Pertussis Toxin-sensitive Activation of Phospholipase C by the C5a and fMet-Leu-Phe Receptors

Signal transduction pathways that mediate C5a and fMet-Leu-Phe (fMLP)-induced pertussis toxin (PTx)-sensitive activation of phospholipase C (PLC) have been investigated using a cotransfection assay system in COS-7 cells. The abilities of the receptors for C5a and fMLP to activate PLC beta 2 and PLC beta 3 through the Gbeta gamma subunits of endogenous Gi proteins in COS-7 cells were tested because both PLC beta 2 and PLC beta 3 were shown to be activated by the beta gamma subunits of G proteins in in vitro reconstitution assays. Neither of the receptors can activate endogenous PLC beta 3 or recombinant PLC beta 3 in transfected COS-7 cells. However, both receptors can clearly activate PLC beta 2 in a PTx-sensitive manner, suggesting that the receptors may interact with endogenous PTx-sensitive G proteins and activate PLC beta 2 probably through the Gbeta gamma subunits. These findings were further corroborated by the results that PLC beta 3 could only be slightly activated by Gbeta 1gamma 1 or Gbeta 1gamma 5 in the cotransfection assay, whereas the Gbeta gamma subunits strongly activated PLC beta 2 under the same conditions. PLC beta 3 can be activated by Galpha q, Galpha 11, and Galpha 16 in the cotransfection assay. In addition, the Ggamma 2 and Ggamma 3 mutants with substitution of the C-terminal Cys residue by a Ser residue, which can inhibit wild type Gbeta gamma -mediated activation of PLC beta 2, were able to inhibit C5a or fMLP-mediated activation of PLC beta 2. These Ggamma mutants, however, showed little effect on m1-muscarinic receptor-mediated PLC activation, which is mediated by the Gq class of G proteins. These results all confirm that the Gbeta gamma subunits are involved in PLC beta 2 activation by the two chemoattractant receptors and suggest that in COS-7 cells activation of PLC beta 3 by Gbeta gamma may not be the primary pathway for the receptors

Activation of phospholipase C beta 2 by the alpha and beta gamma subunits of trimeric GTP-binding protein

Cotransfection assays were used to show that the members of the GTP-binding protein Gq class of alpha subunits could activate phospholipase C (PLC) beta 2. Similar experiments also demonstrated that G beta 1 gamma 1, G beta 1 gamma 5, and G beta 2 gamma 5 could activate the beta 2 isoform of PLC but not the beta 1 isoform, while G beta 2 gamma 1 did not activate PLC beta 2. To determine which portions of PLC beta 2 are required for activation by G beta gamma or G alpha, a number of PLC beta 2 deletion mutants and chimeras composed of various portions of PLC beta 1 and PLC beta 2 were prepared. We identified the N-terminal segment of PLC beta 2 with amino acid sequence extending to the end of the Y box as the region required for activation by G beta gamma and the C-terminal region as the segment containing amino acid sequences required for activation by G alpha. Furthermore, we found that coexpression of G alpha 16 and G beta 1 gamma 1 but not G beta 1 gamma 5 in COS-7 cells was able to synergistically activate recombinant PLC beta 2. We suggest that G alpha 16 may act together with free G beta 1 gamma 1 to activate PLC beta 2, while G alpha 16 may form heterotrimeric complexes with G beta 1 gamma 5 and be stabilized in an inactive form. We conclude that the regions of PLC beta 2 required for activation by G beta gamma and G alpha are physically separate and that the nature of the G beta subunit may play a role in determining the relative specificity of the G beta gamma complex for effector activation while the nature of the G gamma subunit isoform may be important for determining the affinity of the G beta gamma complex for specific G alpha proteins

The Interaction of Phospholipase C-{beta}3 with Shank2 Regulates mGluR-mediated Calcium Signal

Phospholipase C-{beta} isozymes that are activated by G protein-coupled receptors (GPCR) and heterotrimeric G proteins carry a PSD-95/Dlg/ZO-1 (PDZ) domain binding motif at their C terminus. Through interactions with PDZ domains, this motif may endow the PLC-{beta} isozyme with specific roles in GPCR signaling events that occur in compartmentalized regions of the plasma membrane. In this study, we identified the interaction of PLC-{beta}3 with Shank2, a PDZ domain-containing multimodular scaffold in the postsynaptic density (PSD). The C terminus of PLC-{beta}3, but not other PLC-{beta} isotypes, specifically interacts with the PDZ domain of Shank2. Homer 1b, a Shank-interacting protein that is linked to group I metabotropic glutamate receptors and IP3 receptors, forms a multiple complex with Shank2 and PLC-{beta}3. Importantly, microinjection of a synthetic peptide specifically mimicking the C terminus of PLC-{beta}3 markedly reduces the mGluR-mediated intracellular calcium response. These results demonstrate that Shank2 brings PLC-{beta}3 closer to Homer 1b and constitutes an efficient mGluR-coupled signaling pathway in the PSD region of neuronal synapses

Large UK retailers' initiatives to reduce consumers' emissions: a systematic assessment

In the interest of climate change mitigation, policy makers, businesses and non-governmental organisations have devised initiatives designed to reduce in-use emissions whilst, at the same time, the number of energy-consuming products in homes, and household energy consumption, is increasing. Retailers are important because they are at the interface between manufacturers of products and consumers and they supply the vast majority of consumer goods in developed countries like the UK, including energy using products. Large retailers have a consistent history of corporate responsibility reporting and have included plans and actions to influence consumer emissions within them. This paper adapts two frameworks to use them for systematically assessing large retailers’ initiatives aimed at reducing consumers’ carbon emissions. The Framework for Strategic Sustainable Development (FSSD) is adapted and used to analyse the strategic scope and coherence of these initiatives in relation to the businesses’ sustainability strategies. The ISM ‘Individual Social Material’ framework is adapted and used to analyse how consumer behaviour change mechanisms are framed by retailers. These frameworks are used to analyse eighteen initiatives designed to reduce consumer emissions from eight of the largest UK retail businesses, identified from publicly available data. The results of the eighteen initiatives analysed show that the vast majority were not well planned nor were they strategically coherent. Secondly, most of these specific initiatives relied solely on providing information to consumers and thus deployed a rather narrow range of consumer behaviour change mechanisms. The research concludes that leaders of retail businesses and policy makers could use the FSSD to ensure processes, and measurements are comprehensive and integrated, in order to increase the materiality and impact of their initiatives to reduce consumer emissions in use. Furthermore, retailers could benefit from exploring different models of behaviour change from the ISM framework in order to access a wider set of tools for transformative system change

EVALUATION OF PLC PROGRAMME PROVISION. ESRI RESEARCH SERIES NUMBER 61 JANUARY 2018

Post-Leaving Certificate (PLC) courses represent the largest component of full-time further education and training (FET) provision in Ireland, with over 32,000 learners enrolled in such courses in 2015–2016. Recent research on the FET sector as a whole highlighted concerns around its structures and responsiveness to labour market conditions, among other issues (McGuinness et al., 2014). The SOLAS FET Strategy (2014) subsequently pointed to the need for a stronger evidence base in order to inform future policy development in the sector. This study, commissioned by SOLAS, provides a more detailed evaluation of PLC provision. In order to undertake a comprehensive evaluation, our approach has been to combine a variety of research modes in order to examine the underlying processes, experiences and outcomes of PLC provision. This involved three complementary research strands. Firstly, a desk-based analysis of administrative data was used to document the type of provision in terms of field of study and the distribution of PLC places across the country. Secondly, a survey of PLC principals was carried out in order to explore their perceptions of goals, adequacy of existing facilities and the benefits and challenges of PLC provision. Thirdly, a survey of PLC and Leaving Certificate leavers was conducted to assess their labour market outcomes as well as their experiences while taking PLC and higher education courses. Together, these strands provide comprehensive evidence to inform the future development of the sector

Professional Learning Communities in the Expanded Learning Field

This white paper uses twelve evaluation reports of the Professional Learning Community (PLC) initiatives, as well as interviews with PLC participants and facilitators, to better understand how the PLC model is used in the Expanded Learning field, to demonstrate the benefits to participating staff and expanded learning programs, and to share best practices for youth-serving organizations interested in using PLCs

Ezrin-related phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles