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Introduction: Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting. Materials and Methods: Intracellular calcium concentration ([Ca2+]i) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[3H]inositol- (0.5 μCi/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used. Results: Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca2+]i, cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the Gi inhibitor pertussis toxin. Agonists potently stimulated migration of T24 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration. Conclusions: G protein-coupled receptors potently stimulated cell migration in T24 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation–dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3ζ and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation–dephosphorylation cycle of cofilin. We demonstrate that muscarinic receptor-mediated stimulation of phospholipase D1 (PLD1) is controlled by LIM-kinase, slingshot phosphatase as well as 14-3-3ζ, and requires phosphorylatable cofilin. Cofilin directly and specifically interacts with PLD1 and upon phosphorylation by LIM-kinase1, stimulates PLD1 activity, an effect mimicked by phosphorylation-mimic cofilin mutants. The interaction of cofilin with PLD1 is under receptor control and encompasses a PLD1-specific fragment (aa 585–712). Expression of this fragment suppresses receptor-induced cofilin–PLD1 interaction as well as PLD stimulation and actin stress fiber formation. These data indicate that till now designated inactive phospho-cofilin exhibits an active cellular function, and suggest that phospho-cofilin by its stimulatory effect on PLD1 may control a large variety of cellular functions. [ABSTRACT FROM AUTHOR]

Receptor tyrosine kinase regulation of phospholipase C-ε (PLC-ε), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca2+ signaling by the EGF receptor, which activated both PLC-γ1 and PLC-ε, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-ε, and Rap2B-dependent transiocation of PLC-ε to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca2+ and expression of lipase-inactive PLC-γ1 but not of PLC-ε. Expression of RasGRP3, a Ca2+/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca2+ signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-γ1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-ε. [ABSTRACT FROM AUTHOR]

Activation of Rho GTPases, which play pivotal roles in diverse cellular functions, is catalysed by specific guanine nucleotide exchange factors (GEFs). We and others (Souchet et al. (2002)) independently cloned a human cDNA encoding a 580 aa protein (p63RhoGEF), which contains a tandem of Dbl homology and pleckstrin homology domains typical for RhoGEFs. In accordance with Souchet et al., recombinant p63RhoGEF interacted with and catalysed GDP/GTP exchange at RhoA, but not Rac1 or Cdc42. Recently, an N-terminally truncated form of p63RhoGEF, termed GEFT, was described as a Rac/Cdc42-specific GEF (Guo et al. 2003). As judged by RT-PCR with specific primers, we were able to detect mRNA variants encoding p63RhoGEF and GEFT within several tissues and cell lines. Apparently, they co-exist within one cell and are derived from the same gene. When expressed in human embryonic kidney cells, both p63RhoGEF and GEFT caused activation of RhoA, but not Rac1 or Cdc42, and induced serum response factor-mediated gene transcription, which was fully blunted by the Rho-inactivating C3 transferase. In line with these data, expression of either p63RhoGEF or GEFT in J82 human bladder carcinoma cells induced the formation of actin stress fibres. We therefore conclude that p63RhoGEF and GEFT are apparently isoforms derived from the same gene and that GEFT, similar to p63RhoGEF, activates RhoA in several cell types. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca2+ in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca2+ concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca2+ transients induced by photolysis of caged S1P were caused by Ca2+ mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P. [Copyright &y& Elsevier]

1 The lysophospholipids, lysophosphatidic acid and sphingosine 1-phosphate, have been reported to activate platelets. Here we examined effects of the naturally occurring related sphingosylphosphorylcholine (SPC) on human platelet function. 2 Platelet activation was determined as aggregation, elevation of intracellular Ca[sup 2+] concentrations, surface expression of P-selectin, GP 53, and GP IIb/IIIa neoepitope PAC-1, and of fibrinogen binding to the platelet surface. 3 Platelets were activated by ADP (5 and 20 µM), the thrombin receptor-activating peptide TRAP6 (5 and 20 µM), the thromboxane A[sub 2] mimetic U-46619 (1 µM) and collagen (20 and 50 µg ml[sup -1]) but not by SPC (up to 20 µM). 4 SPC concentration-dependently (IC[sub 50] approximately 1-10 µM) inhibited activation of washed human platelets in response to all of the above agonists, with almost complete inhibition occurring at 20 µM SPC. 5 The SPC stereoisomers, D-erythro SPC and L-threo SPC, exhibited similar concentrationresponse curves in inhibiting 20 µM ADP-induced platelet aggregation, suggesting that SPC did not act via specific lysophospholipid receptors. 6 Although SPC slightly activated platelet protein kinase A (as assessed by VASP phosphorylation), this effect could not explain the marked platelet inhibition. Possible protein kinase C inhibition also did not explain the inhibition of platelet activation by SPC. On the other hand, SPC suppressed agonist-induced Ca[sup 2+] mobilization and phospholipase C stimulation. 7 These results indicate that the lysophospholipid SPC is an effective inhibitor of human platelet activation, apparently primarily by uncoupling agonist-activated receptors from their effectors. [ABSTRACT FROM AUTHOR]

We have compared the effectiveness of problem-based learning (PBL) and classical lecture-based learning (LBL) in conveying medical facts in a general pharmacology class of third year medical students (n=107). Three groups with a total of 31 students were randomly assigned to PBL. The PBL groups (9–12 students each) received ten 2-h sessions in which a clinical case was discussed and ten 2-h sessions in which areas of pharmacology not covered by the case discussions were presented in an LBL format (one group with all 31 students). The other students were assigned to groups of 14–15 students and received 20 2-h sessions in an LBL format. At the end of the semester all students received a questionnaire and participated in the same 30-question multiple-choice exam. The mean number of false answers was 7.6±4.0 and 9.7±4.7 in the PBL and LBL groups, respectively (P<0.05 in a two-tailed t-test), and the percentage of failing students (>10 false answers) was 27% and 38%, respectively. Both groups were asked to rate their pharmacology class on a scale of 1 (lowest) to 10 (highest). In this questionnaire, PBL students by average rated generated interest in pharmacology, conveyed knowledge in pharmacology and understanding of medical questions approximately 1 point higher than LBL students. In an additional questionnaire given to the PBL students only, they reported to have prepared themselves 0.9±1.1 h for their lecture sessions but 3.0±1.4 h for their case discussions. The above findings were largely confirmed in the next semester when students were allowed to decide whether to participate in PBL or LBL classes. Moreover, PBL students did not score worse than LBL students in nation-wide pharmacology exams (523±76 vs. 500±91 points, P=0.09). We conclude that a switch from LBL to PBL teaching of pharmacology does not occur at the expense of factual knowledge transmission. [ABSTRACT FROM AUTHOR]

1 We have investigated the vasodilating effects of D-erythro-C2-ceramide (C2-ceramide) in methoxamine-contracted rat mesenteric microvessels. 2 C2-ceramide (10-100 µM) caused a concentration-dependent, slowly developing relaxation which reached maximum values after ≈10 min and partially abated thereafter. 3 Endothelium removal or inhibitors of guanylyl cyclase (3 µM ODQ), protein kinase A (10 µM H7, 1 µM H89) and various types of K[sup +] channels (10 µM BaCl[sub 2], 3 mM tetraethylammonium, 30 nM charybdotoxin, 30 nM iberiotoxin, 300 nM apamine, 10 µM glibenclamide) had only small if any inhibitory effects against C2-ceramide-induced vasodilation, but some of them attenuated vasodilation by sodium nitroprusside or isoprenaline. A combination of ODQ and charybdotoxin almost completely abolished C2-ceramide-induced vasodilation. 4 A second administration of C2-ceramide caused a detectable but weaker relaxation. L-threo-C2ceramide (100 µM), which should not be a substrate to ceramide metabolism, had no biphasic time course. The ceramidase inhibitor (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (100 µM) alone caused some vasodilation, indicating vasodilation by endogenous ceramides, and also hastened relaxation by exogenous C2-ceramide. The late-developing reversal of C2-ceramideinduced vasodilation was absent when α-adrenergic tone was removed by addition of 10 µM phentolamine. 5 We conclude that C2-ceramide relaxes rat resistance vessels in an endothelium-independent manner which is prevented only by combined inhibition of guanylyl cyclase and charybdotoxinsensitive K channels. The vasodilation abates with time partly due to desensitization of the ceramide response and partly due to metabolism of C2-ceramide to an inactive metabolite. [ABSTRACT FROM AUTHOR]

Several studies have shown an activation of adenylyl cyclase by the G protein-inactivating guanine nucleotides, GDP and its phosphate transfer-resistant analog, guanosine 5'-O-(2-thiodiphosphate; GDPβS). Here, we studied the mechanism underlying this unconventional activation. Adenylyl cyclase activity in sarcolemmal membranes from failing human ventricular myocardium, at a low Mg2+ concentration, decreased rapidly during incubation at 37°C. This decrease in enzyme activity was paralleled by a rapid release of GDP from Gi proteins, amounting to 75% release of total Gi-bound GDP within 10 min at 37°C. In contrast, no GDP release was observed at 4°C, and adenylyl cyclase activity remained stable for up to 20 min at 4°C. GDPβS did not alter the initial rates of cyclic AMP formation by the adenylyl cyclase, at either 37°C or 4°C, but almost fully prevented the decrease in enzyme activity occurring at 37°C. Hence, after 10 min of incubation at 37°C, adenylyl cyclase activity in the presence of GDPβS was increased by 150%, while no difference in activity was observed at 4°C. Under conditions where adenylyl cyclase is uncoupled from regulation by the inhibitory Gi proteins, i.e., at high concentrations of Mg2+ or Mn2+, adenylyl cyclase activity remained stable even at 37°C and GDPβS did not stimulate activity. In conclusion, Gi proteins in sarcolemmal membranes from failing human hearts rapidly release bound GDP. The data, furthermore, suggest that this process results in adenylyl cyclase inhibition by the empty but apparently active Gi proteins. [ABSTRACT FROM AUTHOR]

Stimulation of phosphoinositide-hydrolysing phospholipase C (PLC) generating inositol-1,4,5-trisphosphate is a major calcium signalling pathway used by a wide variety of membrane receptors, activating distinct PLC-β or PLC-γ isoforms. Here we report a new PLC and calcium signalling pathway that is triggered by cyclic AMP (cAMP) and mediated by a small GTPase of the Rap family. Activation of the adenylyl cyclase-coupled β2-adrenoceptor expressed in HEK-293 cells or the endogenous receptor for prostaglandin E1 in N1E-115 neuroblastoma cells induced calcium mobilization and PLC stimulation, seemingly caused by cAMP formation, but was independent of protein kinase A (PKA). We provide evidence that these receptor responses are mediated by a Rap GTPase, specifically Rap2B, activated by a guanine-nucleotide-exchange factor (Epac) regulated by cAMP, and involve the recently identified PLC-ε isoform. [ABSTRACT FROM AUTHOR]

The involvement of small GTPases of the Rho family in the control of phosphoinositide metabolism by adhesion signals was examined in NIH 3T3 fibroblasts. Abrogation of adhesion signals by detachment of cells from their substratum resulted in a time-dependent decrease in the cellular level of PtdIns(4,5)P2 by ≈ 50%. This effect could be mimicked by treatment of adherent cells with Clostridium difficile toxin B and toxin B-1470, which inhibit specific subsets of Rho and Ras GTPases. Detachment of cells that had been pretreated with the clostridial toxins did not cause a further reduction in PtdIns(4,5)P2 levels, suggesting that the target GTPases are integrated into the control of phosphoinositide levels by adhesion signals. The reduction in PtdIns(4,5)P2 levels could be attributed to reduced activity of the major PtdIns(4,5)P2-producing enzyme, PtdIns4P 5-kinase. Unexpectedly, both cell detachment and toxin treatment resulted in a twofold to threefold increase in inositol phosphate production in intact cells. In lysates of these cells, in vitro phospholipase C activity was found to be elevated by 30–50%. The effects of cell detachment and toxin treatment on inositol phosphate formation could be mimicked by expression of dominant-negative N17 Rac1. Taken together, these data suggest that adhesion-controlled small GTPases of the Rho family are involved in the regulation of the cellular PtdIns(4,5)P2 levels in NIH 3T3 fibroblasts, by controlling the activities of both PtdIns4P 5-kinase and phospholipase C. [ABSTRACT FROM AUTHOR]

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°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°C, M2 mAChR-mediated [Ca2+]i increases and PLC stimulation desensitized to a similar extent as at 37°C. However, at 16°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. [ABSTRACT FROM AUTHOR]

Phospholipase D (PLD) activity in human embryonic kidney (HEK) cells is stimulated by phorbolester-activated protein kinase C (PKC) and by membrane receptors, the latter apparently cating via the GTP-binding proteins. ADP-ribosylation factor (ARF) and Rho. In the present study, performed in cell-free preparations, we have characterized and compared the regulation of HEK cell PLD activity by the stable GTP analogue, guanosine 5'-O-[γ-thio]triphosphate (GTP[S]), and the phorbol ester, phorbol 12-myristate 13-acetate (PMA). In digitonin-permeabilized HEK cells, prelabeled with [³H]oleic acid.GTP[S] and PMA caused an approximately threefold concentration-dependent increase in the formation of [³H]phosphatidylethanol, measured in the presence of ethanol. Neomycin, which is known to complex with the PLD activities with similar sensitivity. GDP and its analogue, guanosine 5'-O-[β-thio]diphosphate, inhibited the stimulatory effect of GTP[S], whereas the PMA response was prevented by the non-selective PKC inhibitor, staurosporine, but not vice versa. PLD stimulation by GTP[S], but not by PMA, was markedly reduced upon cylosol depletion and reconstituted by purified recombinant ARF1. In HEK cell membranes, addition of purified recombinant ARNO, a guanine-nucleotide-exchange factor for ARF1, potentiated the GTP[S]-stimulated PLD activity. PLD stimulation by PMA in HEK cell membranes required MgATP and was largely prevented by the selective PKC inhibitors Goe 6976 abd bisindolylmaletimide I. Immunoblot analysis demonstrated that both conventional PKC (&alpja;, β, γ) and atypical PKC isozymes (&Zigma;, τ) were present in HEK cell membranes. The results indicate that phorbol ester stimulation of PLD activity in HEK cells apparently occurs by a phosphorylation-dependent mechanism involving membrane-associated PKC isozymes but not ARF proteins, the major targets of GTP[S] action. [ABSTRACT FROM AUTHOR]

The regulation of the cytoskeletal localization of guanine-nucleotide-binding protein αi, subunits by formyl peptide receptors was studied in myeloid differentiated human leukemia (HL-60) cells. Stimulation of formyl peptide receptors with N-formyl-Met-Leu-Phe (fMet-Leu-Phe) transiently increased the amount of α1 subunits in the Triton X-100-insoluble cytoskeleton Similar to the biphasic regulation of the actin content, fMet--Leu-Phe (≥ 10 nM) rapidly increased the cytoskeletal a, content (about threefold at 30 s), which was followed by a rapid reversal to control levels The formyl peptide receptor increased the which was followed by a rapid reversal to control levels The formyl peptide receptor increased the cytoskektal content of both a, subtypes. αi and αi,2, present in HL-60 cells. In cells permeabilized with Staphylococcus aureus α-toxin, fMet-Leu-Phe increased binding of the stable (GTP analogue, guanosine 5'-[gamma;-thio]triphosphate (GTP[S]), to cytoskeletal protein in a pertussis-toxin-sensitive manner, which was completely abolished by the F-actIn-disrupting agent, cytochalasin B, Using the photoreactive GTP analogue m-acetyIaniIido-GTP the formyl peptide receptor-regulated GTP binding sites at the cytoskeleton were identified as 40-kDa proteins, the molecular size of αi subunits are translocated to the cytosekeleton. Finally, in &aflfpha;-toxin-permeabilized HL-60 cells, fMet-Leu-Phe and GTP[S] cooperatively stimulated actin polymerization. In conclusion, evidence is provided that chemattractant receptors cause translocation of activated αi subunit to the cytoskeleton coincidently with F-actin formation. The data therefore argue for a potential role of translocated αisubunits in the process of receptor induced actin polymerization. [ABSTRACT FROM AUTHOR]

The cytoskeletal localization of inhibitory guanine-nucleotide-binding (Gi) proteins and the coupling of these proteins to formyl peptide receptors were studied in myeloid differentiated human leukemia (HL-60) cells. Treatment of HL-60 cells with cytochalasin B or botulinum C2 toxin, which leads to the disruption of microfilaments, increased the binding of the stable GTP analogue guanosine 5′-[γ- thio]triphosphate (GTP[S]) m permeabilized cells by about 30%. In contrast, the microtubule-disrupting agents colchicine and vinblastine, and cytochalasin B treatment of isolated HL-60 membranes did not affect GTP[S] binding. The stimulatory effect of cytochalasin B treatment was concentration and time dependent, with maximal increases observed at 5 μg/ml cytochalasin B and an incubation time of 10 min, and was counteracted by the F-actin-stabilizing toxin phalloidin. Cytochalasin B treatment increased the amount of G proteins activated by chemoattractant receptors by about 25 %. Furthermore, the number of Gi-protein-coupled receptors for the chemoattractant, N-formyl-Met-Leu-Phe, was increased by about 25% upon cytochalasin B treatment. Based on these functional data, which suggest an association of G proteins with actin filaments, the Triton X-100 (1%)-insoluble cytoskeleton was analyzed for the presence of G proteins. Giα subunits were detected in the cytoskeleton preparations, both by specific antisera and by pertussis-toxin-catalyzed ADP-ribosylation. Cytochalasin B pretreatment depleted the cytoskeleton in Giα, with an approximately 20% concomitant increase in membrane Giα content. In conclusion, evidence is presented that part of the cellular Giα is localized at actin filaments in HL-60 cells. After filament disruption, these Giα subunits seem to be translocated to the plasma membrane, where they can productively interact with chemoattractant receptors. [ABSTRACT FROM AUTHOR]

Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine-1- phosphate (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcεRI antigen receptors. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2+]I increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK- 293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling. [ABSTRACT FROM AUTHOR]

The expression of G protein-coupled receptors inducing calcium mobilization and stimulating cell migration was examined in human transitional-cell carcinoma (J82) cells. Measurements of cytoplasmic Ca2+ concentration ([Ca2+]i) and phospholipase C activity indicated that these cells express several calcium-mobilizing receptors, including those for lysophosphatidic acid (LPA), thrombin, bradykinin, bombesin and histamine, of which only the LPA response was sensitive (∼ 50%) to pertussis toxin (PTX). Migration of J82 cells was strongly stimulated by LPA and thrombin, by 5- to 20-fold, whereas bradykinin, bombesin and histamine were ineffective. Migration induced by either LPA or thrombin was inhibited by the actin cytoskeleton-disrupting agent, cytochalasin B, by the Rho protein-inactivating Clostridium difficile toxin B, by preventing [Ca2+]i transients with an intracellular calcium-chelating agent, and by the phorbol ester, phorbol 12-myristate 13-acetate, which also blocked the LPA- and thrombin-induced [Ca2+]i increases. On the other hand, ADP-ribosylation of Gi type G proteins by PTX abrogated the migratory response to LPA, without affecting the thrombin effect. Similarly, raising cAMP levels inhibited, by about 50%, the LPA- but not the thrombin-induced J82 cell migration. In conclusion, human transitional-cell carcinoma (J82) cells express various G protein-coupled, calcium-mobilizing receptors, out of which only those for LPA and thrombin stimulate cell migration, indicating that phospholipase C-derived second messengers per se are not sufficient for initiating this response. The complex signal transduction processes leading to LPA- and thrombin-stimulated motility of these human carcinoma cells apparently involve several common, essential factors, such as [Ca2+]i changes and Rho protein-regulated reorganization of the cytoskeleton, as well as some distinct components, most notably distinct subtypes of heterotrimeric G proteins and apparently also distinct cAMP-sensitive targets. [ABSTRACT FROM AUTHOR]

The non-selective labelled antagonist [H]N-methyl-scopolamine ([H]NMS) was used to identify muscarinic acetylcholine receptors in rat duodenum smooth muscle membranes. Saturation and kinetic experiments revealed a binding site with a K-value of 0.2-0.3 nmol/l and a receptor concentration (B) of 100 fmol/mg protein. The affinities of eight selective muscarinic antagonists were determined and compared with those at M (rat cerebral cortex), M (rat heart), M (rat submandibular gland) and M (data from Dörje et al. 1991) receptors. The 'M-selective' agent AF-DX 116, the group of 'M/M-selective' compounds himbacine, AF-DX 384, AQ-RA 741 and methoctramine but also the 'M3-selective' HHSiD showed affinities corresponding to M and/or M sites. The intermediate affinity of 4-DAMP favours a mixed M/M receptor population mainly containing M receptors. Two compounds, pirenzepine and AQ-RA 741, displayed biphasic displacement curves indicating the presence of a small population of putative M1 receptors. The rat duodenum antagonist binding profile, however, is not consistent with the presence of M3 receptors. We further demonstrate a concentration-dependent stimulation of [S]GTP[S] binding to duodenal G proteins by the muscarinic agonist oxotremorine. Estimation of the binding parameters of GTP[S] in absence and presence of oxotremorine provided evidence for a catalytic activation of G proteins by agonist-activated muscarinic receptors in rat duodenal membranes and a strong signal amplification on the G protein level. [ABSTRACT FROM AUTHOR]

Epinephrine inhibits human platelet adenylate cyclase by an α2-adrenoceptor-mediated and GTP-dependent process. The turn-off reaction for this epinephrineeinhibited enzyme was studied by measuring the rate of cyclic AMP formation upon addition of the α2-adrenoceptor antagonist, yohimbine, or upon addition of an excess of the stable GDP analog, guanosine 5'-O-(2-thiodiphosphate) (GDPβS), which competitively inhibited the action of GTP in the epinephrine-induced inhibition. The decay of the inhibited state of the adenylate cyclase was used to calculate the rate constant of the turn-off reaction. With both methods, almost identical koff values of 0.6 -0.7 min-1 at 25°C were obtained for the epinephrine-inhibited platelet enzyme. Cholera toxin, which does not inhibit the epinephrine- induced GTPase stimulation in platelet membranes, did not affect this turn-off reaction. In contrast, the turn-off rate of the prostaglandin-E1-stimulated human platelet adenylate cyclase, measured with GDPβS, was reduced from about 9 mm ` to 2 mm -` at 25°C by pretreatment of the membranes with cholera toxin, which inhibits the prostaglandin-E1-stimulated GTPase activity. The data strongly suggest that the guaninc nucleotide regulatory site, mediating epinephrine induced adenylate cyclase inhibition, is activated and inactivated by similar mechanisms as is the site mediating adenylate cyclase stimulation, and that cholera toxin affects only the stimulatory site. The findings furthermore suggest that the activity states of these two regulatory sites control the activity of the adenylate cyclase. [ABSTRACT FROM AUTHOR]

In membranes of S49 lymphoma eye- variants, which lack a functional guanine nucleotide-binding component (Ns protein) mediating adenylate cyclase stimulation by hormones, guanine nucleotides decreased the forskolin-stimulated adenylate cyclase activity by maximally 40 - 60%. The potency order of the guanine nucleotides studied was guanosine 5'-[γ-thio]triphosphate(GTP[S]) ≫ guanosine 5'-[β,γ-imido]triphosphate &GT; GTP > guanosine 5'-[β- thio]diphosphate (GDP[S]). GTP and GDP[S] acted as partial inhibitors; they competitively antagonized the GTP[S]-induced inhibition, which was half-maximal and maximal at about 3nM and 100 nM GTP[S], respectively. Cholera toxin did not enhance the inhibitory action of GTP. The eye- adenylate cyclase inhibition by GTP[S] occurred after a short lag period and persisted after washing the membranes. The inhibition was not affected by the pH (6.5 - 9.0) of the incubation medium and was largely independent of the concentrations of MgATP (up to 200 μM) and Mg2+ (up to 10 mM). In contrast, Mn2+ potently reduced the GTP[S]-induced eye- adenylate cyclase inhibition. Similarly as observed with forskolin, GTP[S] decreased the eye- adenylate cyclase activity stimulated by purified. preactivated Ns protein. Apart from Mn2+, inhibition of the eye- adenylate cyclase by GTP[S] was prevented or reversed by various treatments, which have been shown to obliterate hormone-induced adenylate cyclase inhibition in other cell types, such as by N-ethylmaleimide, by limited proteolysis with trypsin and by a factor extracted from bovine sperm particles. The data indicate that in membranes of eye- variants, which lack a functional N, protein, a guanine nucleotide-binding component (N1) is present, which mediates adenylate cyclase inhibition by guanine nucleotides. Such a component apparently also mediates adenylate cyclase inhibition by hormones in other cell types. Comparison of adenylate cyclase stimulation and inhibition by guanine nucleotides suggests that N1 is activated and inactivated by similar but not identical mechanisms as the Ns protein and that the relative activity states of these coupling components determine the activity of the adenylate cyclase. [ABSTRACT FROM AUTHOR]

Many guanine-nucleotide-binding protein-coupled receptors contain consensus sequences for phosphorylation by cAMP-dependent protein kinase (PKA), often located in the membrane proximal regions critically important for receptor signaling. In the present study, we have evaluated by site-directed muta-genesis the role of the putative PKA phosphorylation sites in the m4 muscarinic acetylcholine receptor (mAChR), i.e. Thr145 in the second cytoplasmic loop and Thr399 in the third cytoplasmic loop, and the influence of PKA on m4 mAChR function and internalization. Antagonist binding was unaltered by any of the mutations studied, while the agonist-binding affinity was either not affected (Thr145 alanine), increased (Thr399 alanine) or decreased (Thr399 serine or aspartic acid), m4 mAChR-mediated inhibition of adenylyl cyclase was unaltered by the mutations, except for an approximately tenfold reduced agonist potency of the Thr399 aspartic acid mutated receptor. Agonist-induced receptor internalization was unaltered with Thr399 seine or aspartic acid mutations of the receptors, but was strongly decreased in its rate and extent upon replacement of Thr399, Thr145 or both of these residues with alanine. These mutational effects could not be reproduced by treatment of wild-type receptor-expressing cells with the PKA inhibitor H-8. Furthermore, maximal stimulation of cellular PKA neither affected receptor internalization nor signaling measured as receptor-mediated Ca2+ mobilization. We conclude that the membrane proximal threonine residues of the m4 mAChR are not required for receptor signalling, but replacement by alanine residues can significantly that threonine residues at corresponding positions may be relevant to internalization of other guanine-nucleotide-binding-protein-coupled receptors. [ABSTRACT FROM AUTHOR]

Studies the enhanced cyclic adenylic acid (cAMP) accumulation by the human thyrotropin receptor variant with the Pro52Thr substitution in the extracellular domain. Variant and wild-type receptors; Increase in cAMP accumulation due to thyrotropin; Role of hyperactive thyrotropin receptor in hyperthyroidism of patients with Graves' disease.

In human embryonic kidney cells stably expressing the human m3 muscarinic acetylcholine receptor (mAChR) subtype, agonist (carbachol) activation stimulated phospholipase C, increased cytoplasmic calcium concentration, induced tyrosine phosphorylation of various cellular proteins and activated phospholipase D. Bypassing membrane receptors, phospholipase D was activated in these cells by direct activation of protein kinase C by phorbol esters, by direct activation of GTP-binding proteins by A1F4- and a stable GTP analogue (in permeabilized cells), by increasing cytoplasmic calcium concentration with the calcium ionophore A23187 and also apparently by tyrosine phosphorylation. In order to identify possible mechanisms by which the m3 mAChR couples to phospholipase D, various inhibitors of protein kinase C, tyrosine kinases and calcium-dependent events were studied. Prevention of an agonist-induced increase in cytoplasmic calcium concentration did not alter the mAChR-induced phospholipase D stimulation. The protein kinase C inhibitors, calphostin C and staurosporine, efficiently prevented phospholipase D activation by phorbol 12-myristate 13-acetate but only partially inhibited the activation induced by the mAChR agonist. Additionally, down-regulation of protein kinase C by prolonged exposure to phorbol 12-myristate 13-acetate abrogated phospholipase D activation by this effector but had only minor or no effects on the response to the mAChR agonist and direct activators of GTP-binding proteins. In contrast, the tyrosine kinase inhibitor genistein abolished the carbachol-induced and A1F4(-)-induced phospholipase D activation but had no effect on enzyme activation by phorbol 12-myristate 13-acetate. The data indicate that phospholipase D in m3 mAChR-expressing human embryonic kidney cells can be activated by various different mechanisms, i.e. receptor agonists, GTP-binding proteins, protein kinase C-dependent and calcium-dependent events and [ABSTRACT FROM AUTHOR]

In order to investigate whether coupling to and/or activation of guanine-nucleotide-binding proteins (G proteins) is involved in agonist-induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des-(264–394)mAChR] was constructed that lacks a substantial portion of the putative third intracellular loop. The wild-type receptor and des-(264–394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist-binding affinities. Consistent with the reported importance of the third cytoplasmic loop for Gi protein activation, the des-(264–394)mAChR showed a drastically reduced potential to mediate agonist-induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to Gi proteins was not impaired, as demonstrated by a similar guanine-nucleotide-sensitive and pertussis-toxin-sensitive high-affinity agonist-receptor binding for both mAChRs. In contrast, des-(264–394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of Gi proteins rather than ineffective coupling to Gi proteins. Internalization of wild-type receptor and des-(264–394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des-(264–394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild-type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in Gi-protein coupling and activation are not identical and that activation of, but not coupling to, Gi proteins regulates m4 mAChR internalization. [ABSTRACT FROM AUTHOR]

In order to study whether phosphate transfer reactions are involved in the binding of guanine nucleotide triphosphates to guanine-nucleotide-binding regulatory proteins, binding of the GTP analogues, guanosine 5′-[γ-thio]triphosphate, GTP[S], and guanosine 5′-[β, γ-imino]triphosphate, p[NH]ppG, and the regulation of binding by the formyl-peptide-receptor agonist, fMet-Leu-Phe, were studied in membranes of differentiated HL-60 cells. For fMet-Leu-Phe-stimulated binding of either GTP analogue, a competing nucleotide was required. With GDP as the competing nucleotide, initial rates of fMet-Leu-Phe-stimulated binding of GTP[S] and p[NH]ppG were similar for up to approximately 30 s. Thereafter, receptor-stimulated binding of p[NH]ppG rapidly reached equilibrium, whereas the binding of GTP[S] proceeded further. At equipotent concentrations of p[NH]ppG and GTP[S], maximal fMet-Leu-Phe-stimulated binding of GTP[S] was approximately twofold higher than that of p[NH]ppG. Finally, for half-maximal receptor-stimulated binding of GTP[S], approximately fivefold higher concentrations of both Mg2+ and GDP were required than for p[NH]ppG binding. With p[NH]ppG as the competing nucleotide, the extent of receptor-stimulated binding of GTP[S] as well as its Mg2+ requirement and time course were similar to the receptor-stimulated p[NH]ppG binding observed in the presence of GDP. However, with GTP[S] as the competing nucleotide, fMet-Leu-Phe reduced the binding of p[NH]ppG, a reaction further enhanced when GDP was additionally present. Under similar conditions as used in the binding studies, GTP[S] thiophosphorylated a 35-kDa protein, which is most likely a guanine-nucleotide-binding regulatory protein β subunit [Wieland, T., Nurnberg, B., Ulibarri, I., Kaldenberg-Stasch, S., Schultz, G. & Jakobs, K. H. (1993) J. Biol. Chem. 268, 18111-18118]. [ABSTRACT FROM AUTHOR]

The GTPase activity of membranes isolated from differentiated HL-60 cells was investigated to obtain information about the possible involvement of membrane-bound GTP-binding proteins in the regulation of the NADPH oxidase. A more than tenfold increase in the rate of hydrolysis of membrane-bound GTP was observed when cytosol and arachidonic acid were added simultanously, i.e. under the same conditions where NADPH oxidase becomes activated. There were parallel changes in GTPase and NADPH oxidase activities when the concentration of arachidonic acid or the species of the fatty acid was varied or different detergents were applied. Separation of the GTP-binding proteins of the solubilized membrane by sucrose density gradient centrifugation, allowed us to ascribe the observed effect to the stimulation of the GTPase activity of small GTP-binding proteins by cytosolic component(s). Indirect evidence suggests that, in contrast to the effect upon recombinant ras and ras-GTPase-activating protein, in intact HL-60 membranes the interaction of rap1A with rap-GTPase-activating protein, is strongly enhanced by arachidonic acid. [ABSTRACT FROM AUTHOR]

ADP receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTP[γS]), to guanine-nucleotide-binding proteins (G proteins) was studied in human platelet membranes. The potent ADP receptor agonist, 2-methyl-thio-adenosine 5'-diphosphate (2MeSADP), a non-hydrolyzable analog of ADP, increased the binding of [35S]GTP[γS} without apparent lag phase. Under optimal conditions, i.e. in the presence of GDP (1 - 10 µM), 2MeSADP increased the binding up to about threefold, with half-maximal and maximal increase observed at 10 nM and 1 µM 2MeSADP, respectively. ADP itself increased the binding of [35S]GTP[γS] by maximally about twofold, with half-maximal increase occurring at 0.1 µM ADP. The agonist-induced stimulation was competitively antagonized by the ADP receptor(s) antagonist, (lS)-adenosine 5'-0(1-thiotriphosphate) {(Sp)-ATP[αS]}. Other platelet receptor agonists known to act through receptors coupled to G proteins also increased binding of [35S]GTP[γS] in human platelet membranes, but without being inhibited by (Sp)-ATP[αS]. The data presented indicate that the platelet ADP receptor(s) can interact with and efficiently activate G proteins, the nature of which remains to be identified. [ABSTRACT FROM AUTHOR]

We have previously shown that soluble fractions obtained from human HL-60 granulocytes contain a phospholipase C which is markedly stimulated by the stable GTP analogue guanosine 5'[3-O-thio]triphosphate (Camps, M., Hou, C., Jakobs, K. H. and Gierschik, P. (1990) Biochem. J. 271, 743-748]. To investigate whether this stimulation was due to a soluble α subunit of a heterotrimeric guanine-nucleotide-binding protein or a soluble low-molecular-mass GTP-binding protein, we have examined the effect of purified guanine-nucleotide-binding protein βγ dimers on the phospholipaseC-mediated formation of inositol phosphates by HL-60 cytosol. We found that βγ subunits, purified from bovine retinal transducin (βγt), markedly stimulated the hydrolysis of phosphatidylinositol 4,5bisphosphate by this phospholipase C preparation. The stimulation of phospholipase C by βγt was not secondary to a phospholipase-A2-mediated generation of arachidonic acid, was prevented by the GDP-liganded transducin α subunit and was additive to activation of phospholipase C by guanosine 5'-[3-O-thio]triphosphate. βγt also stimulated soluble phospholipase C from human and bovine peripheral neutrophils, as well as membrane-bound, detergent-solubilized phospholipase C from HL60 cells. Stimulation of soluble HL-60 phospholipase C was not restricted to βγt, but was also observed with highly purified βγ subunits from bovine brain. Fractionation of HL-60 cytosol by anion-exchange chromatography revealed the existence of at least two distinct forms of phospholipase C in HL-60 granulocytes. Only one of these forms was sensitive to stimulation by βγt, demonstrating that stimulation of phospholipase C by βγ subunits is isozyme specific. Taken together, our results suggest that guanine-nucleotide-binding protein βγ subunits may play an important and active role in mediating the stimulation of phospholipase C by heterotrimeric guanine-nucleotide-binding proteins. [ABSTRACT FROM AUTHOR]

Membranes of myeloid differentiated human leukemia (HL 60) cells contain receptors for the chemotactic peptide, fMet-Leu-Phe (fMet, N-formylmethionine), interacting with pertussis-toxin-sensitive guanine-nucleotide-binding proteins (G proteins). Agonist activation of the receptors increases binding of the GTP analog, guanosine 5′-[γ-thio)triphosphate (GTP[S]), to membrane G proteins, at 30°C only in the presence of exogenous GDP. In contrast, at 0°C fMet-Leu-Phe stimulated binding of GTP[S] to G proteins maximally without addition of GDP. Under conditions resulting in marked degradation of membrane-bound GDP, control binding of GTP[S] measured at O°C was significantly increased, whereas the extent of agonist-stimulated binding was reduced. Furthermore, there was a rapid spontaneous release of membrane-bound GDP at 30°C. but not at 0°C. The data suggest that in intact membranes of HL 60 cells G proteins are initially in a GDP-liganded form, which state allows the receptor-induced exchange of bound GDP for GTP[S] at low temperature. In contrast, at or near physiological temperature, bound GDP is rapidly released (and degraded), resulting in unligated G proteins to which GTP[S] will bind independently of agonist-activated receptors. [ABSTRACT FROM AUTHOR]

Binding of the poorly hydrolyzable GTP analog, guanosine 5'-[γ-thio]triphosphate (GTP[S]), to purified guanine-nucleotide-binding regulatory proteins (G proteins) has been shown to be nonreversible in the presence of millimolar concentrations of Mg2+. In porcine atrial membranes, binding of [35S]GTP[S] to G proteins was stable in the presence of 1 mM Mg2+. However, either large dilution or, even more strongly, addition of unlabelled guanine nucleotides, in the potency order, GTP[S] > GTP ≤ guanosine 5'-[β,γ-imino]triphosphate > GDP ≥ guanosine 5'-[β-thio]diphosphate > GMP, markedly enhanced the observed dissociation, with 20-30% of bound [35S]GTP[S] being released by unlabelled guanine nucleotide within 20 min at 25°C. Most interestingly, dissociation of [35S]GTP[S] was rapidly and markedly stimulated by agonist (carbachol) activation of cardiac muscarinic acetycholine receptors. Carbachol-stimulated release of [35S]GTP[S] was strictly dependent on the presence of Mg2+ and an unlabelled guanine nucleotide. Although having different potency and efficiency in releasing [35S]GTP[S] from the membranes by themselves, the guanine nucleoside triphosphates and diphosphates studied, at maximally effective concentrations, promoted the carbachol-induced dissociation to the same extent, while GMP and ATP were ineffective. GTP[S]binding-saturation experiments indicated that one agonist-activated muscarinic acetylcholine receptor can cause release of bound GTP[S] from three to four G proteins. The data presented indicate that binding of GTP[S] to G proteins in intact membranes, in contrast to purified G proteins, is reversible, and that agonist-activated receptors can even, either directly or indirectly, interact with GTP[S]bound G proteins, resulting in release of bound guanine nucleoside triphosphate. [ABSTRACT FROM AUTHOR]

Signal-transducing guanine-nucleotide-binding regulatory proteins (G proteins) are heterotrimers, composed of the nucleotide-binding α subunit and a βγ dimer. The influence of βγ dimer preparations of the retinal G protein transducin (TD) was studied on formylpeptide-receptor-G-protein interactions in membranes of differentiated HL 60 cells. For this, TD was prepared from bovine rod outer segment (ROS) membranes with either GTP or its analogs, guanosine 5'-[γ-thio]triphosphate (GTP[S]) and guanosine 5'-[βγ-imino]triphosphate (Gpp[NH]p). After removal of free nucleotides, TDβγ was separated from TDα and its function analyzed. Addition of TDβγ isolated from TD prepared with GTP[S] (TDβγGTP[S]) to HL 60 membranes abolished high-affinity binding of fMet-Leu[³H]Phe (fMet, N-formylmethionine) to its receptor. In contrast, TDβγ isolated from TD prepared with GTP (TDβγGTP), boiled TDβγGTP[S] and TDα prepared with GTP[S] had no or only slight effects. The inhibitory effect of TDβγGTP[S] on fMet-Leu-[³H]Phe receptor binding was potentiated by GDP at low concentrations but not by GTP[S]. Furthermore, TDβγGTP[S], but not TDβγGTP or TDβγ isolated from TD prepared with Gpp[NH]p (TDβγGpp[NH]p), prevented fMet-Leu-Phe-stimulated binding of [35S]GTP[S] to G proteins in HL 60 membranes, measured in the presence of GDP. When TDβγGTP was incubated with GTP[S] and TD-depleted illuminated ROS membranes, and subsequently separated from the membranes and free GTP[S], this TDβγGTP, similar to TDβγGTP[S], abolished high-affinity binding of fMet-Leu-[³H]Phe to its receptor, fMet-Leu-Phe-stimulated binding of [35S]GTP[S], and fMet-Leu-Phe-stimulated GTP hydrolysis in HL 60 membranes. Inhibition of [35S]GTP[S] binding by TDβγ was not seen in the presence of the metabolically stable GDP analog, guanosine 5'-[β-thio]diphosphate. In order to obtain an insight into the modification of TDβγ apparently caused by GTP[S], and into its mechanism of action in HL 60 membranes, TD, TDα and TDβγ, all prepared in the presence of GTP, were incubated with [35S]GTP[S] and TD-depleted illuminated ROS membranes. Fluorographic analysis of the supernatant proteins revealed 35S labelling of the β band of the G protein. When apparently thiophosphorylated TDβγ was incubated with [³H]GDP in the presence of HL 60 membranes, [³H]GTP[S] was rapidly formed. This formation of [³H]GTP[S] required Mg2+, and was dependent on substrate concentrations, i. e. thiophosphorylated TDβγ and [³H]GDP, and the presence of a factor(s) present in HL 60 membranes, possibly G protein α subunits. Under optimal conditions, about 1 mol [³H]GTP[S] was formed from [³H]GDP by 1 mol added TDβγ. Evidence is presented that TDβγ can undergo a stable conformational change, apparently a thiophosphorylation of the β subunit, which is only obtained with GTP[S], and that thiophosphorylated TDβγ can serve as an intermediate in formation of GTP[S] from GDP exogenously added and/or present in HL 60 membranes, with subsequent G protein activation. As well as the classical GDP/GTP (GTP[S]) exchange at G protein α subunits, information transfer by intermediately (thio)phosphorylated βγ subunits is apparently an additional mechanism of G protein activation, with the two mechanisms possibly acting in a concerted manner. [ABSTRACT FROM AUTHOR]

Receptor-regulated binding of the labeled GTP analog, guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP[S]), to guanine-nucleotide-binding proteins (G-proteins) was studied in porcine atrial membranes enriched in muscarinic acetylcholinc (mACh) receptors. Binding of [35S]GTP[S] to the membranes was not or only slightly affected by the cholinergic agonist, carbachol, unless a second nucleotide was simultaneously present in the binding assay. This additional nucleotide requirement was best fulfilled by GDP, being maximally effective at 0.1-1 µM. In contrast, the GDP analog, guanosine 5'-O-(2-thiodiphosphate), could not replace GDP in promoting carbacholinduced increase in [35S]GTP[S] binding. In addition to GDP, agonist-induced stimulation of [35S]GTP[S] binding to porcine atrial membranes required thc presence of Mg2+, being half-maximally and maximally effective at about 30 µM and 300 µM, respectively. Addition of NaCl, which decreased control binding measured in the presence of GDP alone, had no effect on the maximal extent of agonist-stimulated binding, but reduced the potency of carbachol in stimulating [35S]GTP[S] binding. Under optimal conditions, carbachol increased the binding of [35S]GTP[S] without apparent lag phase up to about 2.5-fold, with half-maximal and maximal increase being observed at 5-10 µM and 100 µM, respectively. The agonist-induced stimulation was competitively antagonized by the mACh receptor antagonist, atropine. The number of GTP[S] binding sites under receptor control was two-three-fold higher than the number of mACh receptors in the porcine atrial membranes used. Pretreatment of thc membranes with pertussis toxin under conditions leading to 95% ADP-ribosylation of the toxin-sensitive G-protein α-subunits markedly reduced agonist-stimulated [35S]GTP[S] binding, with, however, about 30% stimulation still remaining. The data presented indicate that agonist-stimulated binding of [35S]GTP[S] to G-proteins can be a sensitive assay for measuring receptor-regulated G-protein activation in native membranes and, furthermore, suggest that one agonist-activated mACh receptor can activate two or three cardiac G-proteins. being mainly members of the pertussis-toxin-sensitive G-proteins. [ABSTRACT FROM AUTHOR]

Neomycin, an inositol-phospholipid-binding aminoglycoside antibiotic, is known to interfere with signal transduction mechanisms involving phospholipase C as effector enzyme. In this study, we report that neomycin can also markedly influence agonist binding of G-protein-coupled receptors. In membranes of differentiated human leukemia cells (HL 60 cells), neomycin (0.1-10 mM) was found to induce high-affinity binding of the chemotactic tripeptide, N-formyl-methionylleucylphenylalanine (fMet-Leu-Phe), to its receptor sites in a manner similar to magnesium. Gentamycin and streptomycin, two other aminoglycoside antibiotics, were as potent and as effective as neomycin or magnesium in inducing high-affinity agonist receptor binding. Pretreatment of the cells with pertussis toxin reduced the effects of magnesium and neomycin on agonist receptor binding likewise. In contrast, magnesium but not neomycin largely enhanced the potency of guanine nucleotides, particularly of GTP and its analog, guanosine-5'-O-(3-thiotriphosphate), to reduce fMet-Leu-Phe receptor binding, while maximal inhibition of agonist receptor binding by guanine nucleotides was identical with magnesium and neomycin. Furthermore, neomycin could not replace magnesium in providing stimulation of HL 60 membrane high-affinity GTPase by fMet-Leu-Phe. In close agreement to these findings on the pertussis-toxin-sensitive Gi-protein-coupled formyl peptide receptors, neomycin in a manner similar to magnesium induced high-affinity agonist binding of Gs-protein-coupled β-adrenoceptors. Similar to formyl peptide receptor binding, high-affinity binding of isoproterenol to β-adrenoceptors in guinea pig lung membranes induced by magnesium and neomycin was inhibited by the GTP analog, guanosine-5'-O-(3-thiotriphosphate), to a similar maximal extent but with an about 100-fold higher potency in the presence of magnesium than in the presence of neomycin. The data presented thus indicate that neomycin and other aminoglycoside antibiotics can mimic the action of magnesium (or other divalent cations) in inducing high-affinity agonist binding of Gi- and Gs-protein-coupled receptors, but not in inducing subsequent G-protein activation by guanosine triphosphates. The data, furthermore, suggest that neomycin by this selective action will be a powerful tool to dissect the multiple sites of magnesium's action in the agonist receptor-G-protein interaction. [ABSTRACT FROM AUTHOR]

The activity of the adenylate cyclase inhibitory guanine-nucleotide-binding regulatory protein (Gi), measured as inhibition of forskolin-stimulated cyclic AMP formation, and its regulation by various nucleotides and the inhibitory α2-adrenoreceptor agonist epinephrine was studied in membranes of human platelets. When adenylate cyclase activity was measured with ATP as substrate and in the absence of a nucleoside-triphosphate-regenerating system, GTP (0.1–10 μM) by itself potently and efficiently inhibited the enzyme. GDP was almost as potent and as effective as GTP. In the additional presence of epinephrine, the potencies of both GTP and GDP were increased about threefold, while maximal inhibition by these nucleotides was only slightly increased by the receptor agonist. In contrast to GTP and GDP, the metabolically stable GDP analog, guanosine 5′-[β-thio]diphosphate, had only a very small effect, suggesting that GDP but not its stable analog is converted to the active GTP. Addition of UDP (1 mM), used to block the GDP to GTP conversion reaction, completely suppressed the inhibitory effect of GDP, while that caused by GTP was not affected. Most important, the inhibitory receptor agonist epinephrine counteracted the suppressive effect of UDP on GDP's action, suggesting that, while UDP inhibits the formation of GTP from GDP, the activated receptor stimulates this conversion reaction. In the presence of a complete nucleoside-triphosphate-regenerating system, which by itself had no influence on control forskolin-stimulated adenylate cyclase activity, GTP alone, at concentrations up to 10 μM, did not decrease enzyme activity, but required the presence of an inhibitory receptor agonist (epinephrine) to activate the Gi protein. Addition of the regenerating system creatine phosphate plus creatine kinase not only abolished adenylate cyclase inhibition by GTP alone, but also largely reduced both the potency and efficiency of epinephrine to activate the Gi protein in the presence of GTP. Furthermore, the nucleoside-triphosphate-regenerating system also largely delayed the onset of adenylate cyclase inhibition by the GTP analog, guanosine-5′-[β-thio]triphosphate (10 nM), which was accelerated by epinephrine, and it also decreased the final enzyme inhibition caused by this GTP analog. The action of the nucleoside-triphosphate-regenerating system was mimicked by NaCl, which primarily, at low concentrations, suppressed the inhibitory effect of GTP alone, thereby leading to an apparent requirement of NaCl for observing the epinephrine-induced inhibition, and which, finally, at high concentrations, also reduced and abolished the action of hormone plus GTP. The data presented, thus, indicate that the adenylate cyclase inhibitory G protein can apparently be activated by GTP, locally formed from GDP by the action of a membrane-associated nucleoside diphosphokinase, and suggest that this conversion can be stimulated by an agonist-activated inhibitory receptor. The data obtained with GTP and its analog, guanosine-5′-[β-thio]triphosphate, furthermore, suggest that Gi-protein activation by these guanine nucleotides may involve additional, so far uncharacterized, phosphate-transfer reactions. Finally, the data indicate that the use of a nucleoside-triphosphate-regenerating system, which is usually included in adenylate cyclase assays, can mask important regulatory aspects of this multicomponent signal-transduction system. [ABSTRACT FROM AUTHOR]

In neutrophils and several other phagocytic cell types, a pertussis- and cholera-toxin-sensitive form of the guanine-nucleotide-binding protein (G-protein) Gp couples receptors for N-formylmethionine-containing chemotactic peptides to stimulation of phospholipase C. Using membranes of myeloid differentiated HL 60 cells, we have examined the role of Mg2+ and guanine nucleotides in regulating (a) the interaction of the formyl-peptide receptor with the chemotactic agonist N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and (b) the receptor-mediated activation of Gp. Mg2+ markedly enhanced the number of receptors with high affmity for the radiolabeled oligopeptide fMet-Leu-[&sup3H]Phe. At the same time, Mg2+ largely increased the potency of guanosine-5′-(3-O-thio)triphosphate, but not of GDP or guanosine-5′-(2-O-thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mg2+ in eliciting these two effects and analysis of the specificities of the relevant divalent cation sites revealed that Mg2+ interacts with at least two independent sites on the receptor-Gp complex. One site is specific for Mg2+ and exhibits affinity in the micromolar range, the other site interacts with millimolar concentrations of several divalent cations in a non-selective fashion. It is suggested that the former site is located on Gp and that interaction of Mg2+ with this site is necessary for the receptor-mediated G-protein activation, whereas interaction of divalent cations with the latter site is necessary for high affinity agonist binding. The regulation of the formyl-peptide receptor binding properties by guanine nucleotides is independent of Gp activation, since inhibition of peptide binding is achieved by addition of both guanine nucleoside diphosphates and triphosphates and is readily seen both in the presence and in the absence of Mg2+. The latter finding, together with the observation that, at micromolar concentrations of Mg2+, high-affinity GTPase activity is stimulated by fMet-Leu-Phe primarily via low affinity receptors, suggests that, contrary to widely held opinions, (a) divalent cations are not required for a functional receptor—G-protein interaction and (b) high-affinity agonist binding is not a prerequisite for the receptor-mediated activation of the G-protein. [ABSTRACT FROM AUTHOR]

NADPH-oxidase-catalyzed superoxide (O–2) formation in membranes of HL-60 leukemic cells was activated by arachidonic acid in the presence of Mg2+ and HL-60 cytosol. The GTP analogues, guanosine 5′-[γ-thio]triphosphate (GTP[γS] and guanosine 5′-[β,γ-imido]triphosphate, being potent activators of guanine-nucleotide-binding proteins (G proteins), stimulated O–2, formation up to 3.5-fold. The adenine analogue of GTP[γS], adenosine 5′-[γ-thio]triphosphate (ATP[γS]), which can serve as donor of thiophosphoryl groups in kinase-mediated reactions, stimulated O&#82112, formation up to 2.5-fold, whereas the non-phosphorylating adenosine 5′-[β,γ-imidoltriphosphate was inactive. The effect of ATP[γS] was half-maximal at a concentration of 2 μM, was observed in the absence of added GDP and occurred with a lag period two times longer than the one with GTP[γS]. HL-60 membranes exhibited nucleoside-diphosphate kinase activity, catalyzing the thiophosphorylation of GDP to GTP[γS] by ATP[γS]. GTP[γS] formation was half-maximal at a concentration of 3–4 μM ATP[γS] and was suppressed by removal of GDP by creatine kinase/creatine phosphate (CK/CP). The stimulatory effect of ATP[γS] on O–2; formation was abolished by the nucleoside-diphosphate kinase inhibitor UDP. Mg2+ chelation with EDTA and removal of endogenous GDP by CK/CP abolished NADPH oxidase activation by ATP[γS] and considerably diminished stimulation by GTP[γS]. GTP[γS] also served as a thophosphoryl group donor to GDP, with an even higher efficiency than ATP[γS]. Transthiophosphorylation of GDP to GTP[γS] by GTP[γS] was only partially inhibited by CK/CP. Our results suggest that NADPH oxidase is regulated by a G protein, which may be activated either by exchange of bound GDP by guanosine triphosphate or by thiophosphoryl group transfer to endogenous GDP by nucleoside-diphosphate kinase. [ABSTRACT FROM AUTHOR]

The effects of trypsin, acrosin and a recently described trypsin-like protease from bovine sperm were studied on adenylate cyclase activity in membranes of human platelets. These proteases caused an immediate decrease in adenylate cyclase activity, which was independent of the platelet membrane concentration used and which was constant for up to 20 min of incubation at 25°C. When the incubation was prolonged, the proteases eliminated their own inhibitory action as well as that of the inhibitory hormone epinephrine. The adenylate cyclase inhibition caused by the proteases was strictly dependent on the presence of GTP (EC50 ≈ 0.1 µM), whereas in the absence of GTP only minor changes in enzyme activity were observed at the conditions and protease concentrations used. Maximal inhibition caused by the proteases was between 40% and 60%. Half-maximal inhibition by the purified proteases trypsin and acrosin was observed at about 30 ng/ml and 2 µg/ml respectively. Inhibition of platelet adenylate cyclase by the proteases was partially additive with that caused by epinephrine, while with thrombin no additivity was observed. The serine protease inhibitor leupeptin blocked the actions of the proteases when added simultaneously with the enzymes, but was ineffective when added later on. Treatment of platelet membranes with the alkylating N-ethylmaleimide at low concentrations and Mn2+ ions (≥ 1 mM), both agents known to abolish inhibition of adenylate cyclase via the inhibitory guanine-nucleotide-binding protein G1, eliminated the inhibitory action of the proteases. The data indicate that trypsin and trypsin-like proteases have two opposite effects on the platelet adenylate cyclase system, the well-documented elimination of Gi action and, as shown here, an immediate activation of Gi with subsequent adenylate cyclase inhibition. The data are consistent with the hypothesis that the activation of Gi caused by the proteases is due to an interaction of the proteases with specific cell-surface receptor sites in a manner similar to thrombin. [ABSTRACT FROM AUTHOR]

The βγ subunits of guanine nucleotide binding proteins from bovine brain and bovine rod outer segments have different structural and immunochemical properties. In spite of these structural differences, βγ subunits from these sources have been found to be fully interchangeable in terms of their interaction with α subunits of pertussistoxin-sensitive G proteins. In contrast, however, there are striking differences between these βγ subunits with regard to their ability to deactivate fluoride-stimulated Gs. These profound differences were also observed when the interaction of the purified components of the adenylate cyclase system was studied after reconstitution into phospholipid vesicles. Addition of βγ subunits purified from bovine brain to vesicles containing β-receptor and Gs results in a biphasic effect on receptor-stimulated GTPase activity, whereas addition of transducin βγ was virtually without any effect. Likewise, βγ from bovine brain, but not transducin βγ, affected adenylate cyclase activity of a reconstituted system consisting of three purified components (R, Gs, C). Thus, the α subunit of Gs, but not the α subunits of pertussis-toxin-sensitive G proteins discriminate between structurally different βγ subunits. [ABSTRACT FROM AUTHOR]