PLCβ Isoforms Research Articles

Phospholipase Cβ Is Critical for T Cell Chemotaxis

Chemokines acting through G protein-coupled receptors play an essential role in the immune response. PI3K and phospholipase C (PLC) are distinct signaling molecules that have been proposed in the regulation of chemokine-mediated cell migration. Studies with knockout mice have demonstrated a critical role for PI3K in G(alphai) protein-coupled receptor-mediated neutrophil and lymphocyte chemotaxis. Although PLCbeta is not essential for the chemotactic response of neutrophils, its role in lymphocyte migration has not been clearly defined. We compared the chemotactic response of peripheral T cells derived from wild-type mice with mice containing loss-of-function mutations in both of the two predominant lymphocyte PLCbeta isoforms (PLCbeta2 and PLCbeta3), and demonstrate that loss of PLCbeta2 and PLCbeta3 significantly impaired T cell migration. Because second messengers generated by PLCbeta lead to a rise in intracellular calcium and activation of PKC, we analyzed which of these responses was critical for the PLCbeta-mediated chemotaxis. Intracellular calcium chelation decreased the chemotactic response of wild-type lymphocytes, but pharmacologic inhibition of several PKC isoforms had no effect. Furthermore, calcium efflux induced by stromal cell-derived factor-1alpha was undetectable in PLCbeta2beta3-null lymphocytes, suggesting that the migration defect is due to the impaired ability to increase intracellular calcium. This study demonstrates that, in contrast to neutrophils, phospholipid second messengers generated by PLCbeta play a critical role in T lymphocyte chemotaxis.

Tyrosine Phosphorylation-Independent Activation of PLCγ2 Downstream Integrin α2β1 in Platelets: A Possible Role for the Small GTPase Rac.

We have recently documented the existence of a cross-talk between platelet integrins α2β1 and αIIbβ3 involving phospholipase C (PLC)-mediated activation of the small GTPase Rap1b (Bernardi B et al, Blood 2006; 107:2728–35). Here we report that integrin α2β1-mediated platelet adhesion to monomeric collagen and to other specific ligands, such as decorin and collagen-derived peptides, actually induced a robust activation of PLC, evaluated as phosphorylation of pleckstrin, the main platelet substrate for protein kinase C. This was paralleled by a robust tyrosine phosphorylation of the PLCγ2. We found that inhibition of Src kinases by PP2 completely abolished integrin α2β1-promoted PLCγ2 tyrosine phosphorylation, but did not affect either Rap1b stimulation or integrin αIIbβ3 activation in adherent platelets. Surprisingly, phosphorylation of pleckstrin occurred normally under conditions in which tyrosine phosphorylation of PLCγ2 was totally suppressed. The Src-kinase-independent PLC activity in integrin α2β1-adherent cells was not inhibited by pretreatment of platelets with aspirin and apyrase, excluding any possible contribution of Gq-regulated PLCβ isoforms stimulated by released thromboxane A2 or secreted ADP. In addition, PLC-dependent activation of Rap1b and integrin αIIbβ3 were not affected by aspirin and apyrase. Phosphorylation of pleckstrin induced by adhesion to monomeric collagen was not detected in platelets from PLCγ2 knockout mice, confirming that this was the only isoform activated downstream of integrin α2β1. In addition, mouse platelets lacking PLCγ2 showed impaired integrin α2β1-mediated outside-in signaling, and were unable to promote both GTP binding to Rap1b and integrin αIIbβ3 activation. These results indicate that although PLCγ2 is the only PLC isoform stimulated downstream integrin α2β1 and is responsible for the cross-talk to integrin αIIbβ3 through the small GTPase Rap1b, its activation in adherent platelets can occur independently of Src-mediated tyrosine phosphorylation. A recent work has reported that, in vitro, the activity of PLCγ2, but not that of PLCγ1, can be stimulated by the active, GTP-bound form of the small GTPase Rac, with a mechanism independent of phosphorylation on tyrosine residues (Piechlek T el al, J Biol Chem 2005; 208:38923–31). In platelets adherent through integrin α2β1, Rac was found to be activated upstream PLC. Activation of Rac was efficiently prevented by the inhibitor NSC23766, which is able to bind and block Rac-specific GEFs. In integrin α2β1-adherent platelets, inhibition of Rac by NSC23766 did not affect PLCγ2 tyrosine phosphorylation and activation of PLC and Rap1b. However, upon inhibition of Src kinases by PP2 and consequent prevention of PLCγ2 tyrosine phosphorylation, NSC23766 almost completely abolished PLC activation, GTP binding to Rap1b and integrin αIIbβ3 stimulation. These results demonstrate that PLCγ2 is the main PLC isoform involved in integrin α2β1-mediated activation of Rap1b and integrin αIIbβ3, and that its activation can occur through two alternative mechanisms involving either tyrosine phosphorylation or stimulation by Rac GTPase.

Structural Determinants for Phosphatidic Acid Regulation of Phospholipase C-β1

Signaling from G protein-coupled receptors to phospholipase C-beta (PLC-beta) is regulated by coordinate interactions among multiple intracellular signaling molecules. Phosphatidic acid (PA), a signaling phospholipid, binds to and stimulates PLC-beta(1) through a mechanism that requires the PLC-beta(1) C-terminal domain. PA also modulates Galpha(q) stimulation of PLC-beta(1). These data suggest that PA may have a key role in the regulation of PLC-beta(1) signaling in cells. The present studies addressed the structural requirements and the mechanism for PA regulation of PLC-beta(1). We used a combination of enzymatic assays, PA-binding assays, and circular dichroism spectroscopy to evaluate the interaction of PA with wild-type and mutant PLC-beta(1) proteins and with fragments of the Galpha(q) binding domain. The results identify a region that includes the alphaA helix and flexible loop of the Galpha(q)-binding domain as necessary for PA regulation. A mutant PLC-beta(1) with multiple alanine/glycine replacements for residues (944)LIKEHTTKYNEIQN(957) was markedly impaired in PA regulation. The high affinity and low affinity component of PA stimulation was reduced 70% and PA binding was reduced 45% in this mutant. Relative PLC stimulation by PA increased with PLC-beta(1) concentration in a manner suggesting cooperative binding to PA. Similar concentration dependence was observed in the PLC-beta(1) mutant. These data are consistent with a model for PA regulation of PLC-beta(1) that involves cooperative interactions, probably PLC homodimerization, that require the flexible loop region, as is consistent with the dimeric structure of the Galpha(q)-binding domain. PA regulation of PLC-beta(1) requires unique residues that are not required for Galpha(q) stimulation or GTPase-activating protein activity.

Roles for Gα and Gβγ in PLC Activation

There are many isoforms of phospholipase C (PLC), the enzyme that cleaves phosphatidylinositol 4,5 diphosphate (PIP 2 ). PLC-β isoforms are activated by Gα q in response to activation of G protein-coupled receptors (GPCRs). The regulation of the PLC-δ isoforms is less well understood. Guo et al . present evidence to suggest that PLC-β2 maintains PLC-δ1 in an inactive state and that increased concentrations of the Gβγ disrupt the interaction between the two proteins, allowing PLC-δ1 to become active. This provides one model to explain how PLC-δ amplifies calcium signals initiated by PLC-β. Using a fluorescent probe covalently attached to PLC-β2, a direct association between PLC-β2 and PLC-δ1 was detected in vitro, and the affinity of the interaction was increased if the enzymes were immobilized on membranes. In vitro enzymatic activity was less than additive when both isoforms were present, suggesting that one or both enzymes were being inhibited. Using several different assay conditions, the authors determined that the activity of PLC-δ1 was inhibited by the presence of PLC-β2. The addition of Gβγ to the mixture of PLC-β2 and PLC-δ1 disrupted complex formation between the two enzymes in vitro and abolished inhibition of enzyme activity when the enzymes were mixed. Experiments with endogenously expressed PLC-β2 and PLC-δ1, as well as with cells transfected with versions of the proteins for bimolecular fluorescence complementation (BiFC), indicated that both proteins localized at the plasma membrane in unstimulated cells. In the BiFC experiment, stimulation of the cells with acetylcholine, which activates muscarinic GPCRs, decreased the enzymes' interaction. In the transfected cells, inhibition of Gα with pertussis toxin, which allows Gβγ to accumulate, disrupted the interaction between PLC-β2 and PLC-δ1, and this disruption by increasing concentrations of Gβγ was reproducible in isolated membrane preparations from the BiFC-transfected cells. Thus, it appears that in cells expressing both PLC-β2 and PLC-δ1, PLC-β2 maintains PLC-δ1 in an inactive state, and this inhibition is relieved by the Gβγ subunits of the G proteins activated in response to GPCR stimulation. Thus G proteins have a dual role in producing a calcium signal: Gα stimulates PLC-β2 and Gβγ disrupts the PLC-β2 and PLC-δ1 complex, allowing PLC-δ1 to enhance the signal. Y. Guo, M. Rebecchi, S. Scarlata, Phospholipase Cβ 2 binds to and inhibits phospholipase Cδ 1 . J. Biol. Chem . 280 , 1438-1447 (2005). [Abstract] [Full Text]

Phospholipase Cβ2 Binds to and Inhibits Phospholipase Cδ1

Phospholipase Cbeta (PLCbeta) isoforms, which are under the control of Galphaq and Gbetagamma subunits, generate Ca2+ signals induced by a broad array of extracellular agonists, whereas PLCdelta isoforms depend on a rise in cytosolic Ca2+ for their activation. Here we find that PLCbeta2 binds strongly to PLCdelta1 and inhibits its catalytic activity in vitro and in living cells. In vitro, this PLC complex can be disrupted by increasing concentrations of free Gbetagamma subunits. Such competition has consequences for signaling, because in HEK293 cells PLCbeta2 suppresses elevated basal [Ca2+] and inositol phosphates levels and the sustained agonist-induced elevation of Ca2+ levels caused by PLCdelta1. Also, expression of both PLCs results in a synergistic release of [Ca2+] upon stimulation in A10 cells. These results support a model in which PLCbeta2 suppresses the basal catalytic activity of PLCdelta1, which is relieved by binding of Gbetagamma subunits to PLCbeta2 allowing for amplified calcium signals.

PLCβ Is Critical for T-Cell Chemotaxis In Vivo.

Chemokines acting through G-protein coupled receptors play an essential role in both the immune and inflammatory responses. Phosphatidylinositol 3-kinase (PI3K) and phospholipase C (PLC) are two distinct signaling molecules that have been proposed as potential candidates in the regulation of this process. Studies with knockout mice have demonstrated a critical role for PI3Kγ, but not PLCβ, in Gαi-coupled receptor-mediated neutrophil chemotaxis. We compared the chemotactic response of peripheral T-cells derived from wild type mice with mice containing loss-of-function mutations of either PI3Kγ, or both of the two predominant lymphocyte PLCβ isoforms (PLCβ2 and PLCβ3). In contrast to neutrophils, loss of PI3Kγ did not significantly impair T-cell migration in vitro, although PI3K pharmacologic inhibitor experiments suggest that another isoform of this enzyme might contribute to T-cell migration. However, loss of PLCβ2β3 decreased chemokine-stimulated T-cell migration in vitro. Chelation of intracellular calcium by BAPTA-AM and Quin-2 AM decreased the chemotactic response of wild type lymphocytes, but pharmacologic inhibition of PKC isoforms by GF109203x did not impair T-cell migration. This suggests that the T-cell migration defect seen in the PLCβ2β3-null T-cells may be due to an impaired ability to increase the cytoplasmic calcium concentration, while there appears to be little requirement for PKC activity. Indeed, SDF-1α-induced calcium efflux was not detected in the PLCβ2β3-null lymphocytes. Compared to fluorescently labeled wild type T-cells, labeled PLCβ2β3 knockout T-cells migrated less efficiently into secondary lymphoid organs of recipient mice. This demonstrates that PLCβ is also required for migration in vivo. PLCβ2β3-null mice develop spontaneous skin ulcers starting around 3 months of age. Histological examination of the lesions revealed a dense inflammatory infiltrate composed of neutrophils, macrophages, and plasma cells, consistent with acute and chronic inflammation. Remarkably, lymphocytes, typical of chronic inflammation, were rare to absent by histology and by paraffin immunohistochemistry for CD3, also consistent with an in vivo migratory defect of T-cells.These results show that phospholipid second messengers generated by PLCβ and isoforms of PI3K, other than PI3Kγ, play a critical role in lymphocyte chemotaxis. Collectively, our data demonstrate that although PLCβ-mediated signaling plays no role in neutrophil chemotaxis, it makes a substantial contribution to this process within T-lymphocytes.

Chronic morphine-induced loss of the facilitative interaction between vasoactive intestinal polypeptide and δ-opioid: involvement of protein kinase C and phospholipase Cβs

This laboratory recently demonstrated a multiplicative interaction between the pelvic visceral afferent transmitter vasoactive intestinal polypeptide (VIP) and the δ-opioid receptor (DOR)-selective agonist [ d-Pen 2,5] enkephalin (DPDPE) to regulate cAMP levels in spinal cord [Brain Res. 959 (2003) 103]. Although DOR activation is required for the manifestation of the VIP–DPDPE facilitative interaction, its relevance to opioid antinociception remains unclear. The current study investigates whether or not the VIP–DPDPE facilitation of cAMP formation is subject to tolerance formation, a hallmark characteristic of opioid antinociception. Chronic morphine exposure abolishes the VIP–DPDPE facilitative interaction, consistent with its relevance to DOR antinociception. However, acute in vitro inhibition of protein kinase C (PKC) reinstates the VIP–DPDPE multiplicative interaction characteristic of opioid naı̈ve spinal tissue. This suggests that its chronic morphine-induced loss requires a PKC phosphorylation. PKC phosphorylation negatively modulates phospholipase C (PLC)β, enzymes intimately associated with phosphoinositide turnover and calcium trafficking. These are essential determinants of acute and chronic opioid effects. Accordingly, the effect of chronic morphine on their state of phosphorylation was also investigated. Central nervous system opioid tolerance is associated with the reciprocal phosphorylation (regulation) of two PLCβ isoforms, PLCβ1 and PLCβ3. However, although chelerythrine reinstates the chronic morphine-induced loss of the multiplicative VIP–DPDPE interaction, it does not alter the associated changes in PLCβ phosphorylation, possibly indicating different time courses of restitution of function and/or involvement of different kinases for different components of tolerance. These results could provide a mechanistic rubric for understanding positive modulation of opioid antinociception by afferent transmission.

The pleckstrin homology domain of phosphoinositide-specific phospholipase Cdelta4 is not a critical determinant of the membrane localization of the enzyme.

The inositol lipid and phosphate binding properties and the cellular localization of phospholipase Cdelta(4) (PLCdelta(4)) and its isolated pleckstrin homology (PH) domain were analyzed in comparison with the similar features of the PLCdelta(1) protein. The isolated PH domains of both proteins showed plasma membrane localization when expressed in the form of a green fluorescent protein fusion construct in various cells, although a significantly lower proportion of the PLCdelta(4) PH domain was membrane-bound than in the case of PLCdelta(1)PH-GFP. Both PH domains selectively recognized phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), but a lower binding of PLCdelta(4)PH to lipid vesicles containing PI(4,5)P(2) was observed. Also, higher concentrations of inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) were required to displace the PLCdelta(4)PH from the lipid vesicles, and a lower Ins(1,4,5)P(3) affinity of PLCdelta(4)PH was found in direct Ins(1,4,5)P(3) binding assays. In sharp contrast to the localization of its PH domain, the full-length PLCdelta(4) protein localized primarily to intracellular membranes mostly to the endoplasmic reticulum (ER). This ER localization was in striking contrast to the well documented PH domain-dependent plasma membrane localization of PLCdelta(1). A truncated PLCdelta(4) protein lacking the entire PH domain still showed the same ER localization as the full-length protein, indicating that the PH domain is not a critical determinant of the localization of this protein. Most important, the full-length PLCdelta(4) enzyme still showed binding to PI(4,5)P(2)-containing micelles, but Ins(1,4,5)P(3) was significantly less potent in displacing the enzyme from the lipid than with the PLCdelta(1) protein. These data suggest that although structurally related, PLCdelta(1) and PLCdelta(4) are probably differentially regulated in distinct cellular compartments by PI(4,5)P(2) and that the PH domain of PLCdelta(4) does not act as a localization signal.

A New Path to Ca 2+ Influx

Patterson et al. have uncovered a surprising role for phospholipase C-γ (PLC-γ) isoforms in mediating Ca 2+ influx after G protein-coupled receptor (GPCR) stimulation through a mechanism independent of their catalytic activity. PLC proteins are critically involved in mobilizing receptor-mediated Ca 2+ signaling. GPCRs activate PLC-β isoforms, whereas receptor and nonreceptor tyrosine kinases activate PLC-γ. Activated PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate to form inositol 1,4,5-trisphosphate, which triggers Ca 2+ release from intracellular stores, followed by Ca 2+ influx through channels in the plasma membrane. Patterson et al. showed that, when conditionally overexpressed in PC12 cells, both wild-type PLC-γ1 and mutant PLC-γ1 lacking lipase activity enhanced Ca 2+ influx after purinergic receptor stimulation, whereas mutant PLC-γ1 lacking the SH3 domain did not. Using RNA interference to deplete endogenous PLC, the authors found that depleting either PLC-γ1 or PLC-γ2 reduced Ca 2+ entry after purinergic stimulation in PC12 cells or serotonin stimulation in A7r5 cells without affecting release from internal stores. In DT40 B lymphocyte PLC-γ2 knockout cells overexpressing a muscarinic receptor, Ca 2+ entry from extracellular sources after receptor stimulation was abolished, whereas PLC-β-dependent Ca 2+ release from intracellular stores remained intact. Ca 2+ entry in the DT40 knockout cells was rescued by transfection with either PLC-γ2 or a PLC-γ2 lipase-inactive mutant. These data establish a lipase-independent role for PLC-γ in mediating Ca 2+ influx after GPCR stimulation. The inability of the SH3 domain mutant to enhance Ca 2+ influx, together with a requirement for this domain for coimmunoprecipitation of PLC-γ1 with transfected Ca 2+ channels, suggests that the effects of PLC-γ on Ca 2+ entry involve structural interactions with plasma membrane Ca 2+ channels or some protein intermediary. R. L. Patterson, D. B. van Rossum, D. L. Ford, K. J. Hurt, S. S. Bae, P.-G. Suh, T. Kurosaki, S. H. Snyder, D. L. Gill, Phospholipase C-γ is required for agonist-induced Ca 2+ entry. Cell 111 , 529-541 (2002). [Online Journal]

Functional relevance of the disulfide-linked complex of the N-terminal PDZ domain of InaD with NorpA.

In Drosophila, phototransduction is mediated by G(q)-activation of phospholipase C and is a well studied model system for understanding the kinetics of signal initiation, propagation and termination controlled by G proteins. The proper intracellular targeting and spatial arrangement of most proteins involved in fly phototransduction require the multi-domain scaffolding protein InaD, composed almost entirely of five PDZ domains, which independently bind various proteins including NorpA, the relevant phospho lipase C-beta isozyme. We have determined the crystal structure of the N-terminal PDZ domain of InaD bound to a peptide corresponding to the C-terminus of NorpA to 1.8 A resolution. The structure highlights an intermolecular disulfide bond necessary for high affinity interaction as determined by both in vitro and in vivo studies. Since other proteins also possess similar, cysteine-containing consensus sequences for binding PDZ domains, this disulfide-mediated 'dock-and-lock' interaction of PDZ domains with their ligands may be a relatively ubiquitous mode of coordinating signaling pathways.

Association of Heterotrimeric G-Proteins with Bovine Aortic Phospholipase C γ

The widely expressed phospholipase C γ1(PLCγ1) isoform has been implicated in the signalling of cell growth through its ability to hydrolyse phosphatidylinositol 4,5-bisphosphate to give inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. Stimulation of PLCγ1activity occurs upon phosphorylation of specific tyrosine residues, although it is unclear how this phosphorylation actually stimulates catalytic activity. Indeed recent reports suggest that accessory factors such as GTP-binding proteins may also be required for complete activation of PLCγ1in some cells. This may be of importance in vascular smooth muscle where traditionally G-protein linked PLCβ isoforms are often absent. Here, we show that bovine aortic PLCγ1activity is substantially enhanced by both GTPγS and sodium fluoride. Similarly, immunoprecipitated PLCγ1is associated with an approximately 40kDa GTPγS-binding protein and both Gαiand Gαqwere detected in this immunoprecipitate. This data suggests that bovine aortic PLCγ1is both associated with, and may be activated by, heterotrimeric G-proteins.

Ins(1,4,5)P3 and cardiac dysfunction.

Time for primary review 33 days. Cardiac related death occurs rapidly via the development of arrhythmias or more slowly by a progressive decline in mechanical performance reflecting decreased contractile activity as well as loss of viable myocytes. Pathological conditions such as myocardial ischaemia, inflammatory heart diseases and various forms of heart failure are associated with both sudden death and also with a more progressive decrease in cardiac function. Electrophysiological disturbances leading to malignant ventricular arrhythmias are the usual cause of sudden cardiac death, whereas a gradual loss of functional myocytes, or a decline in their performance, underlies heart failure ultimately leading to cardiac decompensation. The mechanisms initiating these scenarios are complex and multifactorial, involving both extracellular and intracellular factors. However, given the continued major contribution of cardiac deaths to overall mortality, understanding the processes which initiate these pathological events is of prime importance. The current review will focus on changes in the intracellular milieu and in particular the potential role of the Ca2+-releasing second messenger inositol(1,4,5) tris phosphate [Ins(1,4,5)P3] in initiating arrhythmias and cell death in cardiac pathologies. Ins(1,4,5)P3 is generated along with sn -1,2-diacylglycerol following the activation of either G protein-coupled receptors and phospholipase C-β (PLC-β) isoforms or receptor tyrosine kinases and PLC-γ isoforms [1]. The Ins(1,4,5)P3 which is rapidly released into the cytoplasm, especially after activation of G protein-coupled receptors, activates Ins(1,4,5)P3 receptors on Ca2+ stores in the endoplasmic reticulum [sarcoplasmic reticulum (SR) in muscle cells] causing a rapid and quantal release of Ca2+ [2]. In most non-cardiac cell types, this Ins(1,4,5)P3-initiated Ca2+ response can be perpetuated by the process of Ca2+-induced Ca2+ release mediated by either the Ins(1,4,5)P3 receptors or the related family of ryanodine receptors, the overall effect … * Corresponding author. Tel.: +61-3-9522-4387; Fax: +61-3-9521-1362; E-mail: liz.woodcock@baker.edu.au

Sticky-end PCR: new method for subcloning.

Sticky-end PCR: new method for subcloning.

Bradykinin Stimulates the Tyrosine Phosphorylation and Bradykinin B2 Receptor Association of Phospholipase Cγ1 in Vascular Endothelial Cells

Bradykinin (BK) B2 receptor signaling involves activation of phospholipase C (PLC). PLC activation by other receptors consists of either allosteric activation of PLCβ isoforms by G-proteins or tyrosine phosphorylation of PLCγ isoforms. Because the B2 receptor is a G-protein-coupled receptor, it has been assumed that the receptor signals through PLCβ. In the present study, however, we have found that BK stimulation of IP3production and the Ca2+signal in endothelial cells is dependent on tyrosine phosphorylation. Furthermore, stimulation of B2 receptors in these cells is accompanied by a transient tyrosine phosphorylation of PLCγ1. Phosphorylation is correlated with increased IP3production and association of PLCγ1 with the C-terminal intracellular domain of the B2 receptor. The B2 receptor can thus physically associate with intracellular proteins other than G-proteins. Activation of PLCγ isoforms, rather than PLCβ isoforms, may, therefore, be primarily responsible for BK-stimulated IP3generation in endothelial cells.

Stimulation by G Protein βγ Subunits of Phospholipase C β Isoforms in Human Platelets

SummaryDifferent phospholipase C (PLC) isoforms were located in human platelet cytosol and membranes. PLCγ2 and PLCβ3b were mainly located in the cytosol and PLCβ2 and PLCβ3a were in both cytosol and membranes by using specific antibodies against PLC isozymes (Banno Y, Nakashima S, Ohzawa M, Nozawa Y. J Biol Chem 1996; 271: 14989-94). Three PLC fractions activated by G protein βγ subunits were purified from human platelet cytosol and membrane fractions. Two PLC fractions from membranes were identified as PLCβ2 and PLCβ3a, and one from cytosol was PLCβ3b. These PLCβ isoforms were activated by the purified βγ subunits of brain G proteins in the order PLCβ3b > PLCβ3a > PLCβ2. Western blot analysis of γ subunits of the purified platelet G proteins with antibodies against various standard γ subunits revealed that the major component of the γ subunit of Gi2 and Gq was γ5, and that γ7 was a minor component. Studies using various subtypes of βγ subunits, βγ2, βγ3, and βγ7 purified from bovine brain, βγ5 from bovine lung, or βγ12 from bovine spleen, failed to show differences in their ability to stimulate the isolated platelet PLCβ isoforms. These results suggest that the βγ subunits of Gi2 and Gq have similar efficacy in regulation of effectors in human platelets.

A β-subclass phosphatidylinositol-specific phospholipase C from squid ( Loligo forbesi) photoreceptors exhibiting a truncated C-terminus

A PCR-based strategy has been used to isolate a full length cDNA encoding a phosphatidylinositol-specific phospholipase C from a sized cDNA squid ( Loligo forbesi) retinal library. The predicted protein sequence contains 875 amino acids, with calculated M r 98,181, and has marked similarity with PLC β-isoforms, including conservation of the ‘X’ and ‘Y’ regions. It is unique in having a major C-terminal truncation. A major protein of apparent M r 120,000 estimated by SDS-PAGE has been isolated from squid photoreceptors and identified by partial protein sequence analysis to correspond to the protein sequence predicted from the cDNA clone. This protein has been shown to hydrolyse phosphatidylinositol 4,5-bisphosphate. It is not yet clear whether this represents the major light-activated PLC in squid vision.