Both receptor tyrosine kinases as exemplified by growth factor receptors and expression of non-receptor kinases such as pp60v-src can stimulate inositol lipid breakdown and generate signals such as diacylglycerol (DAG) and Ca2+ within the cell. The long term goal of the project is to determine how tyrosine kinases interact with the mechanisms regulating inositol lipid signalling. Data in the Progress Report suggest that expression of the v-src tyrosine kinase in Rat-1 fibroblasts causes a dramatic enhancement of the ability of mitogens such as endothelin to stimulate the production of Ins(1,4,5)P3 and DAG from the membrane. Thus, one effect of these changes is to generate increased levels of signalling molecules in the cell. This finding suggests that v-src transformation results in a cell that is hyper-responsive to the hormones and mitogens in serum yielding constant activation of the pathways participating in transformation. The current data points to an effect of v-src transformation on the interaction of the G protein, Gq, with phospholipase C as a primary event causing the hyper-responsive state. Therefore, this proposal is focused on examining the nature of the receptor - Gq - PLC-beta system in Rat-1 cells, determining the site of action of src on these membrane proteins and exploring the possibility that v-src transformation removes feedback inhibition from the system. The project will be conducted via three Specific Aims:
(Aim 1) To define the role of the functional domains of the v-src protein kinase in the dramatic amplification of endothelin-stimulated InsP3 production.
This aim will be approached by using Rat-1 cells transfected with a variety of src constructs designed to allow correlation of src expression with appearance of endothelin hyper-responsiveness, to determine the role of the molecule's SH2 domain in the response and the need for attachment of the v-src kinase to the membrane.
(Aim 2) To determine the site of action of v-src within the endotheline receptor - Gq - PLC-Beta signalling complex.
This aim will be approached by purifying components of the signalling complex from normal and transformed cells prior to assaying Gq-stimulated PLC activity in lipid vesicles containing exogenous [3H]-PIP2 as the substrate. The interaction of the endothelin receptor and Gq will be examined in membranes from Sf9 cells expressing recombinant endothelin receptors.
(Aim 3) To explore the hypothesis that the v-src effect is caused by a loss of feedback control on receptor- stimulated phosphatidylinositol breakdown. Since feedback inhibition of this system potentially occurs via protein kinases, these experiments will be performed by examining whether Gq and/or PLC-Beta can be phosphorylated with various purified protein kinases followed by assay of Gq-stimulated PLC activity in lipid vesicles. The ability of the proteins to be phosphorylated in intact Rat-1 cells will also be examined and compared with the results obtained with purified proteins.
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