Mechanisms of phospholipase C activation: a comparison with the adenylate cyclase system (original) (raw)
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Endocrinology, 2001
The vasoactive intestinal polypeptide/pituitary adenylate cyclaseactivating polypeptide type 2 (VPAC 2 ) receptor was shown to induce both [ 3 H]inositol phosphate ([ 3 H]InsP)and cAMP production in transfected COS7 cells and in GH 3 cells where it is natively expressed. Neither cholera toxin nor forskolin could elicit an equivalent [ 3 H]InsP response, suggesting independent coupling of the two pathways. The VPAC 2 receptor-mediated [ 3 H]InsP response was partially inhibited by pertussis toxin (Ptx) and by the G␥-sequestering C-terminal fragment of GRK2 (GRK2-ct) in COS7 and GH 3 cells, whereas responses of control receptors were unaffected. Blockers of receptor-activated Ca 2ϩ influx pathways (Co 2ϩ and SKF 96365) also partially inhibited VPAC 2 receptor-mediated [ 3 H]InsP responses. This inhibition was not present in the component of the response remaining after Ptx treatment. A range of blockers of voltage-sensitive Ca 2ϩ channels were ineffective, consistent with the reported lack of these channels in COS7 cells. The data suggest that the VPAC 2 receptor may couple to phospholipase C through both Ptx-insensitive and Ptx-sensitive G proteins (G q/11 and G i/o , respectively) to generate [ 3 H]InsP. In addition to G␥, G i/o activation appears to require receptor-activated Ca 2ϩ entry. This is consistent with the possibility that not only G␣ q/11responsive and G␥-responsive isoforms of phospholipase C but also Ca 2ϩ -responsive forms may contribute to the overall [ 3 H]InsP re-
Cellular Signalling, 1989
As previously described, WP, KI plasma membrane possesses a vasopressin-sensitive phospholipase C [G. Guillon et aL, 1986, FEBS Lett. 196, 155-159]. In the present study, we examined the sensitivity of this enzyme to guanylnucleotides. GTP~,S induces a time-and dose-dependent stimulation of Ins(l,4,5)P3 and Ins(l,4)Pz accumulation. No accumulation of InsPt, Ins(l,3,4)P3 or Ins(l,3,4,5)P~ occurred under similar conditions. Gpp(NH)p produced the same effect but was less potent. GTP and a nonhydrolyzable analogue of ATP, App(NH)p, were without effect. Calcium also stimulated the phospholipase C activity in a time-and dose-dependent manner. In the absence of calcium, the activity of GTP?S was considerably reduced. Physiological calcium concentrations (between 10-s and 10 .7 M), allowed maximal GTP~,S stimulation of phospholipase C activity. In this system, the presence of vasopressin alone did not generate inositoi phosphate accumulation. However, this hormone: (i) reduced the lag-time observed during GTPTS stimulation, (ii) increased the sensitivity of phospholipase C to GTP and to GTP~S, and (iii) did not modify the stimulation of phospholipase C induced by maximal doses of GTPTS. Unlike sodium fluoride, GTPyS elicited an irreversible activation of phospholipase C. Calcium, GTP),S and sodium fluoride stimulated the phospholipase C activity via mechanisms sharing a common step, since their maximal effects were not additive. Cholera toxin treatment, known to produce complete ADP-ribosylation of 'as' subunits, partially reduced the basal and the maximal GTP'/S-mediated stimulation of phospholipase C activity as well as that caused by vasopressin. This inhibition was not mimicked by treatment with either forskolin or pertussis toxin.
Proceedings of The National Academy of Sciences, 1990
The mechanism of phospholipase C regulation by inhibitory receptors was analyzed both in intact and in permeabilized rat thyroid cells (FRTL5). In this system, the muscarinic agonist carbachol inhibited phospholipase C, as indicated by the decrease in the basal levels of inositol 1,4,5trisphosphate as well as by the reduced adrenergic stimulation of phosphoinositol accumulation, which was paralleled by a fall in the cytosolic Ca21 levels. This inhibition involved an M2 muscarinic receptor because it was abolished by atropine but not by the Ml antagonist pirenzepine. Cells pretreated with pertussis toxin were not responsive to carbachol, indicating the involvement of a guanine nucleotide-binding protein in this inhibitory process. This possibility was further evaluated in permeabilized cells, where the carbachol inhibition was shown to be completely dependent on GTP. Known second messengers were not involved in this inhibitory process since Ca2 , cAMP, and activators of protein kinases were not able to mimic or prevent the carbachol effect either in intact or in permeabilized FRTL5 cells. In this system, the phospholipases C and A2 are coupled to two classes of muscarinic receptors that display a different sensitivity to pertussis toxin. The carbachol inhibitory effect occurred under conditions that prevented activation of phospholipase A2, excluding a role of the arachidonic acid metabolism in this process. Taken together these data provide the strongest support to date that an inhibitory guanine nucleotide-binding protein sensitive to pertussis toxin can directly mediate receptor-induced inhibition of phospholipase C.
Journal of Biological Chemistry, 2005
Phospholipase Cε (PLCε) is one of the newest members of the phosphatidylinositol-specific phospholipase C (PLC) family. Previous studies have suggested that G-protein-coupled receptors (GPCRs) stimulate phosphoinositide (PI) hydrolysis by activating PLCβ isoforms through G q family G proteins and Gβγ subunits. Using RNA interference to knock down PLC isoforms, we demonstrate that the GPCR agonists endothelin (ET-1), lysophosphatidic acid (LPA), and thrombin, acting through endogenous receptors, couple to both endogenous PLCε and the PLCβ isoform, PLCβ3, in Rat-1 fibroblasts. Examination of the temporal activation of these PLC isoforms, however, reveals agonist-and isoform-specific profiles. PLCβ3 is activated acutely within the first minute of ET-1, LPA, or thrombin stimulation but does not contribute to sustained PI hydrolysis induced by LPA or thrombin and accounts for only part of ET-1 sustained stimulation. PLCε, on the other hand, predominantly accounts for sustained PI hydrolysis. Consistent with this observation, reconstitution of PLCε in knockdown cells dose-dependently increases sustained, but not acute, agonist-stimulated PI hydrolysis. Furthermore, combined knockdown of both PLCε and PLCβ3 additively inhibits PI hydrolysis, suggesting independent regulation of each isoform. Importantly, ubiquitination of inositol 1,4,5-trisphosphate receptors correlates with sustained, but not acute, activation of PLCε or PLCβ3. In conclusion, GPCR agonists ET-1, LPA, and thrombin activate endogenous PLCε and PLCβ3 in Rat-1 fibroblasts. Activation of these PLC isoforms displays agonist-specific temporal profiles; however, PLCβ3 is predominantly involved in acute and PLCε in sustained PI hydrolysis.
Phospholipase C- β 1 is regulated by a pertussis toxin-insensitive G-protein
Biochemical Journal, 1991
Regulation of phospholipase C (PLC) by receptors is mediated either through protein tyrosine phosphorylation or by activation of GTP-binding proteins (Gp). For the latter, pertussis toxin (PT)-sensitive and -insensitive pathways have been described, indicating PLC regulation by at least two types of G-proteins. The identity of PLC isoenzymes which are regulated by either type of Gp remains to be determined. Thyrotropin-releasing hormone stimulates a PLC in GH3 cells via a PT-insensitive Gp. Reconstitution methods for the assay of the GH3-cell Gp were developed. Previously, the membrane PLC was found to be reversibly extracted from membranes by high salt and to be activated by guanosine 5′-[gamma-thio]triphosphate (GTP[S]) only when membrane-associated, suggesting that Gp was retained in salt-extracted membranes. In the present work, Gp was cholate-solubilized from PLC-deficient membranes and incorporated into phospholipid vesicles, which were found to confer GTP[S]- and AlF4(-)-stim...
The Relationship between Phospholipases A 2 and C in Signal Transduction
Ann N Y Acad Sci, 1989
Arachidonic acid can be released from membrane phospholipids of platelets in response to a number of receptor-mediated signals. The enzymes most responsible for this activation are phospholipase A2' and, to a lesser degree, 1,2-diacylgIycerol lipase.' In this paper, we will describe some of the studies on phospholipase A2 activation that we have carried out and also some other published work that has helped us understand some of the physiological control mechanisms of this enzyme. The importance of phospholipase A, in receptor-mediated platelet activation varies with both the type and the strength of agonist used. As examples, collagen and epinephrine are absolutely dependent on the release of arachidonic acid for stimulation of platelet secretion and aggregation, whereas thrombin depends on arachidonic acid release only when used at low concentration; at higher doses the ability of thrombin to activate platelets is independent of arachidonic acid metabolites. The most relevant arachidonic acid metabolites for platelet stimulation are endoperoxides and thromboxane A'.
Biochemical and Biophysical Research Communications, 1987
inositol hydrolyze polyphosphoinositides in a Ca-dependent manner, Membranes prepared from DMSG-differentiated HL6g+cells labeled with generating inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP 1 of ?a*+ Incubation of membranes with GTP or GTPYS reduces the concentration required for activation. This nucleotide effect is potentiated by formyl-Met-Leu-Phe (FMLP). Pertussis toxin inhibits FMLP-induced augmentation, but not the induction of IP2/IP formation by GTP or GTPYS. These results suggest that differentiated HL6 a cells contain a membrane-associated phospholipase C that degrades polyphosphoinositides and that activation of this enzyme is mediated by at least two guanine nucleotide binding proteins, one of which is linked to l?MLP receptors and is pertussis toxin sensitive.