation of [(35)S]-GTPgammaS-G(q/11)alpha binding to human post mortem cortical membranes and (2) the M(1) mAChR-mediated increase in neuronal firing in human neocortical slices. NDMC displayed intrinsic activities of 46+/-9%, compared to oxo-M, at the human recombinant M(1) receptor, in FLIPR studies and 35+/-4% at rat native M(1) receptors in [(35)S]-GTPgammaS-G(q/11)alpha binding studies. In [(35)S]-GTPgammaS-G(q/11)alpha binding studies in human cortex, oxo-M stimulated binding by 240+/-26% above basal with a pEC(50) of 6.56+/-0.05. In contrast, NDMC did not stimulate [(35)S]-GTPgammaS-G(q/11)alpha binding to human cortical membranes but antagonised the response to oxo-M (2microM) showing a pK(B) of 6.8, comparable to its human recombinant M(1) mAChR affinity (pK(i)=6.9) derived from [(3)H]-NMS binding studies. In human, contrary to the rat neocortical slices, NDMC did not elicit a significant increase in M(1) mAChR-mediated neuronal firing, and attenuated a carbachol-induced increase in neuronal firing when pre-applied. These data indicate that, whereas NDMC displays moderate to low levels of partial agonism at the human recombinant and rat native M(1) mAChR, respectively, it acts as an antagonist at the M(1) mAChR in human cortex.

Pharmacology of 5HT(2C) receptor-mediated ERK1/2 phosphorylation: agonist-specific activation pathways and the impact of RNA editing.

We have previously characterized a mechanism of 5HT-stimulated extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation via the non-RNA-edited isoform of the serotonin 5HT(2C) receptor (5HT(2C)R-INI) in a CHO cell line. We have now used CV1 cells, which endogenously express epidermal growth factor receptors (EGFRs), to investigate whether the mechanisms underlying ERK1/2 activation by the 5HT(2C)R change in a time-, agonist-, and cell background-dependent manner. Interrogation of the CV1 5HT(2C)R-INI ERK1/2 signaling pathway, using a variety of pathway-selective inhibitors, revealed a clear time-dependence in the involvement of specific pathway components such as phosphatidylinositol 3-kinase, EGFR, matrix metalloproteases and protein kinase C. The contribution of these components to the overall response also varied with the agonist used to stimulate the receptor, providing further evidence for the ability of 5HT(2C)R-INI to signal in an agonist-specific manner. We also investigated the impact of 5HT(2C)R RNA editing on this phenomenon. Although we found no alteration in antagonist pharmacology, the partially edited VSV and fully edited VGV isoforms of the 5HT(2C)R exhibited altered temporal and pharmacological characteristics, including the degree of dependence on specific effectors, in signaling to ERK1/2 in comparison to the 5HT(2C)R-INI. In conclusion, we provide evidence for remarkable flexibility in 5HT(2C)R-mediated ERK1/2 signaling that can be pharmacologically and mechanistically distinct depending on the agonist or edited isoform involved and on the duration of receptor activation.

The relaxin family peptide receptor 3 activates extracellular signal-regulated kinase 1/2 through a protein kinase C-dependent mechanism.

Human gene 3 relaxin (H3 relaxin) is a member of the relaxin/insulin family of peptides. Neuropeptides mediate behavioral responses to stress and regulates appetite; however, the cell signaling mechanisms that control these events remain to be identified. The relaxin family peptide receptor 3 (RXFP3, formerly GPCR135 or SALPR) was characterized as the receptor for H3 relaxin, functionally coupled to the inhibition of cAMP. We have identified that RXFP3 stably expressed in Chinese hamster ovary (CHO)-K1 (CHO-RXFP3) and human embryonic kidney (HEK) 293 (HEK-RXFP3) cells activates extracellular signal-regulated kinase (ERK) 1/2 when stimulated with H3 relaxin and an H3 relaxin B-chain (dimer) peptide. Using inhibitors of cellular signaling proteins, we subsequently determined the mechanism of ERK1/2 activation by RXFP3. ERK1/2 phosphorylation requires the activation of G(i/o) proteins and seems to require receptor internalization and/or compartmentalization into lipid-rich environments. ERK1/2 activation also predominantly occurred via the activation of a protein kinase C-dependent pathway, although activation of phosphatidylinositol 3-kinase and Src tyrosine kinase were also involved to a lesser extent. The mechanisms underlying ERK1/2 phosphorylation were similar in both CHO-RXFP3 and HEK-RXFP3 cells, although some differences were evident. Phospholipase Cbeta and the transactivation of endogenous epidermal growth factor receptors both played a role in RXFP3-mediated ERK1/2 activation in HEK293 cells; however, they were not involved in RXFP3-mediated ERK1/2 activation in the CHO-K1 cell background. The pathways identified in CHO- and HEK-transfected cells were also used in the murine SN56 neuronal cell line, suggesting that these pathways are also important for RXFP3-mediated signaling in the brain.

Characterization of serotonin 5-HT2C receptor signaling to extracellular signal-regulated kinases 1 and 2.

Serotonin 5-HT2C receptors (5-HT(2C)Rs) are almost exclusively expressed in the CNS, and implicated in disorders such as obesity, depression, and schizophrenia. The present study investigated the mechanisms governing the coupling of the 5-HT(2C)R to the extracellular signal-regulated kinases (ERKs) 1/2, using a Chinese hamster ovary (CHO) cell line stably expressing the receptor at levels comparable to those found in the brain. Using the non-RNA-edited isoform of the 5-HT(2C)R, constitutive ERK1/2 phosphorylation was observed and found to be modulated by full, partial and inverse agonists. Interestingly, agonist-directed trafficking of receptor stimulus was also observed when comparing effects on phosphoinositide accumulation and intracellular Ca2+ elevation to ERK1/2 phosphorylation, whereby the agonists, [+/-]-2,5-dimethoxy-4-iodoamphetamine (DOI) and quipazine, showed reversal of efficacy between the phosphoinositide/Ca2+ pathways, on the one hand, and the ERK1/2 pathway on the other. Subsequent molecular characterization found that 5-HT-stimulated ERK1/2 phosphorylation in this cellular background requires phospholipase D, protein kinase C, and activation of the Raf/MEK/ERK module, but is independent of both receptor- and non-receptor tyrosine kinases, phospholipase C, phosphoinositide 3-kinase, and endocytosis. Our findings underscore the potential for exploiting pathway-selective receptor states in the differential modulation of signaling pathways that play prominent roles in normal and abnormal neuronal signaling.

Cross talk between P2Y2 nucleotide receptors and CXC chemokine receptor 2 resulting in enhanced Ca2+ signaling involves enhancement of phospholipase C activity and is enabled by incremental Ca2+ release in human embryonic kidney cells.

We have shown previously that activation of endogenously expressed, Galphaq/11-coupled P2Y2 nucleotide receptors with UTP reveals an intracellular Ca2+ response to activation of recombinant, Galphai-coupled CXC chemokine receptor 2 (CXCR2) in human embryonic kidney cells. Here, we characterize further this cross talk and demonstrate that phospholipase C (PLC) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent Ca2+ release underlies this potentiation. The putative Ins(1,4,5)P3 receptor antagonist 2-aminoethoxydiphenyl borane reduced the response to CXCR2 activation by interleukin-8, as did sustained inhibition of phosphatidylinositol 4-kinase with wortmannin, suggesting the involvement of phosphoinositides in the potentiation. Against a Li+ block of inositol monophosphatase activity, costimulation of P2Y2 nucleotide receptors and CXCR2 caused phosphoinositide accumulation that was significantly greater than that after activation of P2Y2 nucleotide receptors or CXCR2 alone, and was more than additive. Thus, PLC activity, as well as Ca2+ release, was enhanced. In these cells, agonist-mediated Ca2+ release was incremental in nature, suggesting that a potentiation of Ins(1,4,5)P3 generation in the presence of coactivation of P2Y2 nucleotide receptors and CXCR2 would be sufficient for additional Ca2+ release. Potentiated Ca2+ signaling by CXCR2 was markedly attenuated by expression of either regulator of G protein signaling 2 or the Gbetagamma-scavenger Galphat1 (transducin alpha subunit), indicating the involvement of Galphaq and Gbetagamma subunits, respectively.