Heterotrimeric G proteins are activated by membrane receptors that respond to a diverse set of agonists ranging from hormones, ions, light and neurotransmitters. Activated G proteins in turn can activate a host of intracellular effector proteins, one of which is phospholipase C-beta (PLCb). PLCb catalyzes the hydrolysis of the signaling lipid phosphatidylinositol 4,5 bisphosphate to release two second messengers that cause an increase in intracellular calcium and activation of protein kinase C. The mechanism through which G proteins activate PLCb or other effectors is unknown mainly due to a lack of structural information about PLCb - G protein complexes. PLCb is a multidomain enzyme and we have found that Gbetagamma subunits bind to one of these domains confering activation to the catalytic core.
In Aim 1 we will determine the mechanism through which activator binding changes interdomain contacts that allow for increased catalysis. We have also found that catalysis can be increased to different and distinct levels depending on the activation conditions opening up the possibility that activation of PLCb can befine-tuned to elicit particular cellular responses. This idea will be tested in Aim 2. Activation of PLCb by Gbetagamma subunits is long-lived but can be significantly reduced by changing PLCb-Gbetagammathrough the presence of another protein partner such as Galpha(GDP). This mechanism will be investigated both biophysically and in living cells in Aim 3. Inthe previous funding period, we have found that PLCb will bind to and inhibit the very robust enzyme PLCdelta, and that release of Gbetagamma subunits during signaling will displace PLCb from the complex allowing for both activation of PLCb and reversal of PLCd inhibition.
In Aim 4 we will better define the ability of these two PLCs to regulate calcium signals in cells. Activation of PLCbeta by G proteins causes an increase in the cellular levels of calcium that in turn activates a variety of proteins leading changes in cell growth and division. PLCb - G protein activation is key cellular response for agents such as acetylcholine and a large number of pharmaceutical agents. This proposal seeks to understand on the molecular level howthis activation occurs with the belief that understanding this system on a basic level will allow for the design of more effective and targeted drugs.
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