Heterotrimeric G proteins are activated by membrane receptors that respond to a diverse set of agonists ranging from hormones, ions and neurotransmitters. The alpha and beta-gamma subunits of G proteins can then activate effectors. One of these, phospholipase C-beta (PLC-beta), catalyzes the hydrolysis of the lipid phosphatidylinositol 4,5 bisphosphate (PIP2) to release the two second messengers diacylglycerol and inositol 1,4,5 trisphosphate which in turn result in an increase in intracellular Ca2+ and activation of protein kinase C. The mechanism through which G protein subunits activate PLC-A is unknown. Recently, we have found that if we replace the pleckstrin homology (PH) domain of PLCdelta, which is not G protein regulated, with the PH domain of PLCdelta, we confer G protein activation. Thus, activation of PLCbeta2 by G beta-gamma subunits occurs through residues in the PH domain, which transmit activation through residues in the catalytic core that are conserved among the two families of PLCs. We propose this results in an opening of the active site allowing for a greater release of intermediate and an increased water content which promotes hydrolysis. The crystal structure of the catalytic core of PLCdelta is known and is highly homologous to PLCbeta. Here, we will construct mutant and chimeric proteins of PLCbeta and PLCdelta that will allow us to determine the molecular' pathway through which G beta-gamma activation occurs. After, we will explicitly determine which step(s) in the catalytic pathway is activated by G beta-gamma subunits. This will be the first study to determine the molecular mechanism through which a G protein activates an interfacial effector. Even though PLC-delta is found in all mammalian cells, its role in cell function, and its protein regulators are unknown. We have unexpectedly found that PLC-A binds strongly to PLC-A and inhibits its high basal activity. Thus, release of G beta-gamma subunits from cell stimulation may disrupt PLC-beta2 - PLC-delta1 association causing a synergistic increase in intracellular Ca2+ and implicating PLC-delta as a secondary activator in hormonal regulation. We will test this model by a unique combination of in vitro and in vivo methods.
Showing the most recent 10 out of 58 publications
