Proteins of diverse function and structural features often can be grouped together because they share common methods for controlling function. Such is the case for some proteins involved in signal transduction. For these proteins, turning on the protein's function occurs when the protein is targeted to and binds to the cell membrane. In the absence of a stimulus, the signaling protein is essentially inactive. It is a conformation of the protein itself that maintains it in this inactive state. Binding of the signaling protein to the cell membrane causes a change in conformation that removes the inhibitory block, and allows the protein to effect its action within the cell. Phospholipase C-g1 (PLC-g1A) is a signaling protein. PLC-g1 is located within the interior of the cell and becomes activated when it receives signals from external growth factors. Activated PLC-g1 binds to the membrane and hydrolyzes phosphatidylinositol bisphosphate (PIP2) to generate two-second messengers, soluble inositol 1,4,5-trisphosphate and membrane-associated diacylglycerol. These second messengers then pass the signal along to other proteins and eventually effect an increase in cell division and cell motility. The purpose of this proposal is to determine whether PLC-g1 can be grouped with other signaling proteins that are maintained in an inactive state but become active when a change in conformation occurs upon binding to the cell membrane. This project is designed to determine whether a domain of PLC-g1 keeps it in an inactive state and which part of the protein is responsible for maintaining it in that inactive state. To provide this answer, experiments will be conducted that will determine activity of a modified PLC-g1 that is missing a portion of the inhibitory domain and of a form of PLC-g1 that can no longer receive signals from growth factors. Techniques such as fluorescence, circular dichroism, and electron spin resonance will be used to determine changes in protein conformation. The results of this study will increase our knowledge of how the function of PLC-g1 is regulated and add to our general knowledge of how proteins with divergent functions and structural features develop common ways for controlling protein function.
