Regulation of LPA receptor function by estrogens

17beta-Estradiol induced LPA(1) receptor desensitization in C9 cells stably expressing LPA(1) receptors and transiently expressing estrogen receptor alpha. Such desensitization was evidenced by a reduction in lysophosphatidic acid-mediated Ca(2+)mobilization and it was associated to receptor phosphorylation and internalization. These effects of 17beta-estradiol were rapid (taking place over 5 min) and were blocked by the estrogen receptor antagonist ICI 182780. Similarly, inhibitors of phosphoinositide 3-kinase (wortmannin and LY294002) and of protein kinase C (staurosporine and G? 6976) blocked 17beta-estradiol-induced LPA(1) receptor desensitization and phosphorylation. Confocal microscopy evidenced LPA(1) receptor internalization in response to 17beta-estradiol treatment. Association between LPA(1) receptors and protein kinase C alpha was suggested by co-immunoprecipitation assays. Protein kinase C alpha was associated with LPA(1) receptors in the absence of stimulus and such association further increased in a dynamic fashion in response to 17beta-estradiol. The results demonstrated that in C9 cells estrogens modulate LPA(1) action through estrogen receptor alpha with the participation of protein kinase C alpha and phosphoinositide 3-kinase.     

Sphingosine 1-phosphate-mediated α1B-adrenoceptor desensitization and phosphorylation. Direct and paracrine/autocrine actions

Sphingosine-1-phosphate-induced α1B-adrenergic receptor desensitization and phosphorylation were studied in rat-1 fibroblasts stably expressing enhanced green fluorescent protein-tagged adrenoceptors. Sphingosine-1-phosphate induced adrenoceptor desensitization and phosphorylation through a signaling cascade that involved phosphoinositide 3-kinase and protein kinase C activities. The autocrine/paracrine role of sphingosine-1-phosphate was also studied. It was observed that activation of receptor tyrosine kinases, such as insulin growth factor-1 (IGF-I) and epidermal growth factor (EGF) receptors increased sphingosine kinase activity. Such activation and consequent production of sphingosine-1-phosphate appear to be functionally relevant in IGF-I- and EGF-induced α1B-adrenoceptor phosphorylation and desensitization as evidenced by the following facts: a) expression of a catalytically inactive (dominant-negative) mutant of sphingosine kinase 1 or b) S1P1 receptor knockdown markedly reduced this growth factor action. This action of sphingosine-1-phosphate involves EGF receptor transactivation. In addition, taking advantage of the presence of the eGFP tag in the receptor construction, we showed that S1P was capable of inducing α1B-adrenergic receptor internalization and that its autocrine/paracrine generation was relevant for internalization induced by IGF-I. Four distinct hormone receptors and two autocrine/paracrine mediators participate in IGF-I receptor-α1B-adrenergic receptor crosstalk.     

Activation of extracellular-regulated kinases by normal and mutant EGF receptors

Glioblastoma cells express a mutant EGF receptor (EGFRvIII) that has constitutive tyrosine kinase activity and enhances their tumorigenicity. Here we show that EGFRvIII promotes constitutive phosphorylation of extracellular regulated kinases (ERKs) in glioblastoma cells in the absence of EGF. EGFRvIII also promoted constitutive activation of phosphoinositide 3-kinase in these cells, as assessed by phosphorylation of protein kinase B/akt. As expected, phosphorylation of protein kinase B/akt was blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY294002. Less expectedly, we found that this treatment also blocked EGFRvIII-induced phosphorylation of ERKs. In contrast, ERK phosphorylation induced by EGF-activated normal EGF receptor in the same cells was largely unaffected by treatment with phosphoinositide 3-kinase inhibitors. This difference in behavior between the normal receptor and EGFRvIII was not due to differences in the levels of activated EGFRvIII and wild-type EGF receptor, as the two types of receptor were tyrosine phosphorylated to a similar extent under the experimental conditions used. EGFRvIII activation of ERKs was also sensitive to the phospholipase C inhibitor U73122, whereas ERK activation by normal EGF receptor was not. These results show that EGFRvIII and wild-type EGF receptor preferentially use different signaling pathways to induce ERK phosphorylation. The different mechanisms of ERK activation used by normal and mutant EGF receptors may be important in understanding the potent tumorigenic activity of EGFRvIII.     

Insulin-like growth factor-I induces alpha(1B)-adrenergic receptor phosphorylation through G beta gamma and epidermal growth factor receptor transactivation

IGF-I induces alpha(1B)-adrenoceptor (alpha(1B)-AR) phosphorylation. The effect of IGF-I was rapid and transient, reaching near-maximal values at 10 min and decreasing after 30 min; it was observed at low IGF-I concentrations (EC(50) approximately 10 ng/ml) and was associated to receptor desensitization as evidenced by a decreased alpha(1B)-adrenergic effect on intracellular calcium and production of inositol phosphates. The effect of IGF-I was markedly decreased in cells treated with pertussis toxin suggesting involvement of pertussis toxin-sensitive G proteins. Transfection of the carboxyl terminus of the beta-adrenergic receptor kinase or the Deltap85 mutant of phosphoinositide 3-kinase (PI3K) markedly decreased the alpha(1B)-AR phosphorylation induced by IGF-I without decreasing the receptor phosphorylation induced by noradrenaline. Inhibitors of PI3K and protein kinase C blocked IGF-I-induced alpha(1B)-AR phosphorylation. In addition, it was observed that AG1478, an inhibitor of the epidermal growth factor (EGF) receptor kinase, and BB-94, a metalloproteinase inhibitor, also diminished IGF-I-induced adrenoceptor phosphorylation. The data clearly show that IGF-I triggers a complex signaling pathway, which leads to the phosphorylation and desensitization of a serpentine G protein-coupled receptor, suggesting the following hypothetical model: 1) stimulation of IGF-I receptors activate pertussis toxin-sensitive G proteins; 2) the growth factor action activates metalloproteinases, which catalyze heparin binding-EGF shedding, and transactivation of EGF receptors, and 3) dissociated Gbetagamma subunits and phosphotyrosine residues seem to trigger PI3K activity, which leads to activation of protein kinase C, resulting in alpha(1B)-AR phosphorylation and desensitization.     

Differences in signaling properties of the cytoplasmic domains of the insulin receptor and insulin-like growth factor receptor in 3T3-L1 adipocytes.

Insulin and insulin-like growth factors (IGFs) elicit distinct but overlapping biological effects in vivo. To investigate whether differences in intrinsic signaling capacity of receptors contribute to biological specificity, we constructed chimeric receptors containing the extracellular portion of the neurotrophin receptor TrkC fused to the intracellular portion of the insulin or IGF-I receptors. Chimeras were stably expressed in 3T3-L1 adipocytes at levels comparable to endogenous insulin receptors and were efficiently activated by neurotrophin-3. The wild-type insulin receptor chimera mediated approximately 2-fold greater phosphorylation of insulin receptor substrate 1 (IRS-1), association of IRS-1 with phosphoinositide 3-kinase, stimulation of glucose uptake, and GLUT4 translocation, compared with the IGF-I receptor chimera. In contrast, the IGF-I receptor chimera mediated more effective Shc phosphorylation, association of Shc with Grb2, and activation of mitogen-activated protein kinase compared with the insulin receptor chimera. The two receptors elicited similar activation of protein kinase B, p70S6 kinase, and glycogen synthesis. We conclude that the insulin receptor mediates some aspects of metabolic signaling in adipocytes more effectively than the IGF-I receptor, as a consequence of more efficient phosphorylation of IRS-1 and greater recruitment/activation of phosphoinositide 3-kinase.     

Insulin-induced oxidative neuronal injury in cortical culture: mediation by induced N-methyl-D-aspartate receptors

While effectively attenuating neuronal apoptosis in mouse cortical culture, insulin paradoxically induced neuronal necrosis with 48 h of exposure. The insulin neurotoxicity was blocked by an antioxidant but not by caspase inhibitors. Exposure to insulin led to tyrosine phosphorylation of the insulin receptor and the insulin-like growth factor-1 (IGF-1) receptor and activation of protein kinase C (PKC) and phosphoinositide 3-kinase (PI3-kinase). Inhibitors of tyrosine kinase and PKC, but not PI3-kinase, attenuated the insulin neurotoxicity. Conversely, the inhibitor of PI3-kinase but not PKC reversed the antiapoptotic effect of insulin. Suggesting that the gene activity-dependent emergence of excitotoxicity contributed to insulin neurotoxicity, macromolecule synthesis inhibitors and N-methyl-D-aspartate (NMDA) antagonists blocked it. Consistently, exposure to insulin increased the level of the NR2A subunit of the NMDA receptor without much altering NR1 or NR2B levels. The present study suggests that insulin can be both neuroprotective and neurotoxic in the same cell system but by way of different signaling cascades.     

Muscarinic receptors mediate phospholipase C-dependent activation of protein kinase B via Ca2+, ErbB3, and phosphoinositide 3-kinase in 1321N1 astrocytoma cells.

In 1321N1 astrocytoma cells, heterotrimeric G-protein-coupled receptors that activate phosphoinositide-specific phospholipase Cbeta (PLCbeta) isoforms via G(q), induced a prolonged activation of protein kinase B (PKB) after a short delay. For example, the effect of carbachol acting on M3 muscarinic receptors is blocked by wortmannin, suggesting it is mediated via a phosphoinositide 3-kinase (PI 3-kinase). In support of this, carbachol increased PI 3-kinase activity in PI 3-kinase (p85) immunoprecipitates. The pathway linking PLC-coupled receptors to PI 3-kinase was deduced to involve phosphoinositide hydrolysis and Ca2+-dependent ErbB3 transactivation but not protein kinase C on the basis of the following evidence: (i) inhibition of carbachol stimulated PLC by pretreatment with the phorbol ester phorbol 12-myristate 13-acetate concomitantly reduced PKB activity, whereas stimulation of other PLC-coupled receptors also activated PKB; (ii) Ca2+ ionophores and thapsigargin stimulated PKB activity in a wortmannin-sensitive manner, whereas bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid blocked carbachol-stimulated PKB activity; (iii) phorbol 12-myristate 13-acetate alone did not activate PKB, whereas a protein kinase C inhibitor did not prevent the activation of PKB by carbachol; and (iv) carbachol stimulated ErbB3-tyrosine phosphorylation and association with p85, and both these and PKB activity were blocked by tyrphostin AG1478, an epidermal growth factor receptor-tyrosine kinase inhibitor. These experiments define a novel pathway linking G(q)-coupled G-protein-coupled receptors to the activation of PI 3-kinase and PKB.     

Cross-talk between tyrosine kinase and G-protein-linked receptors. Phosphorylation of beta 2-adrenergic receptors in response to insulin.

Protein kinases play a pivotal role in the propagation and modulation of transmembrane signaling pathways. Two major classes of receptors, G-protein-linked and tyrosine kinase receptors not only propagate signals but also are substrates for phosphorylation in response to stimulation by agonist ligands. Insulin (operating via tyrosine kinase receptors) and catecholamines (operating by G-protein-linked receptors) are counterregulatory with respect to lipid and carbohydrate metabolism. How, on a cellular level, these two distinct classes of receptors may cross-regulate each other remains controversial. In the present work we identify a novel cross-talk between members of two distinct classes of receptors, tyrosine kinase (insulin) and G-protein-linked (beta-adrenergic) receptors. Treatment of DDT1 MF-2 hamster vas deferens smooth muscle cells with insulin promoted a marked attenuation (desensitization) of beta-adrenergic receptor-mediated activation of adenylylcyclase. Measured by immune precipitation of beta 2-adrenergic receptors from cells metabolically labeled with [32P]orthophosphate, the basal state of receptor phosphorylation was increased 2-fold by insulin. Phosphoamino acid analysis revealed that for insulin-stimulated cells, the beta 2-adrenergic receptors showed increased phosphorylation on tyrosyl and decreased phosphorylation on threonyl residues. Phosphorylation of the beta-adrenergic receptor was rapid and peaked at 30 min following stimulation of cells by insulin. beta-Adrenergic receptor phosphorylation and attenuation of catecholamine-sensitive adenylylcyclase provide a biochemical basis for the counterregulatory effects of insulin upon catecholamine action.     

Prostacyclin receptor-mediated activation of extracellular signal-regulated kinases 1 and 2

The prostacyclin mimetic cicaprost increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in Chinese hamster ovary cells transiently expressing human (hIP-CHO) or mouse prostacyclin (mIP-CHO) receptors, but not in human neuroblastoma SK-N-SH cells or rat/mouse neuroblastoma-glioma NG108-15 cells which endogenously express IP receptors. Cicaprost stimulated ERK1/2 activity in hIP-CHO and mIP-CHO cells with EC50 values of 60 and 83 nM, respectively, and this response was significantly inhibited by protein kinase C inhibitors and agents which elevate cyclic AMP. A poor correlation was discovered between the level of ERK1/2 activity and the ability of agents to increase or decrease cyclic AMP production. The potent inhibitory effect of 3-isobutyl-1-methyl xanthine on cicaprost-stimulated phospho-ERK1/2 may be due to inhibition of phosphoinositide 3-kinase. Therefore, IP receptor-mediated activation of ERK1/2 in CHO cells occurs through a Gq/11/protein kinase C-dependent and a phosphoinoside 3-kinase-dependent process which is insensitive to IP receptor-generated cyclic AMP.     

Ligand-induced alpha2-adrenoceptor endocytosis: relationship to Gi protein activation

Most G protein-coupled receptors are desensitized by a uniform two-step mechanism: phosphorylation followed by arrestin binding and internalization. In this study we explored the time-, ligand-, and concentration dependence of alpha2-adrenoceptor internalization in human embryonal kidney (HEK-293) cells expressing alpha2A- and alpha2B-adrenoceptors. We also explored the relationship between ligand-induced receptor internalization and agonist efficacy, determined with a [35S]GTPgammaS binding assay. The results showed rapid dose-dependent internalization of both alpha2A- and alpha2B-receptors; the extent of internalization was directly proportional to agonist efficacy. The agonist UK 14,304 had a subtype-specific high efficacy at alpha2A-AR and dexmedetomidine at alpha2B-AR. Agonist-induced [35S]GTPgammaS binding was totally blocked by pretreatment with pertussis toxin (PTX) for both receptor subtypes, while only about 50% of the internalization was blocked by PTX. The results indicate that the extent of internalization of alpha2A-AR and alpha2B-AR is proportional to agonist efficacy, but only partly dependent on Gi protein coupling.     

Insulin-induced beta-arrestin1 Ser-412 phosphorylation is a mechanism for desensitization of ERK activation by Galphai-coupled receptors

Beta-arrestin1 is an adapter/scaffold for many G protein-coupled receptors during mitogen-activated protein kinase signaling. Phosphorylation of beta-arrestin1 at position Ser-412 is a regulator of beta-arrestin1 function, and in the present study, we showed that insulin led to a time- and dose-dependent increase in beta-arrestin1 Ser-412 phosphorylation, which blocked isoproterenol- and lysophosphatidic acid-induced Ser-412 dephosphorylation and impaired ERK signaling by these G protein-coupled receptor ligands. Insulin treatment also led to accumulation of Ser-412-phosphorylated beta-arrestin1 at the insulin-like growth factor 1 receptor and prevented insulin-like growth factor 1/Src association. Insulin-induced Ser-412 phosphorylation was partially dependent on ERK as treatment with the MEK inhibitor PD98059 inhibited the insulin effect (62% reduction, p = 0.03). Inhibition of phosphatidylinositol 3-kinase by wortmannin did not have a significant effect (9% reduction, p = 0.41). We also found that the protein phosphatase 2A (PP2A) was in a molecular complex with beta-arrestin1 and that the PP2A inhibitor okadaic acid increased Ser-412 phosphorylation. Concomitant addition of insulin and okadaic acid did not produce an additive effect on Ser-412 phosphorylation, suggesting a common mechanism. Small t antigen specifically inhibited PP2A, and in HIRcB cells expressing small t antigen, beta-arrestin1 Ser-412 phosphorylation was increased, and insulin had no further effect. Insulin treatment caused increased beta-arrestin1 Ser-412 phosphorylation, which blocked mitogen-activated protein kinase signaling and internalization by beta-arrestin1-dependent receptors with no effect on beta-adrenergic receptor Gs-mediated cAMP production. These findings provide a new mechanism for insulin-induced desensitization of ERK activation by Galphai-coupled receptors.     

Angiotensin II subtype 2 receptor activation inhibits insulin-induced phosphoinositide 3-kinase and Akt and induces apoptosis in PC12W cells

In the present study, we identified novel negative cross-talk between the angiotensin II subtype 2 (AT2) receptor and insulin receptor signaling in the regulation of phosphoinositide 3-kinase (PI3K), Akt, and apoptosis in rat pheochromocytoma cell line, PC12W cells, which exclusively express AT2 receptor. We demonstrated that insulin-mediated insulin receptor substrate (IRS)-2-associated PI3K activity was inhibited by AT2 receptor stimulation, whereas IRS-1-associated PI3K activity was not significantly influenced. AT2 receptor stimulation did not change insulin-induced tyrosine phosphorylation of IRS-2 or its association with the p85alpha subunit of PI3K, but led to a significant reduction of insulin-induced p85alpha phosphorylation. AT2 receptor stimulation increased the association of a protein tyrosine phosphatase, SHP-1, with IRS-2. Moreover, we demonstrated that AT2 receptor stimulation inhibited insulin-induced Akt phosphorylation and that insulin-mediated antiapoptotic effect was also blocked by AT2 receptor activation. Overexpression of a catalytically inactive dominant negative SHP-1 markedly attenuated the AT2 receptor- mediated inhibition of IRS-2-associated PI3K activity, Akt phosphorylation, and antiapoptotic effect induced by insulin. Taken together, these results indicate that AT2 receptor-mediated activation of SHP-1 and the consequent inhibition IRS-2-associated PI3K activity contributed at least partly to the inhibition of Akt phosphorylation, thereby inducing apoptosis.     

Stimulation of an Insulin Receptor Activates and Down-Regulates the Ca2+-Independent Protein Kinase C, Apl II, Through a Wortmannin-Sensitive Signaling Pathway in Aplysia

Abstract: Activation of tyrosine kinase-linked receptors has been shown to stimulate Ca²⁺-independent protein kinase C isoforms in nonneuronal cells. We have examined this signaling pathway in the nervous system. Incubating bag cell neurons from the marine mollusk Aplysia californica with concentrations of insulin known to stimulate a tyrosine kinase-linked receptor in these cells persistently activated and down-regulated the Ca²⁺-independent protein kinase C (Apl II), whereas insulin only transiently activated and did not down-regulate the Ca²⁺-activated protein kinase C (Apl I). The effects of insulin may be mediated by activation of phosphoinositide 3-kinase because (a) diC₁₆phosphatidylinositol 3,4,5-trisphosphate, a synthetic phosphoinositide 3-kinase product, stimulated autophosphorylation of baculovirus-expressed Apl II, but not of Apl I, and (b) wortmannin, an inhibitor of phosphoinositide 3-kinase, blocked the activation and down-regulation of Apl II by insulin but not the transient activation of Apl I. These results suggest that activators of tyrosine kinase-linked receptors may mediate some of their effects in neurons through activation of Ca²⁺-independent protein kinase C isoforms.     

Lysophosphatidic acid induces alpha1B-adrenergic receptor phosphorylation through G beta gamma,phosphoinositide 3-kinase,protein kinase C and epidermal growth factor receptor transactivation

Lysophosphatidic acid (LPA) induces alpha(1B)-adrenoceptor phosphorylation through pertussis toxin-sensitive G proteins, phosphoinositide 3-kinase (PI3K) and protein kinase C (PKC). Here we showed that transfection of the carboxyl terminus of the beta-adrenergic receptor kinase (betaARK) or the Deltap85 mutant of PI3K markedly decreased the alpha(1B)-adrenoceptor phosphorylation induced by LPA without decreasing the receptor phosphorylations induced by active phorbol esters or noradrenaline. In addition, it was observed that inhibitors of epidermal growth factor (EGF) receptor kinase and of metalloproteinases and an anti-heparin binding-EGF antibody also diminish LPA-induced phosphorylation; such partial inhibitions were not additive, indicating that they occur through a common process.Our data indicate that stimulation of LPA receptors activates pertussis-toxin-sensitive G proteins. Dissociated Gbetagamma subunits initiate two processes: one of them involving activation of metalloproteinases, heparin binding-EGF shedding and transactivation of EGF receptors and another independent of these events. Both processes triggered PI3K activity, which lead to activation of PKC and this to alpha(1B)-adrenoceptor phosphorylation. This is the first demonstration of a role of EGF receptor transactivation in the phosphorylation of a G protein-coupled receptor.     

PDGF-induced vascular smooth muscle cell proliferation is associated with dysregulation of insulin receptor substrates

In vascular smooth muscle cells (VSMCs), platelet-derived growth factor (PDGF) plays a major role in inducing phenotypic switching from contractile to proliferative state. Importantly, VSMC phenotypic switching is also determined by the phosphorylation state/expression levels of insulin receptor substrate (IRS), an intermediary signaling component that is shared by insulin and IGF-I. To date, the roles of PDGF-induced key proliferative signaling components including Akt, p70S6kinase, and ERK1/2 on the serine phosphorylation/expression of IRS-1 and IRS-2 isoforms remain unclear in VSMCs. We hypothesize that PDGF-induced VSMC proliferation is associated with dysregulation of insulin receptor substrates. Using human aortic VSMCs, we demonstrate that prolonged PDGF treatment led to sustained increases in the phosphorylation of protein kinases such as Akt, p70S6kinase, and ERK1/2, which mediate VSMC proliferation. In addition, PDGF enhanced IRS-1/IRS-2 serine phosphorylation and downregulated IRS-2 expression in a time- and concentration-dependent manner. Notably, phosphoinositide 3-kinase (PI 3-kinase) inhibitor (PI-103) and mammalian target of rapamycin inhibitor (rapamycin), which abolished PDGF-induced Akt and p70S6kinase phosphorylation, respectively, blocked PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. In contrast, MEK1/ERK inhibitor (U0126) failed to block PDGF-induced IRS-1 serine phosphorylation and IRS-2 downregulation. PDGF-induced IRS-2 downregulation was prevented by lactacystin, an inhibitor of proteasomal degradation. Functionally, PDGF-mediated IRS-1/IRS-2 dysregulation resulted in the attenuation of insulin-induced IRS-1/IRS-2-associated PI 3-kinase activity. Pharmacological inhibition of PDGF receptor tyrosine kinase with imatinib prevented IRS-1/IRS-2 dysregulation and restored insulin receptor signaling. In conclusion, strategies to inhibit PDGF receptors would not only inhibit neointimal growth but may provide new therapeutic options to prevent dysregulated insulin receptor signaling in VSMCs in nondiabetic and diabetic states.     

Inhibition of insulin-induced activation of Akt by a kinase-deficient mutant of the epsilon isozyme of protein kinase C

Akt, also known as protein kinase B, is a protein-serine/threonine kinase that is activated by growth factors in a phosphoinositide (PI) 3-kinase-dependent manner. Although Akt mediates a variety of biological activities, the mechanisms by which its activity is regulated remain unclear. The potential role of the epsilon isozyme of protein kinase C (PKC) in the activation of Akt induced by insulin has now been examined. Expression of a kinase-deficient mutant of PKCepsilon (epsilonKD), but not that of wild-type PKCepsilon or of kinase-deficient mutants of PKCalpha or PKClambda, with the use of adenovirus-mediated gene transfer inhibited the phosphorylation and activation of Akt induced by insulin in Chinese hamster ovary cells or L6 myotubes. Whereas the epsilonKD mutant did not affect insulin stimulation of PI 3-kinase activity, the phosphorylation and activation of Akt induced by a constitutively active mutant of PI 3-kinase were inhibited by epsilonKD, suggesting that epsilonKD affects insulin signaling downstream of PI 3-kinase. PDK1 (3'-phosphoinositide-dependent kinase 1) is thought to participate in Akt activation. Overexpression of PDK1 with the use of an adenovirus vector induced the phosphorylation and activation of Akt; epsilonKD inhibited, whereas wild-type PKCepsilon had no effect on, these actions of PDK1. These results suggest that epsilonKD inhibits the insulin-induced phosphorylation and activation of Akt by interfering with the ability of PDK1 to phosphorylate Akt.     

Differential signalling of purinoceptors in HeLa cells through the extracellular signal-regulated kinase and protein kinase C pathways

We have previously shown that HeLa cells express P2Y2 and P2Y6 receptors endogenously and determined the pathways by which the P2Y2 controls proliferation and Na+/K+ATPase activity. Our objective in this study was to investigate the hypothesis that P2Y6 also controls proliferation and Na+/K+ATPase activity; the pathways used in these actions were partially characterised. We found that P2Y6 activation controlled cell proliferation but not the activity of the Na+/K+ATPase. UDP activation of P2Y6 provoked: (a) an increase in free cytosolic calcium; (b) the activation of protein kinase C-alpha, -beta, -delta, -epsilon, and -zeta but not of PKC-iota and -eta; (c) the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2); (d) the expression of c-Fos protein. The P2Y6 induced cell proliferation was blocked by the mitogen-activated protein kinase kinase (MAPKK) inhibitor PD098059, thereby indicating that the ERK pathway mediates the mitogenic signalling of P2Y6. PKC and phosphoinositide 3-kinase (PI3K) inhibitors were tested at two different time points of ERK1/2 phosphorylation (10 and 60 min). The results suggest that novel PKCs and PI3K initiate the response but both conventional and atypical PKCs are required for the maintenance of the UDP-induced phosphorylation of ERK1/2. The induction of c-Fos was greatly diminished by conventional or atypical PKC-zeta inhibition, suggesting that it may be due to PKC-alpha/beta and -zeta activity. These observations demonstrate that UDP acts as a proliferative agent in HeLa cells activating multiple signalling pathways involving conventional, novel, and atypical PKCs, PI3K, and ERK. Of these pathways, conventional and atypical PKCs appear responsible for the induction of c-Fos, while ERK is responsible for cell proliferation and depends upon both novel and atypical PKCs and PI3K activities.     

Differential regulation of the phosphatidylinositol 3-kinase/Akt and p70 S6 kinase pathways by the alpha(1A)-adrenergic receptor in rat-1 fibroblasts

Phosphatidylinositol (PI) 3-kinase and its downstream effector Akt are thought to be signaling intermediates that link cell surface receptors to p70 S6 kinase. We examined the effect of a G(q)-coupled receptor on PI 3-kinase/Akt signaling and p70 S6 kinase activation using Rat-1 fibroblasts stably expressing the human alpha(1A)-adrenergic receptor. Treatment of the cells with phenylephrine, a specific alpha(1)-adrenergic receptor agonist, activated p70 S6 kinase but did not activate PI 3-kinase or any of the three known isoforms of Akt. Furthermore, phenylephrine blocked the insulin-like growth factor-I (IGF-I)-induced activation of PI 3-kinase and the phosphorylation and activation of Akt-1. The effect of phenylephrine was not confined to signaling pathways that include insulin receptor substrate-1, as the alpha(1)-adrenergic receptor agonist also inhibited the platelet-derived growth factor-induced activation of PI 3-kinase and Akt-1. Although increasing the intracellular Ca(2+) concentration with the ionophore A23187 inhibited the activation of Akt-1 by IGF-I, Ca(2+) does not appear to play a role in the phenylephrine-mediated inhibition of the PI 3-kinase/Akt pathway. The differential ability of phenylephrine and IGF-I to activate Akt-1 resulted in a differential ability to protect cells from UV-induced apoptosis. These results demonstrate that activation of p70 S6 kinase by the alpha(1A)-adrenergic receptor in Rat-1 fibroblasts occurs in the absence of PI 3-kinase/Akt signaling. Furthermore, this receptor negatively regulates the PI 3-kinase/Akt pathway, resulting in enhanced cell death following apoptotic insult.     

Agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane by beta-adrenergic receptor kinase 1. A role in receptor sequestration

Agonist-dependent desensitization of the beta-adrenergic receptor requires translocation and activation of the beta-adrenergic receptor kinase1 by liberated Gbetagamma subunits. Subsequent internalization of agonist-occupied receptors occurs as a result of the binding of beta-arrestin to the phosphorylated receptor followed by interaction with the AP2 adaptor and clathrin proteins. Receptor internalization is known to require D-3 phosphoinositides that are generated by the action of phosphoinositide 3-kinase. Phosphoinositide 3-kinases form a family of lipid kinases that couple signals via receptor tyrosine kinases and G-protein-coupled receptors. The molecular mechanism by which phosphoinositide 3-kinase acts to promote beta-adrenergic receptor internalization is not well understood. In the present investigation we demonstrate a novel finding that beta-adrenergic receptor kinase 1 and phosphoinositide 3-kinase form a cytosolic complex, which leads to beta-adrenergic receptor kinase 1-mediated translocation of phosphoinositide 3-kinase to the membrane in an agonist-dependent manner. Furthermore, agonist-induced translocation of phosphoinositide 3-kinase results in rapid interaction with the receptor, which is of functional importance, since inhibition of phosphoinositide 3-kinase activity attenuates beta-adrenergic receptor sequestration. Therefore, agonist-dependent recruitment of phosphoinositide 3-kinase to the membrane is an important step in the process of receptor sequestration and links phosphoinositide 3-kinase to G-protein-coupled receptor activation and sequestration.