5-HT1A receptors transactivate the platelet-derived growth factor receptor type beta in neuronal cells

In the absence of ligand, certain growth factor receptors can be activated via G-protein coupled receptor (GPCR) activation in a process termed transactivation. Serotonin (5-HT) receptors can transactivate platelet-derived growth factor (PDGF) β receptors in smooth muscle cells, but it is not known if similar pathways occur in neuronal cells. Here we show that 5-HT can transiently increase the phosphorylation of PDGFβ receptors through 5-HT_1A receptors in a time- and dose-dependent manner in SH-SY5Y neuroblastoma cells. 5-HT also transactivates PDGFβ receptors in primary cortical neurons. This transactivation pathway is pertussis-toxin sensitive and Src tyrosine kinase-dependent. This pathway is also dependent on phospholipase C activity and intracellular calcium signaling. Several studies involving PDGFβ receptor transactivation by GPCRs have also demonstrated a PDGFβ receptor-dependent increase in the phosphorylation of ERK1/2. Yet in SH-SY5Y cells, 5-HT treatment causes a PDGFβ receptor-independent increase in ERK1/2 phosphorylation. This crosstalk between 5-HT and PDGFβ receptors identifies a potentially important signaling link between the serotonergic system and growth factor signaling in neurons.     

Cannabinoid 1 receptor-dependent transactivation of fibroblast growth factor receptor 1 emanates from lipid rafts and amplifies extracellular signal-regulated kinase 1/2 activation in embryonic cortical neurons

G-protein coupled receptors may mediate their effects on neuronal growth and differentiation through activation of extracellular signal-regulated kinases 1/2 (ERK1/2), often elicited by transactivation of growth factor receptor tyrosine kinases. This elaborate signaling process includes inducible formation and trafficking of multiprotein signaling complexes and is facilitated by pre-ordained membrane microdomains, in particular lipid rafts. In this study, we have uncovered novel signaling interactions of cannabinoid receptors with fibroblast growth factor receptors, which depended on lipid rafts and led to ERK1/2 activation in primary neurons derived from chick embryo telencephalon. More specifically, the cannabinoid 1 receptor (CB1R) agonist methanandamide induced tyrosine phosphorylation and transactivation of fibroblast growth factor receptor (FGFR)1 via Src and Fyn, which drove an amplification wave in ERK1/2 activation. Transactivation of FGFR1 was accompanied by the formation of a protein kinase C ε-dependent multiprotein complex that included CB1R, Fyn, Src, and FGFR1. Recruitment of molecules increased with time of exposure to methanandamide, suggesting that in addition to signaling it also served trafficking of receptors. Upon agonist stimulation we also detected a rapid incorporation of CB1R, as well as activated Src and Fyn, and FGFR1 in lipid rafts. Most importantly, lipid raft integrity was a pre-requisite for CB1R-dependent complex formation. Our data provide evidence that lipid rafts may organize CB1 receptor proximal signaling events, namely activation of Src and Fyn, and transactivation of FGFR1 towards activation of ERK1/2 and induction of neuronal differentiation.     

Fibroblast growth factor receptor signaling in Xenopus retinal axon extension

Fibroblast growth factor receptors (FGFRs) and N-cadherin both regulate axon extension in developing Xenopus retinal ganglion cells (RGCs). Cultured cerebellar neurons have been shown to require FGFR activity for N-cadherin–stimulated neurite outgrowth, raising the possibility that N-cadherin is a FGFR ligand. To investigate this possibility in the developing visual system, retinal neurons were transfected with a dominant-negative FGFR (XFD) and plated on purified N-cadherin substrates. XFD-expressing neurons extended markedly shorter processes than control GFP-expressing neurons, implicating a role for FGFRs in N-cadherin–stimulated neurite outgrowth. To examine whether N-cadherin and FGFRs share the same pathway or use distinct second messenger pathways, specific inhibitors of implicated signaling molecules were added to neurons stimulated by N-cadherin, basic fibroblast growth factor (bFGF), or brain-derived nerve factor (BDNF) (which stimulates RGC outgrowth by a FGFR-independent mechanism). Diacylglycerol (DAG) lipase and Ca²⁺/calmodulin kinase II inhibitors both significantly reduced outgrowth stimulated by N-cadherin or bFGF but not by BDNF. Furthermore, we show that inhibiting DAG lipase activity in RGC axons extending in vivo toward the optic tectum reversibly slows axon extension without collapsing their growth cones. Thus, a common second-messenger signaling pathway mediating both N-cadherin– and bFGF-stimulated neurite extension is consistent with a model in which N-cadherin directly modulates the FGFR or a model whereby both FGFR and N-cadherin regulate the same second-messenger system. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 633–641, 1998     

AFDX-116 discriminates between muscarinic M2 receptors of the heart and the iris smooth muscle

Summary Studies with the atypical muscarinic antagonist pirenzepine provide convincing evidence for the classification of muscarinic acetylcholine receptors (mAChRs) into two subtypes, M₁ and M₂. The present study examines the heterogeneity of the M₂ subtype employing the newly developed competitive muscarinic antagonist, AFDX-116. Comparison of the binding affinities of pirenzepine, atropine, and AFDX-116 to mAChRs in microsomes from the rabbit cerebral cortex, heart, and iris smooth muscle shows that iris mAChRs, which are pharmacologically of the M₂ subtype, can be distinguished from M₂ cardiac receptors based on their affinity for AFDX-116. These results are consistent with the hypothesis that the M₂ receptor subtype consists of a heterogeneous population of receptors.Abbreviations mAChRs Muscarinic Acetylcholine Receptors - CCh Carbachol - NMS N-Methylscopolamine - AFDX-116 11-[[2-[(diethylamino)methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6Hpyrido[2,3-b][1,4]benzodiazepine-6-one     

Src homology 2 (SH2) domain containing protein tyrosine phosphatase-1 (SHP-1) dephosphorylates VEGF Receptor-2 and attenuates endothelial DNA synthesis, but not migration*

Background

Sustained agonist-promoted ubiquitination of β-arrestin has been correlated with increased stability of the GPCR – β-arrestin complex. Moreover, abrogation of β-arrestin ubiquitination has been reported to inhibit receptor internalization with minimal effects on receptor degradation.

Results

Herein we report that agonist activation of M₁ mAChRs produces a sustained β-arrestin ubiquitination but no stable co-localization with β-arrestin. In contrast, sustained ubiquitination of β-arrestin by activation of M₂ mAChRs does result in stable co-localization between the M₂ mAChR and β-arrestin. Internalization of receptors was unaffected by proteasome inhibitors, but down-regulation was significantly reduced, suggesting a role for the ubiquitination machinery in promoting down-regulation of the receptors. Given the ubiquitination status of β-arrestin following agonist treatment, we sought to determine the effects of β-arrestin ubiquitination on M₁ and M₂ mAChR down-regulation. A constitutively ubiquitinated β-arrestin 2 chimera in which ubiquitin is fused to the C-terminus of β-arrestin 2 (YFP-β-arrestin 2-Ub) significantly increased agonist-promoted down-regulation of both M₁ and M₂ mAChRs, with the effect substantially higher on the M₂ mAChR. Based on this observation, we were interested in examining the effects of disruption of potential ubiquitination sites in the β-arrestin sequence on receptor down-regulation. Agonist-promoted internalization of the M₂ mAChR was not affected by expression of β-arrestin lysine mutants lacking putative ubiquitination sites, β-arrestin 2^{K18R, K107R, K108R, K207R, K296R}, while down-regulation and stable co-localiztion of the receptor with this β-arrestin lysine mutant were significantly reduced. Interestingly, expression of β-arrestin 2^{K18R, K107R, K108R, K207R, K296R} increased the agonist-promoted down-regulation of the M₁ mAChR but did not result in a stable co-localiztion of the receptor with this β-arrestin lysine mutant.

Conclusion

These findings indicate that ubiquitination of β-arrestin has a distinct role in the differential trafficking and degradation of M₁ and M₂ mAChRs.     

Activation of Trk neurotrophin receptor signaling by pituitary adenylate cyclase-activating polypeptides.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide that acts through G protein-coupled receptors, exerts neuroprotective effects upon many neuronal populations. However, the intracellular signaling mechanisms that account for PACAP's trophic effects are not well characterized. Here we have tested the possibility that PACAP uses neurotrophin signaling pathways. We have found that PACAP treatment resulted in an increase in TrkA tyrosine kinase activity in PC12 cells and TrkB activity in hippocampal neurons. The activation of TrkA receptors by PACAP required at least 1 h of treatment and did not involve binding to nerve growth factor. Moreover, PACAP induced an increase in activated Akt through a Trk-dependent mechanism that resulted in increased cell survival after trophic factor withdrawal. The increases in Trk and Akt were blocked by K252a, an inhibitor of Trk receptor activity. In addition, transactivation of TrkA receptors by PACAP could be inhibited with PP1, an inhibitor of Src family kinases or BAPTA/AM, (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester), an intracellular calcium chelator. Therefore, PACAP can exert trophic effects through a mechanism involving Trk receptors and utilization of tyrosine kinase signaling. This ability may explain several neuroprotective actions of PACAP upon neuronal populations after injury, nerve lesion, or neurotrophin deprivation.     

Use of M1–M5 Muscarinic Receptor Knockout Mice as Novel Tools to Delineate the Physiological Roles of the Muscarinic Cholinergic System

In this review we report recent findings on the physiological role of the five known muscarinic acetylcholine receptors (mAChRs) as shown by gene targeting technology. Using knockout mice for each mAChRs subtype, the role of mAChRs subtypes in a number of physiological functions was confirmed and new activities were discovered. The M₁ mAChRs modulate neurotransmitter signaling in cortex and hippocampus. The M₃ mAChRs are involved in exocrine gland secretion, smooth muscle contractility, pupil dilation, food intake, and weight gain. The role of the M₅ mAChRs involves modulation of central dopamine function and the tone of cerebral blood vessels. mAChRs of the M₂ subtype mediate muscarinic agonist-induced bradycardia, tremor, hypothermia, and autoinhibition of release in several brain regions. M₄ mAChRs modulate dopamine activity in motor tracts and act as inhibitory autoreceptors in striatum. Thus, as elucidated by gene targeting technology, mAChRs have widespread and manifold functions in the periphery and brain.     

Dynamic modulation of FGFR1–5-HT1A heteroreceptor complexes. Agonist treatment enhances participation of FGFR1 and 5-HT1A homodimers and recruitment of β-arrestin2

New findings show that neurotrophic and antidepressant effects of 5-HT in brain can, in part, be mediated by activation of the 5-HT1A receptor protomer in the hippocampal and raphe FGFR1–5-HT1A heteroreceptor complexes enhancing the FGFR1 signaling. The dynamic agonist modulation of the FGFR1–5-HT1A heteroreceptor complexes and their recruitment of β-arrestin is now determined in cellular models with focus on its impact on 5-HT1AR and FGFR1 homodimerization in the heteroreceptor complexes based on BRET² assays. The findings show that coagonist treatment with 8-OH-DPAT and FGF2 but not treatment with the 5-HT1A agonist alone markedly increases the BRETmax values and significantly reduces the BRET50 values of 5HT1A homodimerization. The effects of FGF2 or FGF20 with or without the 5-HT1A agonist were also studied on the FGFR1 homodimerization of the heteroreceptor complexes. FGF2 produced a marked and rapid increase in FGFR1 homodimerization which partially declined over a 10 min period. Cotreatment with FGF2 and 5-HT1A agonist blocked this decline in FGFR1 homodimerization. Furthermore, FGF2 alone produced a small increase in the BRET² signal from the 5-HT1A-β-arrestin2 receptor–protein complex which was additive to the marked effect of 8-OH-DPAT alone. Taken together, the participation of 5-HT1A and FGFR1 homodimers and recruitment of β-arrestin2 was demonstrated in the FGFR1–5-HT1A heteroreceptor complexes upon agonist treatments.     

Serotonin (5-HT) induces glial cell line-derived neurotrophic factor (GDNF) mRNA expression via the transactivation of fibroblast growth factor receptor 2 (FGFR2) in rat C6 glioma cells

We previously reported that serotonin (5-HT) increased glial cell line-derived neurotrophic factor (GDNF) release in a 5-HT₂ receptor (5-HT₂R) and mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK)-dependent manner in rat C6 glioma cells (C6 cells), a model of astrocytes. We herein found that 5-HT-induced rapid ERK phosphorylation was blocked by 5-HT₂R antagonists in C6 cells. We therefore examined 5-HT-induced ERK phosphorylation to reveal the mechanism of 5-HT-induced GDNF mRNA expression. As 5-HT-induced ERK phosphorylation was blocked by inhibitors for Gα_q/11 and fibroblast growth factor receptor (FGFR), but not for second messengers downstream of Gα_q/11, 5-HT₂R-mediated FGFR transactivation was suggested to be involved in the ERK phosphorylation. Although FGFR1 and 2 were functionally expressed in C6 cells, 5-HT selectively phosphorylated FGFR2. Indeed, small interfering RNA for FGFR2, but not for FGFR1, blocked 5-HT-induced ERK phosphorylation. As Src family tyrosine kinase inhibitors and microtubule depolymerizing agents blocked 5-HT-induced FGFR2 phosphorylation, Src family tyrosine kinase and stabilized microtubules were suggested to act upstream of FGFR2. Finally, 5-HT-induced GDNF mRNA expression was also inhibited by the blockade of 5-HT₂R, FGFR, and Src family tyrosine kinase. In conclusion, our findings suggest that 5-HT induces GDNF mRNA expression via 5-HT₂R-mediated FGFR2 transactivation in C6 cells.     

Discovery of N-substituted 7-azaindoline derivatives as potent,orally available M1 and M4 muscarinic acetylcholine receptors selective agonists

We designed and synthesized novel N-substituted 7-azaindoline derivatives as selective M₁ and M₄ muscarinic acetylcholine receptors (mAChRs) agonists. Hybridization of compound 2 with the HTS hit compound 5 followed by optimization of the N-substituents of 7-azaindoline led to identification of compound 1, which showed highly selective M₁ and M₄ mAChRs agonistic activity, weak human ether-a-go-go related gene inhibition, and good bioavailability in multiple animal species.     

Diverse FGF receptor signaling controls astrocyte specification and proliferation

During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.     

Agonist mediated internalization of M2 mAChR is β-arrestin-dependent

Background

Muscarinic acetylcholine receptors (mAChRs) undergo agonist-promoted internalization, but evidence suggesting that the mechanism of internalization is β-arrestin dependent has been contradictory and unclear. Previous studies using heterologous over-expression of wild type or dominant-negative forms of β-arrestins have reported that agonist-promoted internalization of M₂ mAChRs is a β-arrestin- and clathrin-independent phenomenon. In order to circumvent the complications associated with the presence of endogenous β-arrestin that may have existed in these earlier studies, we examined agonist-promoted internalization of the M₂ mAChR in mouse embryonic fibroblasts (MEFs) derived from β-arrestin knockout mice that lack expression of either one or both isoforms of β-arrestin (β-arrestin 1 and 2).

Results

In wild type MEF cells transiently expressing M₂ mAChRs, 40% of surface M₂ mAChRs underwent internalization and sorted into intracellular compartments following agonist stimulation. In contrast, M₂ mAChRs failed to undergo internalization and sorting into intracellular compartments in MEF β-arrestin double knockout cells following agonist stimulation. In double knockout cells, expression of either β-arrestin 1 or 2 isoforms resulted in rescue of agonist-promoted internalization. Stimulation of M₂ mAChRs led to a stable co-localization with GFP-tagged β-arrestin within endocytic structures in multiple cell lines; the compartment to which β-arrestin localized was determined to be the early endosome. Agonist-promoted internalization of M₂ mAChRs was moderately rescued in MEF β-arrestin 1 and 2 double knockout cells expressing exogenous arrestin mutants that were selectively defective in interactions with clathrin (β-arrestin 2 ΔLIELD), AP-2 (β-arrestin 2-F391A), or both clathrin/AP-2. Expression of a truncated carboxy-terminal region of β-arrestin 1 (319–418) completely abrogated agonist-promoted internalization of M₂ mAChRs in wild type MEF cells.

Conclusion

In summary, this study demonstrates that agonist-promoted internalization of M₂ mAChRs is β-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with β-arrestin in early endosomal vesicles.     

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M₁ muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M₁ mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M₁ mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M₁ mAChRs, we show that once internalized, M₁ mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M₁ mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.     

Discovery and structure-activity relationships study of positive allosteric modulators of the M3 muscarinic acetylcholine receptor

The M₃ muscarinic acetylcholine receptor (mAChR) is a member of the family of mAChRs, which are associated with a variety of physiological functions including the contraction of various smooth muscle tissues, stimulation of glandular secretion, and regulation of a range of cholinergic processes in the central nerve system. We report here the discovery and a comprehensive structure­-activity relationships (SARs) study of novel positive allosteric modulators (PAMs) of the M₃ mAChR through a high throughput screening (HTS) campaign. Compound 9 exhibited potent in vitro PAM activity towards the M₃ mAChR and significant enhancement of muscle contraction in a concentration-dependent manner when applied to isolated smooth muscle strips of rat bladder. Compound 9 also showed excellent subtype selectivity over other subtypes of mAChRs including M₁, M₂, and M₄ mAChRs, and moderate selectivity over the M₅ mAChR, indicating that compound 9 is an M₃-preferring M₃/M₅ dual PAM. Moreover, compound 9 displayed acceptable pharmacokinetics profiles after oral dosing to rats. These results suggest that compound 9 may be a promising chemical probe for the M₃ mAChR for further investigation of its pharmacological function both in vitro and in vivo.     

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists

Background and Objective

Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M₁ mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).

Methods

Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M₁ mAChR, respectively. The optimized FRET receptor construct (M₁-cam5) was expressed stably in HEK293 cells.

Results

The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα_q/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M₁ FRET (F_EYFP/F_ECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.

Conclusion

The M₁ FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M₁ mAChR activation.     

Vasopressin-induced neurotrophism in cultured hippocampal neurons via V1 receptor activation

Structural enhancement of nerve cell morphology has been postulated to be an integral step in the cellular process leading to information storage in the nervous system. To investigate this postulate, we determined whether vasopressin (AVP), a neural peptide that can enhance memory function, would enhance the cytoarchitectural features of hippocampal neurons in culture. Results of these studies demonstrated that in the presence of serum, vasopressin (1 μM), induced a significant in crease in the number of neurites, in neuritic length, and in neurite diameter following 48 h of exposure. Morphological complexity was also enhanced following vasopressin exposure as indicated by a significant increase in the number of filopodia/branches, in the sum of branch lengths, and in the number of branch bifurcation points. The number of microspikes decorating neuritic branches was also significantly increased following vasopressin exposure. To determine whether the neurotrophic effects of vasopressin was dependent upon factors present in serum, hippocampal nerve cells were cultured in serum-free media and exposed to 100–1000 nM AVP. Results of these studies demonstrated that in the absence of serum, AVP induced significant enhancement of hippocampal nerve cell growth and that the minimally effective concentration was reduced from 1 μM, as required in the presence serum, to 100 nM. In addition, the time required for a significant increase in nerve cell growth to become apparent decreased from 48 to 24 h. These results demonstrate that AVP-induced neurotrophism is not dependent upon unidentified factors in serum. AVP-induced neurotrophism was found to be mediated by V₁ receptor activation. Significant enhancement of nerve cell growth occurred following exposure to V₁ receptor agonist (100–1000 nM), whereas exposure to V₂ receptor agonist (100–1000 nM) did not increase any of the morphological parameters measured. Considered together, these data indicate that vasopressin can exert a significant neurotrophic effect upon hippocampal nerve cells in culture. Moreover, AVP-induced neurotrophism is a direct effect and not dependent upon unidentified factors present in serum. Enhancement of hippocampal nerve cell growth occurred in the presence of a specific V₁ receptor agonist and not following exposure to a V₂ agonist, suggesting that activation of the phosphatidyl inositol pathway via V₁ receptor activation mediates AVP-induced neurotrophism. Results of these studies are discussed with respect to their implications for understanding vasopressin involvement during neural development and induction of cytoarchitectual modifications associated with memory formation. 1994 John Wiley & Sons, Inc.     

Neurite outgrowth induced by a synthetic peptide ligand of neural cell adhesion molecule requires fibroblast growth factor receptor activation

The neural cell adhesion molecule NCAM is involved in axonal outgrowth and target recognition in the developing nervous system. In vitro, NCAM-NCAM binding has been shown to induce neurite outgrowth, presumably through an activation of fibroblast growth factor receptors (FGFRs). We have recently identified a neuritogenic ligand, termed the C3 peptide, of the first immunoglobulin (lg) module of NCAM using a combinatorial library of synthetic peptides. Here we investigate whether stimulation of neurite outgrowth by this synthetic ligand of NCAM involves FGFRs. In primary cultures of cerebellar neurons from wild-type mice, the C3 peptide stimulated neurite outgrowth. This response was virtually absent in cultures of cerebellar neurons from transgenic mice expressing a dominant-negative form of the FGFR1. Likewise, in PC12E2 cells transiently expressing a dominant-negative form of the mouse FGFR1, induction of neurites by the C3 peptide was abrogated. These findings suggest that the neuritogenic effect of the C3 peptide requires the presence of functional FGFRs and support the hypothesis that FGFRs are essential in cell adhesion molecule-stimulated neurite outgrowth. The C3 peptide appears to stimulate neurite outgrowth by specifically activating an NCAM-FGFR-dependent signaling cascade and may therefore be of considerable interest as a tool for the determination of NCAM-dependent neurite outgrowth as well as a potential drug capable of promoting outgrowth and regeneration of NCAM-responsive axons.     

The M3-muscarinic acetylcholine receptor stimulates glucose uptake in L6 skeletal muscle cells by a CaMKK–AMPK–dependent mechanism

The role of muscarinic acetylcholine receptors (mAChRs) in regulating glucose uptake in L6 skeletal muscle cells was investigated. [³H]-2-Deoxyglucose uptake was increased in differentiated L6 cells by insulin, acetylcholine, oxotremorine-M and carbachol. mAChR-mediated glucose uptake was inhibited by the AMPK inhibitor Compound C. Whole cell radioligand binding using [³H]-N-methyl scopolamine chloride identified mAChRs in differentiated but not undifferentiated L6 cells and M₃ mAChR mRNA was detected only in differentiated cells. M₃ mAChRs are Gq-coupled, and cholinergic stimulation by the mAChR agonists acetylcholine, oxotremorine-M and carbachol increased Ca²⁺ in differentiated but not undifferentiated L6 cells. This was due to muscarinic but not nicotinic activation as responses were antagonised by the muscarinic antagonist atropine but not the nicotinic antagonist tubocurarine. Western blotting showed that both carbachol and the AMPK activator AICAR increased phosphorylation of the AMPKα subunit at Thr172, with responses to carbachol blocked by Compound C and the CaMKK inhibitor STO609 but not by the PI3K inhibitor wortmannin. AICAR-stimulated AMPK phosphorylation was not sensitive to STO-609, confirming that this compound inhibits CaMKK but not the classical AMPK kinase LKB1. The TAK1 inhibitor (5Z)-7-oxozeaenol and the G_i inhibitor pertussis toxin both failed to block AMPK phosphorylation in response to carbachol. Using CHO-K1 cells stably expressing each of the mAChR subtypes (M₁–M₄), it was determined that only the M₁ and M₃ mAChRs phosphorylate AMPK, confirming a G_q-dependent mechanism. This study demonstrates that activation of M₃ mAChRs in L6 skeletal muscle cells stimulates glucose uptake via a CaMKK–AMPK-dependent mechanism, independent of the insulin-stimulated pathway.     

RasGAP Shields Akt from Deactivating Phosphatases in Fibroblast Growth Factor Signaling but Loses This Ability Once Cleaved by Caspase-3

Fibroblast growth factor receptors (FGFRs) are involved in proliferative and differentiation physiological responses. Deregulation of FGFR-mediated signaling involving the Ras/PI3K/Akt and the Ras/Raf/ERK MAPK pathways is causally involved in the development of several cancers. The caspase-3/p120 RasGAP module is a stress sensor switch. Under mild stress conditions, RasGAP is cleaved by caspase-3 at position 455. The resulting N-terminal fragment, called fragment N, stimulates anti-death signaling. When caspase-3 activity further increases, fragment N is cleaved at position 157. This generates a fragment, called N2, that no longer protects cells. Here, we investigated in Xenopus oocytes the impact of RasGAP and its fragments on FGF1-mediated signaling during G₂/M cell cycle transition. RasGAP used its N-terminal Src homology 2 domain to bind FGFR once stimulated by FGF1, and this was necessary for the recruitment of Akt to the FGFR complex. Fragment N, which did not associate with the FGFR complex, favored FGF1-induced ERK stimulation, leading to accelerated G₂/M transition. In contrast, fragment N2 bound the FGFR, and this inhibited mTORC2-dependent Akt Ser-473 phosphorylation and ERK2 phosphorylation but not phosphorylation of Akt on Thr-308. This also blocked cell cycle progression. Inhibition of Akt Ser-473 phosphorylation and entry into G₂/M was relieved by PHLPP phosphatase inhibition. Hence, full-length RasGAP favors Akt activity by shielding it from deactivating phosphatases. This shielding was abrogated by fragment N2. These results highlight the role played by RasGAP in FGFR signaling and how graded stress intensities, by generating different RasGAP fragments, can positively or negatively impact this signaling.     

Differential Regulation of Primary Afferent Input to Spinal Cord by Muscarinic Receptor Subtypes Delineated Using Knockout Mice

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M₂/M₄ antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M₂/M₄ double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M₂/M₄ double-KO mice. In M₂ single-KO and M₄ single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M₃ single-KO and M₁/M₃ double-KO mice were similar to those in wild-type mice. In M₅ single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M₂ and M₄ subtypes reduces glutamate release from primary afferents. Activation of the M₅ subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs.