A broad range of hormones, neurotransmitters, growth factors, and other stimuli produce their physiological effects by stimulation of inositol lipid breakdown. This response for most receptors involves activation of a G-protein, which in turn activates phospholipase C (PLC). The long- term goal of this research is to identify the component proteins of this signalling pathway and to delineate the molecular details of their receptor-promoted interaction. In the previous funding period this laboratory used the turkey erythrocyte as a model system to identify and purify a G-protein-regulated PLC and used this enzyme in a reconstitution assay to purify its activating G-protein, Galpha(11). The full range of effector PLCs activated by Galpha(11), by other members of the Gq class of G-proteins, and by pertussis toxin-sensitive G-proteins has not been delineated. As such, PLC-(Beta(1), PLC-(Beta(2), PLC-(Beta(3), and potentially other G-protein-regulated PLCs will be purified and the specificity of their activation by G(alpha(11) and G(alpha)(g) will be determined with native and recombinant proteins. The possibility that one or more of the PLC isoenzymes is activated by pertussis toxin- sensitive G-proteins will be examined by reconstitution with recombinant G(alpha(i1), G(alpha(i2, G(alpha(i3, and G(alpha(0). This laboratory recently has observed that G-protein-Beta/gamma-subunits activate the avian G-protein-regulated PLC. This work will be extended to determine whether other PLC-(Beta) class isoenzymes are regulated directly by (Beta/gamma-subunits, to establish the specificity of activation by (Beta/gamma) dimers of defined composition made from various (Beta)- and gamma-subunits, and to establish the relative activities of (alpha)- versus (Beta/gamma)-subunits for each isoenzyme. The components of the pertussis toxin-sensitive inositol lipid signalling cascade will be identified in HT29 human carcinoma cells. The PLC involved in pertussis toxin-sensitive purinergic receptor regulation of inositol lipid signalling will be purified, and the G-protein will be identified by receptor-promoted in situ labelling with [32P] azidoanilido GTP and immunoprecipitation. The relative contribution of alpha-versus Beta/gamma-subunits to activation of the involved PLC in the pertussis toxin-sensitive pathway of HT29 cells will be established. The molecular details of activation of the avian G-protein-regulated PLC by G(alpha11) and by G-protein Beta/gamma-subunits will be defined using model lipid monolayers of various composition at an air water interface. This and other knowledge accrued for G(alpha11) and the G-protein-regulated PLC of avian erythrocytes will be used to delineate the molecular basis of the heterologous desensitization of the inositol lipid signalling pathway that occurs during exposure of these cells to various stimuli. Completion of the work described in this proposal should considerably extend knowledge of the identify, specificity, and mechanism of interaction of the component proteins of the receptor-regulated inositol lipid signalling pathway.
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