Phosphorylation of serine 1105 by protein kinase A inhibits phospholipase Cbeta3 stimulation by Galphaq

The mechanism by which protein kinase A (PKA) inhibits Galphaq -stimulated phospholipase C activity of the beta subclass (PLCbeta ) is unknown. We present evidence that phosphorylation of PLCbeta3 by PKA results in inhibition of Galphaq -stimulated PLCbeta3 activity, and we identify the site of phosphorylation. Two-dimensional phosphoamino acid analysis of in vitro phosphorylated PLCbeta3 revealed a single phosphoserine as the putative PKA site, and peptide mapping yielded one phosphopeptide. The residue was identified as Ser1105 by direct sequencing of reverse-phase high pressure liquid chromatography-isolated phosphopeptide and by site-directed mutagenesis. Overexpression of Galphaq with PLCbeta3 or PLCbeta (Ser1105--> Ala) mutant in COSM6 cells resulted in a 5-fold increase in [3H]phosphatidylinositol 1,4,5-trisphosphate formation compared with expression of Galphaq, PLCbeta3, or PLCbeta3 (Ser1105 --> Ala mutant alone. Whereas Galpha1-stimulated PLCbeta3, activity was inhibited by 58-71% by overexpression of PKA catalytic subunit, Galphaq-stimulated PLCbeta3 (Ser1105 --> Ala) mutant activity was not affected. Furthermore, phosphatidylinositide turnover stimulated by presumably Galpha1-coupled M1 muscarinic and oxytocin receptors was completely inhibited by pretreating cells with 8-[4-chlorophenythio]-cAMP in RBL-2H3 cells expressing only PLCbeta3. These data establish that direct phosphorylation by PKA of Ser1105 in the putative G-box of PLCbeta3 inhibits Galphaq-stimulated PLCbeta3 activity. This can at least partially explain the inhibitory effect of PKA on Galphaq-stimulated phosphatidylinositide turnover observed in a variety of cells and tissues.     

Differential regulation of formyl peptide and platelet-activating factor receptors. Role of phospholipase Cbeta3 phosphorylation by protein kinase A

Formylated peptides (e.g. n-formyl-Met-Leu-Phe (fMLP)) and platelet-activating factor (PAF) mediate chemotactic and cytotoxic responses in leukocytes through receptors coupled to G proteins that activate phospholipase C (PLC). In RBL-2H3 cells, fMLP utilizes a pertussis toxin (ptx)-sensitive G protein to activate PLC, whereas PAF utilizes a ptx-insensitive G protein. Here we demonstrate that fMLP, but not PAF, enhanced intracellular cAMP levels via a ptx-sensitive mechanism. Protein kinase A (PKA) inhibition by H-89 enhanced inositol phosphate formation stimulated by fMLP but not PAF. Furthermore, a membrane-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) inhibited phosphoinositide hydrolysis and secretion stimulated by fMLP but not PAF. Both cpt-cAMP and fMLP stimulated PLCbeta3 phosphorylation in intact RBL cells. The purified catalytic subunit of PKA phosphorylated PLCbeta3 immunoprecipitated from RBL cell lysate. Pretreatment of intact cells with cpt-cAMP and fMLP, but not PAF, resulted in an inhibition of subsequent PLCbeta3 phosphorylation by PKA in vitro. These data demonstrate that fMLP receptor, which couples to a ptx-sensitive G protein, activates both PLC and cAMP production. The resulting PKA activation phosphorylates PLCbeta3 and appears to block the ability of Gbetagamma to activate PLC. Thus, both fMLP and PAF generate stimulatory signals for PLCbeta3, but only fMLP produces a PKA-dependent inhibitory signal. This suggests a novel mechanism for the bidirectional regulation of receptors which activate PLC by ptx-sensitive G proteins.     

Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin

Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.     

Opioid modulation of extracellular signal-regulated protein kinase activity is ras-dependent and involves Gbetagamma subunits

Although it is well-established that G protein-coupled receptor signaling systems can network with those of tyrosine kinase receptors by several mechanisms, the point(s) of convergence of the two pathways remains largely undelineated, particularly for opioids. Here we demonstrate that opioid agonists modulate the activity of the extracellular signal-regulated protein kinase (ERK) in African green monkey kidney COS-7 cells transiently cotransfected with mu-, delta-, or kappa-opioid receptors and ERK1- or ERK2-containing plasmids. Recombinant proteins in transfected cells were characterized by binding assay or immunoblotting. On treatment with corresponding mu- ([D-Ala2,Me-Phe4,Gly-ol5]enkephalin)-, delta- ([D-Pen2,D-Pen5]enkephalin)-, or kappa- (U69593)-selective opioid agonists, a dose-dependent, rapid stimulation of ERK1 and ERK2 activity was observed. This activation was inhibited by specific antagonists, suggesting the involvement of opioid receptors. Pretreatment of cells with pertussis toxin abolished ERK1 and ERK2 activation by agonists. Cotransfection of cells with dominant negative mutant N17-Ras or with a betagamma scavenger, CD8- beta-adrenergic receptor kinase-C, suppressed opioid stimulation of ERK1 and ERK2. When epidermal growth factor was used to activate ERK1, chronic (>2-h) opioid agonist treatment resulted in attenuation of the stimulation by the growth factor. This inhibition was blocked by the corresponding antagonists and CD8- beta-adrenergic receptor kinase-C cotransfection. These results suggest a mechanism involving Ras and betagamma subunits of Gi/o proteins in opioid agonist activation of ERK1 and ERK2, as well as opioid modulation of epidermal growth factor-induced ERK activity.     

Somatostatin receptor-mediated signaling in smooth muscle. Activation of phospholipase C-beta3 by Gbetagamma and inhibition of adenylyl cyclase by Galphai1 and Galphao

In COS-7 cells, all five cloned somatostatin receptors are coupled via inhibitory G proteins to activation of an unidentified phospholipase C-beta (PLC-beta) isozyme and inhibition of adenylyl cyclase. In the present study, intestinal smooth muscle cells (SMC) that express only one receptor type, sstr3, and possess a full complement of G proteins and PLC-beta isozymes were used to identify the PLC-beta isozyme and the G proteins coupled to it and to adenylyl cyclase. Somatostatin-14 bound with high affinity to intestinal SMC; stimulated D-myo-inositol-1,4,5-trisphosphate (IP3) formation, Ca2+ release, and contraction; and inhibited forskolin-stimulated cAMP formation in a pertussis toxin-sensitive fashion. Somatostatin also stimulated phosphoinositide hydrolysis in plasma membranes. Only those somatostatin analogs that shared a high affinity for sstr3 receptors elicited muscle contraction. IP3 formation, Ca2+ release, and contraction in permeabilized SMC and phosphoinositide hydrolysis in plasma membranes were inhibited (approximately 80%) by pretreatment with antibodies to PLC-beta3 but not other PLC-beta isozymes, and by antibodies to Gbeta but not Galpha. Inhibition of cAMP formation was partially blocked by antibody to Galphai1 or Galphao and additively blocked by a combination of both antibodies. Somatostatin-stimulated [35S]GTPgammaS-Galpha complexes in plasma membranes were bound selectively by Galphai1 and Galphao antibodies. We conclude that in smooth muscle sstr3 is coupled to Gi1 and Go; the alpha subunits of both G proteins mediate inhibition of adenylyl cyclase, while the betagamma subunits mediate activation of PLC-beta3.     

Dopamine regulates phosphate uptake by opossum kidney cells through multiple counter-regulatory receptors

The purpose of this study was to determine the mechanisms of dopamine regulation of phosphate uptake in opossum kidney (OK) cells, a model of proximal renal tubules. Dopamine stimulated cAMP generation and inhibited radiolabeled phosphate uptake into OK cell monolayers by 14.4 +/- 1.8%. The effect of dopamine was transient, as phosphate uptake returned toward control level by 3 h despite the continued presence of dopamine. Pretreatment with pertussis toxin increased dopamine inhibition of phosphate uptake to 25 +/- 3%, increased the duration of the dopamine effect to at least 3 h, and enhanced cAMP generation. In an OK cell clone that overexpressed cAMP phosphodiesterase, dopamine did not inhibit phosphate uptake, but pharmacologic inhibition of protein kinase A activation did not prevent dopamine inhibition of phosphate uptake. A DA1 receptor agonist inhibited phosphate uptake more potently than dopamine (29.5 +/- 1.1%) or a DA2 receptor agonist (7.9 +/- 2%). However, both DA1 and DA2 receptor antagonists completely blocked dopamine inhibition of phosphate uptake. DA1, but not the DA2, antagonists blocked dopamine-stimulated cAMP generation. Treatment with alpha-adrenergic receptor antagonists potentiated dopamine inhibition of phosphate uptake to the same extent as pertussis toxin and was not additive with pertussis toxin. It is concluded that dopamine inhibits phosphate uptake through DA1 and DA2 receptor stimulation by cAMP-dependent and -independent pathways and activates a pertussis toxin-sensitive counter-regulatory pathway that attenuates this response through alpha-adrenergic receptor stimulation.     

Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells

Members of the beta isozyme subfamily of phosphatidylinositol-specific phospholipase C (PLC) are stimulated by alpha subunits and betagamma dimers of heterotrimeric guanine-nucleotide-binding proteins (G proteins). Myeloid differentiated human HL-60 granulocytes and bovine neutrophils contain a soluble phospholipase C, which is stimulated by the metabolically stable GTP analogue guanosine (5'-->O)-3-thiotriphosphate (GTP[S]). To identify the component(s) involved in mediating this stimulation, the relevant polypeptide(s) was resolved from endogenous phospholipase C and purified from bovine neutrophil cytosol by measuring its ability to confer GTP[S] stimulation to exogenous recombinant PLCbeta2. The resolved factor, which behaved as 48-kDa protein upon gel filtration, stimulated PLCbeta2 but not PLCbeta1 or PLCdelta1. Activation of phosphatidylinositol 4-phosphate 5-kinase was not involved in this stimulation. The purified stimulatory factor consisted of two polypeptides of molecular masses of approximately 23 kDa and 26 kDa. The protein stimulated a deletion mutant of PLCbeta2 that lacked a carboxyl-terminal region necessary for stimulation by members of the alpha(q) subfamily of the G-protein alpha subunits. The results of this study suggest that a GTP-binding protein distinct from alpha(q) subunits, probably a low-molecular-mass GTP-binding protein associated with a regulatory protein, is involved in isozyme-specific activation of PLCbeta2.     

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus

In normoxic conditions, myocardial glucose utilization is inhibited when alternative oxidizable substrates are available. In this work we show that this inhibition is relieved in the presence of cAMP, and we studied the mechanism of this effect. Working rat hearts were perfused with 5.5 mM glucose alone (controls) or together with 5 mM lactate, 5 mM beta-hydroxybutyrate, or 1 mM palmitate. The effects of 0.1 mM chlorophenylthio-cAMP (CPT-cAMP), a cAMP analogue, were studied in each group. Glucose uptake, flux through 6-phosphofructo-1-kinase, and pyruvate dehydrogenase activity were inhibited in hearts perfused with alternative substrates, and addition of CPT-cAMP completely relieved the inhibition. The mechanism by which CPT-cAMP induced a preferential utilization of glucose was related to an increased glucose uptake and glycolysis, and to an activation of phosphorylase, pyruvate dehydrogenase, and 6-phosphofructo-2-kinase, the enzyme responsible for the synthesis of fructose 2,6-bisphosphate, the well-known stimulator of 6-phosphofructo-1-kinase. In vitro phosphorylation of 6-phosphofructo-2-kinase by cAMP-dependent protein kinase increased the Vmax of the enzyme and decreased its sensitivity to the inhibitor citrate. Therefore, in hearts perfused with various oxidizable substrates, cAMP induces a preferential utilization of glucose by a concerted stimulation of glucose transport, glycolysis, glycogen breakdown, and glucose oxidation.     

Multiple signaling pathways of human interleukin-8 receptor A. Independent regulation by phosphorylation

Interleukin-8 (IL-8) receptor A (CXCR1) couples to a pertussis toxin-sensitive G protein to mediate phospholipase Cbeta (PLCbeta) activation and cellular responses. Responses to CXCR1 are attenuated by prior exposure of neutrophils to either IL-8, a cleavage product of the fifth component of complement (C5a) or n-formylated peptides (formylmethionylleucylphenylalanine, fMLP). To characterize the role of receptor phosphorylation in the regulation of the CXCR1, a phosphorylation-deficient mutant, M2CXCR1, was constructed. This receptor, stably expressed in RBL-2H3 cells, coupled more efficiently to G protein and stimulated enhanced phosphoinositide hydrolysis, cAMP production, exocytosis, and phospholipase D activation, and was resistant to IL-8-induced receptor internalization. The rate and total amount of ligand stimulated actin polymerization remained unchanged, but interestingly, chemotaxis was decreased by approximately 30% compared with the wild type receptor. To study the role of receptor phosphorylation in cross-desensitization of chemoattractant receptors, M2CXCR1 was coexpressed with cDNAs encoding receptors for either fMLP (FR), C5a (C5aR), or platelet-activating factor (PAFR). Both C5aR and PAFR were cross-phosphorylated upon M2CXCR1 activation, resulting in attenuated guanosine 5'-3'-O-(thio)triphosphate (GTPgammaS) binding in membranes. In contrast, FR and M2CXCR1 were resistant to cross-phosphorylation and cross-inhibition of GTPgammaS binding by other receptors. Despite the resistance of M2CXCR1 to cross-phosphorylation and receptor/G protein uncoupling, its susceptibility to cross-desensitization of its Ca2+ response by fMLP and C5a, was equivalent to CXCR1. Regardless of the enhancement in certain receptor functions in M2CXCR1 compared with the wild type CXCR1, the mutated receptors mediated equivalent PLCbeta3 phosphorylation and cross-desensitization of Ca2+ mobilization by FR, C5aR, and PAFR. The results herein indicate that phosphorylation of CXCR1 regulates some, but not all of the receptors functions. While receptor phosphorylation inhibits G protein turnover, PLC activation, Ca2+ mobilization and secretion, it is required for normal chemotaxis and receptor internalization. Since phosphorylation of CXCR1 had no effect on its ability to induce phosphorylation of PLCbeta3 or to mediate class-desensitization, these activities may be mediated by independently regulated pathways.     

A membrane-permeant, bioactivatable derivative of Ins(1,3,4)P3 and its effect on Cl(-)-secretion from T84 cells

The synthesis of rac-2,5,6-tri-O-butyryl-myo-inositol 1,3,4-trisphosphate hexakis(acetoxymethyl) ester [Bt3-Ins(1,3,4)P3/AM, 1], a membrane-permeant derivative of myo-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] is reported. 1 inhibited calcium-mediated chloride secretion of T84 cells, suggesting a regulatory link of Ins(1,3,4)P3 and the biosynthesis of the known inhibitor myo-inositol 3,4,5,6-tetrakisphosphate.     

Parathyroid hormone raises cytosolic calcium in pancreatic islets: study on mechanisms

The pancreatic islets of Langerhans are targets for PTH and the action of the hormone on the islet is most likely mediated through the ability of PTH to increase cytosolic calcium ([Ca2+]i) of the islet cells. Although direct evidence for such an effect has been clearly demonstrated, the mechanisms through which the hormone exerts such an action are not elucidated. The present study examined these questions using pancreatic islets isolated from normal rats. Both 1-34 and 1-84 PTH produced a dose dependent increase in [Ca2+]i of the islets but the effect of the latter was significantly (P < 0.01) greater than that of the former. This action of PTH was significantly (P < 0.01) decreased by the use of PTH antagonist or by verapamil. The G protein activator (GTP gamma S) mimicked the effect of PTH while pertussis toxin and the G protein inhibitor (GDP beta S) significantly reduced the PTH-induced rise in [Ca2+]i. Dibutyryl cAMP, and phorbol ester 12-myristate 13 acetate increased [Ca2+]i of pancreatic islets in a dose dependent manner and the effect was inhibited (P < 0.01) by verapamil. Staurosporine inhibited the effect of TPA as well as of 1-84 PTH on [Ca2+]i of the islets. These data indicate that: (1) PTH increases [Ca2+]i of pancreatic islets, (2) this action is partly receptor mediated and is produced by activation of L-type calcium channels through stimulation of G protein(s), and (3) the rise in [Ca2+]i is due to both stimulation of cAMP generation and activation of protein kinase C.     

The Mel1a melatonin receptor is coupled to parallel signal transduction pathways

The recent cloning of a family of high affinity melatonin receptors has provided us with a unique opportunity to define the signal transduction pathways used by these receptors. We have studied signaling through the human Mel1a receptor subtype by stable expression of receptor complementary DNA in NIH 3T3 cells. Our data indicate that the human Mel1a receptor is coupled to inhibition of forskolin-stimulated cAMP accumulation by a pertussis toxin-sensitive G protein. Although melatonin alone is without effect on phosphoinositide hydrolysis, it potentiates the effects of PGF2 alpha stimulation on phospholipase C activation. Melatonin potentiates arachidonate release stimulated by PGF2 alpha and by ionomycin. The effects of melatonin on arachidonate release are sensitive to inhibition of protein kinase C. They are independent of the effects of melatonin on cAMP and do not appear to involve activation of mitogen-activated protein kinase. The effects of melatonin on both phosphoinositide hydrolysis and arachidonate release are sensitive to pertussis toxin treatment. Thus, we show that the melatonin signal is transduced by parallel pathways involving inhibition of adenylyl cyclase and potentiation of phospholipase activation.     

Differential intracellular signaling of the GalR1 and GalR2 galanin receptor subtypes

The diverse physiological functions exerted by the neuropeptide galanin may be regulated by multiple G protein-coupled receptor subtypes and intracellular signaling pathways. Three galanin receptor subtypes (GalRs) have been recently cloned, but the G protein coupling profiles of these receptors are not completely understood. We have generated GalR1- and GalR2-expressing Chinese hamster ovary (CHO) cell lines and systematically examined the potential for these two receptors to couple to the Gs, Gi, Go, and Gq proteins. Galanin did not stimulate an increase in cAMP levels in GalR1/CHO or GalR2/CHO cells, suggesting an inability of either receptor to couple to Gs. Galanin inhibited forskolin-stimulated cAMP production in GalR1/CHO cells by 70% and in GalR2/CHO cells by 30%, suggesting a strong coupling of GalR1 to Gi and a more modest coupling between GalR2 and Gi. GalR1 and GalR2 both mediated pertussis toxin-sensitive MAPK activity (2-3-fold). The stimulation mediated by GalR1 was inhibited by expression of the C-terminus of beta-adrenergic receptor kinase (beta ARKct), which specifically inhibits G beta gamma signaling, but was not affected by the protein kinase C (PKC) inhibitor, bis[indolylmaleimide], or cellular depletion of PKC. In contrast, GalR2-mediated MAPK activation was not affected by beta ARKct expression but was abolished by inhibition of PKC activity. The data demonstrate that GalR1 is coupled to a Gibetagamma signaling pathway to mediate MAPK activation. In contrast, GalR2 utilizes a distinct signaling pathway to mediate MAPK activation, which is consistent with Go-mediated MAPK activation in CHO cells. Galanin was unable to stimulate inositol phosphate (IP) accumulation in CHO or COS-7 cells expressing GalR1. In contrast, galanin stimulated a 7-fold increase in IP production in CHO or COS-7 cells expressing GalR2. The GalR2-mediated IP production was not affected by pertussis toxin, suggesting a linkage of GalR2 with Gq/G11. Thus, the GalR1 receptor appears to activate only the Gi pathway. By contrast, GalR2 is capable of stimulating signaling which is consistent with activation of Go, Gq/G11, and Gi. The differential signaling profiles and the tissue distribution patterns of GalR1 and GalR2 may underlie the functional spectra of galanin action mediated by these galanin receptors and regulate the diverse physiological functions of galanin.     

Human metabotropic glutamate receptor 2 couples to the MAP kinase cascade in chinese hamster ovary cells

We have examined the functional coupling of the human metabotropic glutamate receptor type 2 (mGluR2) with the regulation of the mitogen activated protein kinase (MAP kinase) signal transduction cascade. We demonstrated that L-glutamate stimulation of the human mGluR2 receptor transiently expressed in chinese hamster ovary (CHO) cells leads to a rapid increase in the activity of p42/p44 MAP kinase (also known as the extracellular signal regulated kinases, ERK1 and ERK2). Activation of p42/p44 MAP kinase has been demonstrated in a peptide phosphorylation assay and through the demonstration of a shift in electrophoretic mobility of p42 MAP kinase following activation. In both assay systems L-glutamate stimulation of MAP kinase was inhibited by pertussis toxin and by the MEK (MAP/ERK activating kinase) inhibitor PD 98059. We conclude that L-glutamate stimulation of the mGluR2 receptor in CHO cells mediated regulation of p42/p44 MAP kinase following the activation of pertussis toxin-sensitive G alpha(i) G-proteins via a distinct protein kinase signalling pathway that utilizes MEK.     

Sites important for PLCbeta2 activation by the G protein betagamma subunit map to the sides of the beta propeller structure

The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., I?iguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.     

Functional parameters calculated by 99mTc-MAG3 in transplanted kidney

In rat anterior pituitary tumour cells (GH3/B6) thyrotropin-releasing hormone (TRH) elicits a biphasic response. First, a release of intracellularly stored Ca2+ induces a hyperpolarization of the cell. Second, a depolarization thought to be induced by a reduction of the inward-rectifying K+ current (KIR) causes an increase in action potential frequency and a plateau-like increase in [Ca2+]i. It has been proposed that the two phases are induced by the actions of inositol 1,4,5-trisphosphate (InsP3) and protein kinase C (PKC), respectively, but we demonstrate here that PKC is not responsible for the second phase increase in [Ca2+]i and suggest that the pathways diverge at the level of receptor and G protein coupling. Both phases of the TRH response were insensitive to pertussis toxin, but cholera toxin (CTX) selectively affected the second phase. After CTX pretreatment cells had a high spontaneous spiking frequency and smaller KIR amplitude. In these cells TRH failed to increase the action potential frequency after the first phase hyperpolarization, elicited only a transient peak increase in [Ca2+]i with no plateau phase and could only slightly reduce KIR. These effects of CTX are not mediated by its ability to increase cAMP via activation of GS, as increased cAMP levels neither inhibit KIR nor prevent its reduction by TRH. In addition, inhibition of protein kinase A activation did not block the second phase increase in [Ca2+]i induced by TRH, suggesting that the CTX-sensitive G protein mediating the second phase of the TRH response is not GS.     

Angiotensin II inhibits glomerular adenylate cyclase via the angiotensin II receptor subtype 1 (AT1)

We examined the role of angiotensin II (AII) receptor subtypes in the regulation of hormone-stimulated cyclic AMP (cAMP) accumulation in isolated rat glomeruli. All inhibited cAMP formation induced by histamine, serotonin and parathyroid hormone, but not by prostaglandin E2 or calcitonin gene-related peptide. Angiotensin III but not the angiotensin fragments (1-7) and (3-8) also showed inhibitory activity. The inhibition of histamine-induced cAMP accumulation by AII was concentration-dependent and was absent in glomeruli isolated from pertussis toxin-treated rats. The effect of AII on histamine-induced cAMP levels was not mimicked by the protein kinase C activator, phorbol-12-myristate-13-acetate, nor was the effect of AII inhibited by the protein kinase C inhibitors, staurosporine and H-7. The angiotensin II receptor subtype 1 (AT1) antagonists, SK&F 108566 and losartan, attenuated the inhibitory effect of AII on histamine-stimulated cAMP accumulation, whereas the AT2 selective antagonists, CGP 42112A, WL-19 and PD 123319, had no effect. Displacement of [125I]AII from glomerular membrane using the subtype-selective antagonists confirmed that the glomerular AII receptor has characteristics of an AT1 subtype. The results suggest that AII, through activation of the AT1 receptor, may act to maintain the contractile state of glomerular mesangial cells by attenuating the increase in cAMP levels induced by some hormones.     

A novel mitogenic signaling pathway of bradykinin in the human colon carcinoma cell line SW-480 involves sequential activation of a Gq/11 protein, phosphatidylinositol 3-kinase beta, and protein kinase Cepsilon

The signaling routes connecting G protein-coupled receptors to the mitogen-activated protein kinase (MAPK) pathway reveal a high degree of complexity and cell specificity. In the human colon carcinoma cell line SW-480, we detected a mitogenic effect of bradykinin (BK) that is mediated via a pertussis toxin-insensitive G protein of the Gq/11 family and that involves activation of MAPK. Both BK-induced stimulation of DNA synthesis and activation of MAPK in response to BK were abolished by two different inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY 294002, as well as by two different inhibitors of protein kinase C (PKC), bisindolylmaleimide and Ro 31-8220. Stimulation of SW-480 cells by BK led to increased formation of PI3K lipid products (phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 4-bisphosphate) and to enhanced translocation of the PKCepsilon isoform from the cytosol to the membrane. Both effects of BK were inhibited by wortmannin, too. Using subtype-specific antibodies, only the PI3K subunits p110beta and p85, but not p110alpha and p110gamma, were detected in SW-480 cells. Finally, p110beta was found to be co-immunoprecipitated with PKCepsilon. Our data suggest that in SW-480 cells, (i) dimeric PI3Kbeta is activated via a Gq/11 protein; (ii) PKCepsilon is a downstream target of PI3Kbeta mediating the mitogenic signal to the MAPK pathway; and (iii) PKCepsilon associates with the p110 subunit of PI3Kbeta. Thus, these results add a novel possibility to the emerging picture of multiple pathways linking G protein-coupled receptors to MAPK.     

Identification of a structural element in phospholipase C beta2 that interacts with G protein betagamma subunits

To delineate the specific regions of phospholipase C beta2 (PLC beta2) involved in binding and activation by G protein betagamma subunits, we synthesized peptides corresponding to segments of PLC beta2. Two overlapping peptides corresponding to Asn-564-Lys-583 (N20K) and Glu-574-Lys-593 (E20K) inhibited the activation of PLC beta2 by betagamma subunits (IC50 50 and 150 microM, respectively), whereas two control peptides did not. N20K and E20K, but not the control peptides, inhibited betagamma-dependent ADP-ribosylation of Galphai1 by pertussis toxin and betagamma-dependent activation of phosphoinositide 3-kinase. To demonstrate direct binding of the peptides to betagamma subunits, the peptides were chemically cross-linked to purified beta1gamma2. N20K and E20K cross-linked to both beta1 and gamma2 subunits, whereas the control peptides did not. Cross-linking to beta and gamma was inhibited by incubation with excess PLC beta2 or PLC beta3, whereas cross-linking to gamma but not beta was inhibited by r-myr-alphai1. These data together demonstrate specificity of N20K and E20K for G betagamma binding and inhibition of effector activation by betagamma subunits. The results suggest that an overlapping region of the two active peptides, Glu-574-Lys-583, mimics a region of PLC beta2 that is involved in binding to betagamma subunits. Changing a tyrosine to a glutamine in this overlapping region of the peptides inhibited binding of the peptide to betagamma subunits. Alignment of these peptides with the three-dimensional structure from PLC delta1 identifies a putative alpha helical region on the surface of the catalytic domain of PLC beta2 that could interact with betagamma subunits.     

A tyrosine kinase signaling pathway accounts for the majority of phosphatidylinositol 3,4,5-trisphosphate formation in chemoattractant-stimulated human neutrophils

The signaling pathway leading from G protein-coupled chemoattractant receptors to the generation of oxidants by NADPH oxidase in human neutrophils requires the formation of the lipid mediator phosphatidylinositol 3,4,5-trisphosphate (PIP3). Two mechanisms through which PIP3 can be generated have been described in human leukocytes. One pathway involves the coupling of the src-related tyrosine kinase Lyn to the "classical" p85/p110 form of phosphatidylinositol 3-kinase. The second paradigm utilizes a novel form of phosphatidylinositol 3-kinase whose activity is directly regulated by G protein betagamma subunits. In this paper, we show that formation of PIP3 in chemoattractant-stimulated neutrophils is substantially attenuated by inhibitors that specifically block tyrosine kinase activity. These data suggest that the Lyn activation pathway plays a major role in the formation of this important lipid messenger during chemoattractant stimulation of human neutrophils.