Ginger, Rebecca Susan; (1994) Studies on the regulation of inositol lipid-specific phospholipase C. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (19MB)

Abstract

Several inositol-lipid-specific phospholipase C (PtdIns-PLC) isozymes have been cloned and divided into 3 classes, β, γ and δ, on the basis of structural homology. The γ isozymes are activated through stimulation of tyrosine kinase receptors, however the β and δ isozymes do not appear to be activated in this way. Stimulation of 7-transmembrane receptors also activates phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2) hydrolysis and this receptor-coupled PtdIns-PLC activation is sensitive to GTP, implying that G-proteins are involved in receptor mediated PtdIns-PLC activation. This G-protein coupled activation of PtdIns-PLC is both sensitive and insensitive to Pertussis toxin (PTX), implying the existence of more than one G-protein-coupled PtdIns-PLC activation pathway. A family of PTX insensitive G-proteins, Gq, has been cloned, and purified α subunits from this family activate PtdIns-PLCβ1 in vitro. To investigate the mechanisms involved in the regulation of the β and δ isozymes, both full length proteins and domains of the β isozymes have been expressed as fusion proteins in E.coli and purified. Using these proteins, the role of phosphorylation in the regulation of the different isozymes has been assessed and it is demonstrated that these isozymes are not targets of Protein kinase C (PKC) phosphorylation. PtdIns-PLCβ1 activation by Gqα subunits has been reconstituted in vitro and the ability of the different PtdIns-PLCβ domains to disrupt this activation has been assessed. Thus the carboxy terminus has been identified as the Gqα subunit-binding region of PtdIns-PLCβ1. This result is confirmed using an in vivo COS-1 cell expression system in which the binding of G-protein βγ subunits to the aminoterminus of PtdIns-PLCβ2 is indirectly demonstrated. These findings are discussed in the context of results from other laboratories.

Download activity - last month

Export as CSVXMLJSON

Download activity - last 12 months

Export as JSONCSVXML

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item