Regulation of mTOR and p70 S6 kinase by the muscarinic M4 receptor in PC12 cells

The G_i-coupled M₄ muscarinic acetylcholine receptor (mAChR) has recently been shown to stimulate the survival of PC12 cells through the PI3K/Akt/tuberin pathway. Since mTOR and p70S6K are critical components in activating translation which lie downstream of tuberin, we examined the ability of M₄ mAChR to regulate these targets in PC12 cells. Carbachol (CCh) dose-dependently stimulated both mTOR and p70S6K phosphorylations and these responses were abolished by pertussis toxin pretreatment, indicating the involvement of the G_i-coupled M₄ mAChR. Phosphorylations of both mTOR and p70S6K were effectively blocked upon inhibition of PI3K by wortmannin. As compared to similar responses elicited by the nerve growth factor (NGF), the M₄ mAChR-induced activation of Akt/tuberin/mTOR/p70S6K occurred in a relatively transient manner. Although inhibition of protein phosphatase 2A by okadaic acid augmented the transient effects of CCh on Akt/tuberin phosphorylations, it failed to significantly prolong these responses. The total protein level of PTEN (tumor suppressor gene phosphatase and tensin homologue deleted on chromosome ten) was attenuated upon NGF, but not CCh treatment. This indicates that downregulation of PTEN may help to sustain the phosphorylation of Akt/tuberin by NGF. Collectively, these findings suggest that PP2A and PTEN may be involved in fine tuning the regulation of Akt/tuberin/mTOR/p70S6K in PC12 cells by M₄ mAChR and TrkA, respectively.     

Molecular events associated with the regulation of signaling by M2 muscarinic receptors

Multiple events are associated with the regulation of signaling by the M2 muscarinic cholinergic receptors (mAChRs). Desensitization of the attenuation of adenylyl cyclase by the M2 mAChRs appears to involve agonist-dependent phosphorylation of M2 mAChRs by G-protein coupled receptor kinases (GRKs) that phosphorylate the receptors in a serine/threonine rich motif in the 3rd intracellular domain of the receptors. Mutation of residues 307-311 from TVSTS to AVAAA in this domain of the human M2 mAChR results in a loss of receptor/G-protein uncoupling and a loss of arrestin binding. Agonist-induced sequestration of receptors away from their normal membrane environment is also regulated by agonist-induced phosphorylation of the M2 mAChRs on the 3rd intracellular domain, but in HEK cells, the predominant pathway of internalization is not regulated by GRKs or arrestins. This pathway of internalization is not inhibited by a dominant negative dynamin, and does not appear to involve either clathrin coated pits or caveolae. The signaling of the M2 mAChR to G-protein regulated inwardly rectifying K channels (GIRKs) can be modified by RGS proteins. In HEK cells, expression of RGS proteins leads to a constitutive activation of the channels through a mechanism that depends on Gbetagamma. RGS proteins appear to increase the concentration of free Gbetagamma in addition to acting as GAPs. Thus multiple mechanisms acting at either the level of the M2 mAChRs or the G-proteins can contribute to the regulation of signaling via the M2 mAChRs.     

Muscarinic activation of mitogen-activated protein kinase in PC12 cells

Muscarinic acetylcholine receptors (mAChRs) activate many downstream signaling pathways, some of which can lead to mitogen-activated protein kinase (MAPK) phosphorylation and activation. MAPKs play roles in regulating cell growth, differentiation, and synaptic plasticity. Here, the activation of MAPK was examined in PC12 cells endogenously expressing mAChRs. Western blot analysis using a phosphospecific MAPK antibody revealed a dose-dependent and atropine-sensitive increase in MAPK phosphorylation in cells stimulated with carbachol (CCh). The maximal response occurred after 5 min and was rapidly reduced to baseline. To investigate the receptors responsible for CCh activation of MAPK in PC12 cells, the mAChR subtypes present were determined using RT-PCR and immunoprecipitation. RT-PCR was used to amplify fragments of the appropriate sizes for m1, m4, and m5, and the identities of the bands were confirmed with restriction digests. Immunoprecipitation using subtype-specific antibodies showed that approximately 95% of the expressed receptors were m4, whereas the remaining approximately 5% were m1 and m5. A highly specific m1 toxin completely blocked MAPK phosphorylation in response to CCh stimulation. The mAChR-induced MAPK activation was abolished by protein kinase C down-regulation and partially inhibited by pertussis toxin. Although m1 represents a small proportion of the total mAChR population, pharmacological evidence suggests that m1 is responsible for MAPK activation in PC12 cells.     

Essential role of dynamin in internalization of M2 muscarinic acetylcholine and angiotensin AT1A receptors

Most G protein-coupled receptors (GPCRs), including the M(1) muscarinic acetylcholine receptor (mAChR), internalize in clathrin-coated vesicles, a process that requires dynamin GTPase. The observation that some GPCRs like the M(2) mAChR and the angiotensin AT(1A) receptor (AT(1A)R) internalize irrespective of expression of dominant-negative K44A dynamin has led to the proposal that internalization of these GPCRs is dynamin-independent. Here, we report that, contrary to what is postulated, internalization of M(2) mAChR and AT(1A)R in HEK-293 cells is dynamin-dependent. Expression of N272 dynamin, which lacks the GTP-binding domain, or K535M dynamin, which is not stimulatable by phosphatidylinositol 4, 5-bisphosphate, strongly inhibits internalization of M(1) and M(2) mAChRs and AT(1A)Rs. Expression of kinase-defective K298M c-Src or Y231F,Y597F dynamin (which cannot be phosphorylated by c-Src) reduces M(1) mAChR internalization. Similarly, c-Src inhibitor PP1 as well as the generic tyrosine kinase inhibitor genistein strongly inhibit M(1) mAChR internalization. In contrast, M(2) mAChR internalization is not (or is only slightly) reduced by expression of these constructs or treatment with PP1 or genistein. Thus, dynamin GTPases are not only essential for M(1) mAChR but also for M(2) mAChR and AT(1A)R internalization in HEK-293 cells. Our findings also indicate that dynamin GTPases are differentially regulated by c-Src-mediated tyrosine phosphorylation.     

Transient hypoxia induces sequestration of M1 and M2 muscarinic acetylcholine receptors

Oxidative stress has been implicated in impairing muscarinic acetylcholine receptor (mAChR) signaling activity. It remains unclear, however, whether alterations in the cell surface distribution of mAChRs following oxidative stress contribute to the diminished mAChR signaling activity. We report here that M1 and M2 mAChRs, stably expressed in Chinese hamster ovary cells, undergo sequestration following transient hypoxic-induced oxidative stress (2% O2). Sequestration of M1 and M2 mAChRs following transient hypoxia was associated with an increase in phosphorylation of these receptors. Over-expression of a catalytically inactive G protein-coupled receptor kinase 2 (GRK2 K220R) blocked the increased phosphorylation and sequestration of the M2, but not M1, mAChRs following transient hypoxia. Hypoxia induced phosphorylation and sequestration of the M1 mAChR was, however, blocked by over-expression of a catalytically inactive casein kinase 1 alpha (CK1alpha K46R). These results are the first demonstration that M1 and M2 mAChRs undergo sequestration following transient hypoxia. The data suggest that increased phosphorylation of M1 and M2 mAChRs underlies the mechanism responsible for sequestration of these receptors following transient hypoxia. We report here that distinct pathways involving CK1alpha and GRK2 mediated sequestration of M1 and M2 mAChRs following transient hypoxic-induced oxidative stress.     

Novel signalling events mediated by muscarinic receptor subtypes

The M2 muscarinic acetylcholine receptor (mAChR) activates Gi protein coupled pathways, such as stimulation of G-protein activated inwardly rectifying K channels (GIRKs). Here we report a novel heterologous desensitization of these GIRK currents, which appeared to be specifically induced by M2/M4 mAChR stimulation, but not via adenosine (Ado) and alpha2-adrenergic receptors (AR). This heterologous desensitization reflected an inhibition of the GIRK signalling pathway downstream of G-protein activation. It was mediated in a membrane-delimited fashion via a PTX insensitive GTP dependent pathway and could be competed with exogenous Gbetagamma. The activation of M3 mAChR/Gq coupled receptors potently inhibited GIRK currents similar as M2 mAChR. By monitoring simultaneously the response of A1 adenosine receptor (AdoR) activation on N-type Ca2+ channels and GIRK channels, the stimulation of M3 mAChR was found to cause an inhibition of the Ado response in both effector systems, suggesting that the inhibition occurred at the level of the G-protein common to both effectors. These results indicated that Gq proteins inhibit pathways that are commonly regulated by Gbetagamma proteins.     

Phospholipase C, protein kinase C, Ca(2+)/calmodulin-dependent protein kinase II, and tyrosine phosphorylation are involved in carbachol-induced phospholipase D activation in Chinese hamster ovary cells expressing muscarinic acetylcholine receptor of Caenorhabditis elegans

Recently, we have isolated a cDNA encoding a muscarinic acetylcholine receptor (mAChR) from Caenorhabditis elegans. To investigate the regulation of phospholipase D (PLD) signaling via a muscarinic receptor, we generated stable transfected Chinese hamster ovary (CHO) cells that overexpress the mAChR of C. elegans (CHO-GAR-3). Carbachol (CCh) induced inositol phosphate formation and a significantly higher Ca(2+) elevation and stimulated PLD activity through the mAChR; this was insensitive to pertussis toxin, but its activity was abolished by the phospholipase C (PLC) inhibitor U73122. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after CCh treatment. The CCh-induced PLD activation and tyrosine phosphorylation were significantly reduced by the protein kinase C (PKC) inhibitor calphostin C and down-regulation of PKC and the tyrosine kinase inhibitor genistein. Moreover, the Ca(2+)-calmodulin-dependent protein kinase II (CaM kinase II) inhibitor KN62, in addition to chelation of extracellular or intracellular Ca(2+) by EGTA and BAPTA/AM, abolished CCh-induced PLD activation and protein tyrosine phosphorylation. Taken together, these results suggest that the PLC/PKC-PLD pathway and the CaM kinase II/tyrosine kinase-PLD pathway are involved in the activation of PLD through mAChRs of C. elegans.     

G protein-coupled receptor-induced sensitization of phospholipase C stimulation by receptor tyrosine kinases

Activation of stably expressed M(2) and M(3) muscarinic acetylcholine receptors (mAChRs) as well as of endogenously expressed lysophosphatidic acid and purinergic receptors in HEK-293 cells can induce a long lasting potentiation of phospholipase C (PLC) stimulation by these and other G protein-coupled receptors (GPCRs). Here, we report that GPCRs can induce an up-regulation of PLC stimulation by receptor tyrosine kinases (RTKs) as well and provide essential mechanistic characteristics of this sensitization process. Pretreatment of HEK-293 cells for 2 min with carbachol, a mAChR agonist, lysophosphatidic acid, or ATP, followed by agonist washout, strongly increased (by 2-3-fold) maximal PLC stimulation (measured >/=40 min later) by epidermal growth factor and platelet-derived growth factor, but not insulin, and largely enhanced PLC sensitivity to these RTK agonists. The up-regulation of RTK-induced PLC stimulation was cycloheximide-insensitive and was observed for up to approximately 90 min after removal of the GPCR agonist. Sensitization of receptor-induced PLC stimulation caused by prior M(2) mAChR activation was fully prevented by pertussis toxin and strongly reduced by expression of Gbetagamma scavengers. Furthermore, inhibition of conventional protein kinase C (PKC) isoenzymes and chelation of intracellular Ca(2+) suppressed the sensitization process, while overexpression of PKC-alpha, but not PKC-betaI, further enhanced the M(2) mAChR-induced sensitization of PLC stimulation. None of these treatments affected acute PLC stimulation by either GPCR or RTK agonists. Taken together, short term activation of GPCRs can induce a strong and long lasting sensitization of PLC stimulation by RTKs, a process apparently involving G(i)-derived Gbetagammas as well as increases in intracellular Ca(2+) and activation of a PKC isoenzyme, most likely PKC-alpha.     

Distinct internalization of M2 muscarinic acetylcholine receptors confers selective and long-lasting desensitization of signaling to phospholipase C

Although M1-M4 muscarinic acetylcholine receptors (mAChRs) in HEK-293 cells internalize on agonist stimulation, only M1, M3, and M4 but not M2 mAChRs recycle to the plasma membrane. To investigate the functional consequences of this phenomenon, we compared desensitization and resensitization of M2 versus M4 mAChRs. Treatment with 1 mM carbachol for 1 h at 37 degrees C reduced numbers of cell surface M2 and M4 mAChRs by 40-50% and M2 and M4 mAChR-mediated inhibition of adenylyl cyclase, intracellular Ca2+ concentration ([Ca2+]i) increases, and phospholipase C (PLC) activation by 60-70%. Receptor-mediated inhibition of adenylyl cyclase and [Ca2+]i increases significantly resensitized within 3 h. However, M4 but not M2 mAChR-mediated PLC activation resensitized. At 16 degrees C, M2 mAChR-mediated [Ca2+]i increases and PLC stimulation desensitized to a similar extent as at 37 degrees C. However, at 16 degrees C, where M2 mAChR internalization is negligible, both M2 mAChR responses resensitized, demonstrating that M2 mAChR resensitization proceeds at the plasma membrane. Examination of M2 mAChR responses following inactivation of cell surface mAChRs by quinuclidinyl benzilate revealed substantial receptor reserve for coupling to [Ca2+]i increases but not to PLC. We conclude that M2 mAChR internalization induces long-lasting PLC desensitization predominantly because receptor loss is not compensated for by receptor recycling or receptor reserve.     

Betagamma subunits of G(i/o) suppress EGF-induced ERK5 phosphorylation, whereas ERK1/2 phosphorylation is enhanced

Extracellular signal-regulated kinases (ERKs) play important physiological roles in proliferation, differentiation and gene expression. ERK5 is twice the size of ERK1/2, the amino-terminal half contains the kinase domain that shares the homology with ERK1/2 and TEY activation motif, whereas the carboxy-terminal half is unique. In this study, we examined the cross-talk mechanism between G-protein-coupled receptors (GPCRs) and receptor tyrosine kinases, focusing on ERK1/2 and 5. The pretreatment of rat pheochromocytoma cells (PC12) with pertussis toxin (PTX) specifically enhanced epidermal growth factor (EGF)-induced ERK5 phosphorylation. In addition, lysophosphatidic acid (LPA) attenuated the EGF-induced ERK5 phosphorylation in LPA(1) receptor- and G(i/o)-dependent manners. On the other hand, LPA alone activated ERK1/2 via Gbetagamma subunits and Ras and potentiated EGF-induced ERK1/2 phosphorylation at late time points. These results suggest G(i/o) negatively regulates ERK5, while it positively regulates ERK1/2. LPA did not affect cAMP levels after EGF treatment, and the reagents promoting cAMP production such as forskolin and cholera toxin also attenuated the EGF-induced ERK5 phosphorylation, indicating that the inhibitory effect of LPA on ERK5 inhibition via G(i/o) is not due to inhibition of adenylyl cyclase by Galpha(i/o). However, the inhibitory effect of LPA on ERK5 was abolished in PC12 cells stably overexpressing C-terminus of GPCR kinase2 (GRK2), and overexpression of Gbeta(1) and gamma(2) subunits also suppressed ERK5 phosphorylation by EGF. In response to LPA, Gbetagamma subunits interacted with EGF receptor in a time-dependent manner. These results strongly suggest that LPA negatively regulates the EGF-induced ERK5 phosphorylation through Gbetagamma subunits.     

Cyclic AMP induces transactivation of the receptors for epidermal growth factor and nerve growth factor,thereby modulating activation of MAP kinase,Akt, and neurite outgrowth in PC12 cells

In PC12 cells, a well studied model for neuronal differentiation, an elevation in the intracellular cAMP level increases cell survival, stimulates neurite outgrowth, and causes activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2). Here we show that an increase in the intracellular cAMP concentration induces tyrosine phosphorylation of two receptor tyrosine kinases, i.e. the epidermal growth factor (EGF) receptor and the high affinity receptor for nerve growth factor (NGF), also termed Trk(A). cAMP-induced tyrosine phosphorylation of the EGF receptor is rapid and correlates with ERK1/2 activation. It occurs also in Panc-1, but not in human mesangial cells. cAMP-induced tyrosine phosphorylation of the NGF receptor is slower and correlates with Akt activation. Inhibition of EGF receptor tyrosine phosphorylation, but not of the NGF receptor, reduces cAMP-induced neurite outgrowth. Expression of dominant-negative Akt does not abolish cAMP-induced survival in serum-free media, but increases cAMP-induced ERK1/2 activation and neurite outgrowth. Together, our results demonstrate that cAMP induces dual signaling in PC12 cells: transactivation of the EGF receptor triggering the ERK1/2 pathway and neurite outgrowth; and transactivation of the NGF receptor promoting Akt activation and thereby modulating ERK1/2 activation and neurite outgrowth.     

Arrestin binding to the M(2) muscarinic acetylcholine receptor is precluded by an inhibitory element in the third intracellular loop of the receptor

Desensitization of G protein-coupled receptors (GPCRs) involves the binding of members of the family of arrestins to the receptors. In the model system involving the visual GPCR rhodopsin, activation and phosphorylation of rhodopsin is thought to convert arrestin from a low to high affinity binding state. Phosphorylation of the M(2) muscarinic acetylcholine receptor (mAChR) has been shown to be required for binding of arrestins 2 and 3 in vitro and for arrestin-enhanced internalization in intact cells (Pals-Rylaarsdam, R., and Hosey, M. M. (1997) J. Biol. Chem. 272, 14152-14158). For the M(2) mAChR, arrestin binding requires phosphorylation at multiple serine and threonine residues at amino acids 307-311 in the third intracellular (i3) loop. Here, we have investigated the molecular basis for the requirement of receptor phosphorylation for arrestin binding. Constructs of arrestin 2 that can bind to other GPCRs in a phosphorylation-independent manner were unable to interact with a mutant M(2) mAChR in which the Ser/Thr residues at 307-311 were mutated to alanines. However, although phosphorylation-deficient mutants of the M(2) mAChR that lacked 50-157 amino acids from the i3 loop were unable to undergo agonist-dependent internalization when expressed alone in tsA201 cells, co-expression of arrestin 2 or 3 restored agonist-dependent internalization. Furthermore, a deletion of only 15 amino acids (amino acids 304-319) was sufficient to allow for phosphorylation-independent arrestin-receptor interaction. These results indicate that phosphorylation at residues 307-311 does not appear to be required to activate arrestin into a high affinity binding state. Instead, phosphorylation at residues 307-311 appears to facilitate the removal of an inhibitory constraint that precludes receptor-arrestin association in the absence of receptor phosphorylation.     

Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.     

Tuberin: a stimulus-regulated tumor suppressor protein controlled by a diverse array of receptor tyrosine kinases and G protein-coupled receptors

Tuberin, a tumor suppressor protein, is involved in various cellular functions including survival, proliferation, and growth. It has emerged as an important effector regulated by receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). Regulation of tuberin by RTKs and GPCRs is highly complex and dependent on the type of receptors and their associated signaling molecules. Apart from Akt, the first kinase recognized to phosphorylate and inactivate tuberin upon growth factor stimulation, an increasing number of kinases upstream of tuberin have been identified. Furthermore, recruitment of different scaffolding adaptor components to the activated receptors appears to play an important role in the regulation of tuberin activity. More recently, the differential regulation of tuberin by various G protein family members have also been intensively studied, it appears that G proteins can both facilitate (e.g., G(i/o)) as well as inhibit (e.g., G(q)) tuberin phosphorylation. In the present review, we attempt to summarize our emerging understandings of the roles of RTKs, GPCRs, and their cross-talk on the regulation of tuberin.     

Activation of delta-, kappa-, and mu-opioid receptors induces phosphorylation of tuberin in transfected HEK 293 cells and native cells

A number of G protein-coupled receptors have been shown to stimulate tuberin phosphorylation, which is critical for the regulation of translation and is apparently involved in neurotrophin-promoted survival of serum-deprived cells. Here, in HEK 293 cells transiently expressing the delta-, kappa-, or mu-opioid receptors, Western blotting analysis using a phosphospecific anti-tuberin antibody revealed a dose- and time-dependent increase in tuberin phosphorylation upon stimulation by specific opioid agonists. In NG108-15, PC12, and SH-SY5Y cells that endogenously express delta-, kappa-, and mu-opioid receptors, respectively, specific opioid agonists also stimulated tuberin phosphorylation in a dose- and time-dependent manner. Pretreatment of cells with pertussis toxin or PI3K inhibitor wortmannin blocked the opioid-stimulated tuberin phosphorylation, implicating the possible involvement of the G(i/o) proteins and the phosphatidylinositol-3 kinase/Akt pathway in opioid-induced tuberin phosphorylation. This is the first study that demonstrates the regulatory role of opioid receptors on tuberin.     

Cooperative regulation of p70S6 kinase by receptor tyrosine kinases and G protein-coupled receptors augments airway smooth muscle growth

We have previously demonstrated that concomitant activation of receptor tyrosine kinases and certain G protein-coupled receptors (GPCRs) can promote a synergistic increase in the rate of airway smooth muscle cell (ASM) proliferation. Here we clarify the role of p70S6 kinase (p70S6K) as an integrator of receptor tyrosine kinase and GPCR signaling that augments ASM DNA synthesis by demonstrating that specific p70S6K phosphorylation sites receive distinct regulatory input from GPCRs that promotes sustained kinase activity critical to mitogenesis. Prolonged stimulation of ASM cells with EGF and thrombin induced a greater than additive effect in levels of p70S6K phosphorylated at residue T389, whereas a significant but more modest increase in the level of T229 and T421/S424 phosphorylation was also observed. The augmenting effects of thrombin could be dissociated from p42/p44 MAPK activation, as selective inhibition of thrombin-stimulated p42/p44 failed to alter the profile of cooperative p70S6K T389 phosphorylation, p70S6K kinase activity, or ASM [(3)H]thymidine incorporation. Thrombin stimulated a sustained increase in the level of Akt phosphorylation and also augmented EGF-stimulated Akt phosphorylation. The cooperative effects of thrombin on Akt/p70S6K phosphorylation and [(3)H]thymidine incorporation were all attenuated by heterologous expression of Gbetagamma sequestrants. These data suggest that PI3K-dependent T389/T229 phosphorylation is limiting in late-phase p70S6K activation by EGF and contributes to the cooperative effect of GPCRs on p70S6K activity and cell growth.     

Nerve growth factor-induced stimulation of p38 mitogen-activated protein kinase in PC12 cells is partially mediated via G(i/o) proteins

Differentiation of PC12 cells by nerve growth factor (NGF) requires the activation of various mitogen-activated protein kinases (MAPKs) including p38 MAPK. Accumulating evidence has suggested cross-talk regulation of NGF-induced responses by G protein-coupled receptors, thus we examined whether NGF utilizes G(i/o) proteins to regulate p38 MAPK in PC12 cells. Induction of p38 MAPK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). NGF-dependent p38 MAPK phosphorylation became insensitive to PTX treatment upon transient expressions of Galpha(z) or the PTX-resistant mutants of Galpha(i2) and Galpha(oA). Moreover, Galpha(i2) was co-immunoprecipitated with the TrkA receptor from PC12 cell lysates. To discern the participation of various signaling intermediates, PC12 cells were treated with a panel of specific inhibitors prior to the NGF challenge. NGF-induced p38 MAPK phosphorylation was abolished by inhibitors of Src (PP1, PP2, and SU6656) and MEK1/2 (U0126). Inhibition of the p38 MAPK pathway also suppressed NGF-induced PC12 cell differentiation. In contrast, inhibitors of JAK2, phospholipase C, protein kinase C and Ca(2+)/calmodulin-dependent kinase II did not affect the ability of NGF to activate p38 MAPK. Collectively, these studies indicate that NGF-dependent p38 MAPK activity may be mediated via G(i2) protein, Src, and the MEK/ERK cascade.     

M2 muscarinic receptors induce airway smooth muscle activation via a dual, Gbetagamma-mediated inhibition of large conductance Ca2+-activated K+ channel activity

Airway smooth muscle is richly endowed with muscarinic receptors of the M(2) and M(3) subtype. Stimulation of these receptors inhibits large conductance calcium-activated K(+) (BK) channels, a negative feed back regulator, in a pertussis toxin-sensitive manner and thus facilitates contraction. The underlying mechanism, however, is unknown. We therefore studied the activity of bovine trachea BK channels in HEK293 cells expressing the M(2) or M(3) receptor (M(2)R or M(3)R). In M(2)R- but not M(3)R-expressing cells, maximal effective concentrations of carbamoylcholine (CCh) inhibited whole cell BK currents by 53%. This M(2)R-induced inhibition was abolished by pertussis toxin treatment or overexpression of the Gbetagamma scavenger transducin-alpha. In inside-out patches, direct application of 300 nm purified Gbetagamma decreased channel open probability by 55%. The physical interaction of Gbetagamma with BK channels was confirmed by co-immunoprecipitation. Interestingly, inhibition of phospholipase C as well as protein kinase C activities also reversed the CCh effect but to a smaller (approximately 20%) extent. Mouse tracheal cells responded similarly to CCh, purified Gbetagamma and phospholipase C/protein kinase C inhibition as M(2)R-expressing HEK293 cells. Our results demonstrate that airway M(2)Rs inhibit BK channels by a dual, Gbetagamma-mediated mechanism, a direct membrane-delimited interaction, and the activation of the phospholipase C/protein kinase C pathway.