Phospholipase C β4 is involved in modulating the visual response in mice

Expression of G protein-regulated phospholipase C (PLC) β4 in the retina, lateral geniculate nucleus, and superior colliculus implies that PLC β4 may play a role in the mammalian visual process. A mouse line that lacks PLC β4 was generated and the physiological significance of PLC β4 in murine visual function was investigated. Behavioral tests using a shuttle box demonstrated that the mice lacking PLC β4 were impaired in their visual processing abilities, whereas they showed no deficit in their auditory abilities. In addition, the PLC β4-null mice showed 4-fold reduction in the maximal amplitude of the rod a- and b-wave components of their electroretinograms relative to their littermate controls. However, recording from single rod photoreceptors did not reveal any significant differences between the PLC β4-null and wild-type littermates, nor were there any apparent differences in retinas examined with light microscopy. While the behavioral and electroretinographic results indicate that PLC β4 plays a significant role in mammalian visual signal processing, isolated rod recording shows little or no apparent deficit, suggesting that the effect of PLC β4 deficiency on the rod signaling pathway occurs at some stage after the initial phototransduction cascade and may require cell–cell interactions between rods and other retinal cells.

A heterotrimeric G protein complex couples the muscarinic m1 receptor to phospholipase C-beta.

We addressed the question as to which subtypes of G protein subunits mediate the activation of phospholipase C-beta by the muscarinic m1 receptor. We used the rat basophilic leukemia cell line RBL-2H3-hm1 stably transfected with the human muscarinic m1 receptor cDNA. We microinjected antisense oligonucleotides into the nuclei of the cells to inhibit selectively the expression of G protein subunits; 48 hr later muscarinic receptors were activated by carbachol, and the increase in free cytosolic calcium concentration ([Ca2+]i) was measured. Antisense oligonucleotides directed against the mRNA coding for alpha(q) and alpha11 subunits both suppressed the carbachol-induced increase in [Ca2+]i. In cells injected with antisense oligonucleotides directed against alpha(o1) and alpha14 subunits, the carbachol effect was unchanged. A corresponding reduction of Galpha(q), and Galpha11 proteins by 70-80% compared to uninjected cells was immunochemically detected 2 days after injection of a mixture of alpha(q) and alpha11 antisense oligonucleotides. Expression of Galpha(q) and Galpha11 completely recovered after 4 days. Cells injected with antisense oligonucleotides directed against the mRNAs encoding for beta1, beta4, and gamma4 subunits showed a suppression of the carbachol-induced increase in [Ca2+]i compared to uninjected cells measured at the same time from the same coverslip, whereas in cells injected with antisense oligonucleotides directed against the beta2, beta3, gamma1, gamma2, gamma3, gamma5, and gamma7 subunits, no suppression of carbachol effect was observed. In summary, the results from RBL-2H3-hm1 cells indicate that the m1 receptor utilizes a G protein complex composed of the subunits alpha(q), alpha11, beta1, beta4, and gamma4 to activate phospholipase C.

Phosphatidylcholine-specific phospholipase C regulates glutamate-induced nerve cell death

Phosphatidylcholine-specific phospholipase C (PC-PLC) is a necessary intermediate in transducing apoptotic signals for tumor necrosis factor and Fas/Apo-1 ligands in nonneuronal cells. The data presented here show that PC-PLC also is required in oxidative glutamate-induced programmed cell death of both immature cortical neurons and a hippocampal nerve cell line, HT22. In oxidative glutamate toxicity, which is distinct from excitotoxicity, glutamate interferes with cystine uptake by blocking the cystine/glutamate antiporter, indirectly causing a depletion of intracellular glutathione. A PC-PLC inhibitor blocks oxidative glutamate toxicity, and exogenous PC-PLC potentiates glutamate toxicity. The inhibition of PC-PLC uncouples the cystine uptake from glutamate inhibition, allowing the maintenance of glutathione synthesis and cell viability. These data suggest that PC-PLC modulates neuronal cell death through a mechanism that is distinct from that involved in nonneuronal apoptosis.

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

Altered peptide ligands act as partial agonists by inhibiting phospholipase C activity induced by myasthenogenic T cell epitopes

Myasthenia gravis (MG) is a T cell-regulated, antibody-mediated autoimmune disease. Two peptides representing sequences of the human acetylcholine receptor α-subunit, p195–212 and p259–271, previously were shown to stimulate the proliferation of peripheral blood lymphocytes of patients with MG and were found to be immunodominant T cell epitopes in SJL and BALB/c mice, respectively. Single amino acid-substituted analogs of p195–212 and p259–271, as well as a dual analog composed of the tandemly arranged two single analogs, were shown to inhibit, in vitro and in vivo, MG-associated autoimmune responses. Stimulation of T cells through the antigen-specific T cell receptor activates tyrosine kinases and phospholipase C (PLC). Therefore, in attempts to understand the mechanism of action of the analogs, we first examined whether the myasthenogenic peptides trigger tyrosine phosphorylation and activation of phospholipase C. For that purpose, we measured generation of inositol phosphates and tyrosine phosphorylation of PLC after stimulation of the p195–212- and p259–271-specific T cell lines with these myasthenogenic peptides. Both myasthenogenic peptides stimulated generation of inositol phosphates as well as tyrosine phosphorylation of PLC. However, the single and dual analogs, although inducing tyrosine phosphorylation of PLC, could not induce PLC activity. Furthermore, the single and dual analogs inhibited the induced PLC activity whereas they could not inhibit tyrosine phosphorylation of PLC that was caused by the myasthenogenic peptides. Thus, the altered peptides and the dual analog act as partial agonists. The down-regulation of PLC activity by the analogs may account for their capacity to inhibit in vitro MG-associated T cell responses.

Phospholipase C-linked receptors regulate the ATP-sensitive potassium channel by means of phosphatidylinositol 4,5-bisphosphate metabolism

In the COS7 cells transfected with cDNAs of the Kir6.2, SUR2A, and M1 muscarinic receptors, we activated the ATP-sensitive potassium (KATP) channel with a K+ channel opener and recorded the whole-cell KATP current. The KATP current was reversibly inhibited by the stimulation of the M1 receptor, which is linked to phospholipase C (PLC) by the Gq protein. The receptor-mediated inhibition was observed even when protein kinase C (PKC) was inhibited by H-7 or by chelating intracellular Ca2+ with 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA) included in the pipette solution. However, the receptor-mediated inhibition was blocked by U-73122, a PLC inhibitor. M1-receptor stimulation failed to inhibit the KATP current activated by the injection of exogenous phosphatidylinositol 4,5-bisphosphate (PIP2) through the whole-cell patch pipette. The receptor-mediated inhibition became irreversible when the replenishment of PIP2 was blocked by wortmannin (an inhibitor of phosphatidylinositol kinases), or by including adenosine 5′-[β,γ–imido]triphosphate (AMPPNP, a nonhydrolyzable ATP analogue) in the pipette solution. In inside-out patch experiments, the ATP sensitivity of the KATP channel was significantly higher when the M1 receptor in the patch membrane was stimulated by acetylcholine. The stimulatory effect of pinacidil was also attenuated under this condition. We postulate that stimulation of PLC-linked receptors inhibited the KATP channel by increasing the ATP sensitivity, not through PKC activation, but most probably through changing PIP2 levels.

The Small Mr Ras-like GTPase Rap1 and the Phospholipase C Pathway Act to Regulate Phagocytosis in Dictyostelium discoideum

The function of the small-Mr Ras-like GTPase Rap1 remains largely unknown, but this protein has been demonstrated to regulate cortical actin-based morphologic changes in Dictyostelium and the oxidative burst in mammalian neutrophils. To test whether Rap1 regulates phagocytosis, we biochemically analyzed cell lines that conditionally and modestly overexpressed wild-type [Rap1 WT(+)], constitutively active [Rap1 G12T(+)], and dominant negative [Rap1 S17N(+)] forms of D. discoideum Rap1. The rates of phagocytosis of bacteria and latex beads were significantly higher in Rap1 WT(+) and Rap1 G12T(+) cells and were reduced in Rap1 S17N(+) cells. The addition of inhibitors of protein kinase A, protein kinase G, protein tyrosine kinase, or phosphatidylinositide 3-kinase did not affect phagocytosis rates in wild-type cells. In contrast, the addition of U73122 (a phospholipase C inhibitor), calphostin C (a protein kinase C inhibitor), and BAPTA-AM (an intracellular Ca2+ chelator) reduced phagocytosis rates by 90, 50, and 65%, respectively, suggesting both arms of the phospholipase C signaling pathways played a role in this process. Other protein kinase C–specific inhibitors, such as chelerythrine and bisindolylmaleimide I, did not reduce phagocytosis rates in control cells, suggesting calphostin C was affecting phagocytosis by interfering with a protein containing a diacylglycerol-binding domain. The addition of calphostin C did not reduce phagocytosis rates in Rap1 G12T(+) cells, suggesting that the putative diacylglycerol-binding protein acted upstream in a signaling pathway with Rap1. Surprisingly, macropinocytosis was significantly reduced in Rap1 WT(+) and Rap1 G12T(+) cells compared with control cells. Together our results suggest that Rap1 and Ca2+ may act together to coordinate important early events regulating phagocytosis.

Phospholipase Cβ4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum

Elimination of excess climbing fiber (CF)–Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, Gαq, and the γ isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLCβ4, one of two major isoforms expressed in Purkinje cells. PLCβ4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLCβ4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLCβ4 mRNA but reciprocally high levels of PLCβ3 mRNA are found. These results indicate that PLCβ4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.

Ca2+/calmodulin-binding peptides block phototransduction in Limulus ventral photoreceptors: Evidence for direct inhibition of phospholipase C

Phototransduction in Limulus photoreceptors involves a G protein-mediated activation of phospholipase C (PLC) and subsequent steps involving InsP3-mediated release of intracellular Ca2+. While exploring the role of calmodulin in this cascade, we found that intracellular injection of Ca2+/calmodulin-binding peptides (CCBPs) strongly inhibited the light response. By chemically exciting the cascade at various stages, we found the primary target of this effect was not in late stages of the cascade but rather at the level of G protein and PLC. That PLCδ1 contains a calmodulin-like structure raised the possibility that PLC might be directly affected by CCBPs. To test this possibility, in vitro experiments were conducted on purified PLC. The activity of this enzyme was strongly inhibited by CCBPs and also inhibited by calmodulin itself. Our results suggest that the calmodulin-like region of PLC has an important role in regulating this enzyme.

Molecular and Enzymatic Characterization of Three Phosphoinositide-Specific Phospholipase C Isoforms from Potato1

Many cellular responses to stimulation of cell-surface receptors by extracellular signals are transmitted across the plasma membrane by hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2), which is cleaved into diacylglycerol and inositol-1,4,5-tris-phosphate by phosphoinositide-specific phospholipase C (PI-PLC). We present structural, biochemical, and RNA expression data for three distinct PI-PLC isoforms, StPLC1, StPLC2, and StPLC3, which were cloned from a guard cell-enriched tissue preparation of potato (Solanum tuberosum) leaves. All three enzymes contain the catalytic X and Y domains, as well as C2-like domains also present in all PI-PLCs. Analysis of the reaction products obtained from PIP2 hydrolysis unequivocally identified these enzymes as genuine PI-PLC isoforms. Recombinant StPLCs showed an optimal PIP2-hydrolyzing activity at 10 μm Ca2+ and were inhibited by Al3+ in equimolar amounts. In contrast to PI-PLC activity in plant plasma membranes, however, recombinant enzymes could not be activated by Mg2+. All three stplc genes are expressed in various tissues of potato, including leaves, flowers, tubers, and roots, and are affected by drought stress in a gene-specific manner.

Haemonchus contortus GA1 antigens: related, phospholipase C-sensitive, apical gut membrane proteins encoded as a polyprotein and released from the nematode during infection.

It was previously shown that the Haemonchus contortus apical gut surface proteins p46, p52, and p100 induced protective immunity to challenge infections in goats. Here, it is shown that the three proteins are all encoded by a single gene (GA1) and initially expressed in adult parasites as a polyprotein (p100GA1). p46GA1 and p52GA1 are related proteins with 47% sequence identity, including a cysteine-containing region, which appears to confer secondary structure to these proteins, and a region with sequence similarity to bacterial Tolb proteins. GA1 protein expression is regulated during the life cycle at the level of transcript abundance. Only p52GA1 has characteristics of a glycosylinositolphospholipid membrane-anchored protein. However, both p46GA1 and p52GA1 were released from the gut membrane by phosphatidylinositol specific-phospholipase C, suggesting that p46GA1 membrane association depends on interactions with a glycosylinositolphospholipid gut membrane protein. Finally, GA1 proteins occur in abomasal mucus of infected lambs, demonstrating possible presentation to the host immune system during H. contortus infection. The results identify multiple characteristics of the GA1 proteins that should be considered for design of recombinant antigens for vaccine trials and that implicate a series of cellular processes leading to modification and expression of GA1 proteins at the nematode apical gut surface.

Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.

The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2.

The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.