PROJECT SUMMARY / ABSTRACT Phosphatidylinositol 4,5-bisphosphate (PIP2)-Ca2+ signaling is activated following stimulation of growth factor receptors, antigen receptors, and G protein-coupled receptors by ligands including neurotransmitters and histamine. The activation of phospholipase C triggers hydrolysis of the PIP2 lipid at the plasma membrane (PM) to generate diacylglycerol (DAG) and inositol trisphosphate (IP3). IP3 subsequently releases Ca2+ stored in the endoplasmic reticulum (ER) to activate cytosolic Ca2+ effectors for downstream signaling events such as secretion, proliferation, and migration. The consumed PM PIP2 and ER Ca2+ must be quickly restored to sustain signaling responses and to maintain cellular homeostasis. This homeostatic regulation of PIP2-Ca2+ signaling requires transport of phosphatidylinositol (PI) from the ER to the PM for PIP2 resynthesis and store-operated Ca2+ entry (SOCE) that activates Ca2+ influx to refill the ER Ca2+ store. SOCE has been studied extensively; however, the mechanisms underlying PIP2 replenishment during PIP2-Ca2+ signaling are not well understood. Recently, we and others discovered the lipid transfer protein Nir2 that localizes at ER-PM junctions, where the ER is in contact with the PM, to mediate PM PIP2 replenishment. The objective of this proposal is to define the mechanisms regulating PIP2 replenishment by Nir2 at ER-PM junctions during receptor-induced Ca2+ signaling. Our preliminary studies identified Nir1, an Nir protein lacking a lipid transfer protein domain, as a binding partner and positive regulator of Nir2 at ER-PM junctions. In addition, our recent data revealed that the membrane- shaping hairpin sequence present in all extended synaptotagmins (E-Syts) is important for regulating PIP2 replenishment at ER-PM junctions. Moreover, we found that conversion of DAG into phosphatidic acid (PA) by DAG kinases (DGKs) is crucial for Nir2 localization at ER-PM junctions. Among the ten DGKs in mammalian cells, the epsilon-isoform of DGK (DGKε) is recently shown to localize at ER-PM junctions. Our central hypothesis is that PM PIP2 replenishment during receptor-induced Ca2+ signaling is mediated by the oligomers of Nir1 and Nir2 formed following PA production from DAG by DGKε at ER-PM junctions shaped by E-Syts. We propose three specific aims to test our central hypothesis using biochemical analysis and advanced imaging techniques to determine the mechanisms by which Nir1, E-Syts and DGKε regulate PIP2 replenishment. We expect that successfully completion of the proposed studies will establish the molecular and cellular mechanisms regulating PIP2 replenishment following hydrolysis induced by receptor stimulation. Restoring PM PIP2 is critical to sustain Ca2+ signaling and maintain PM PIP2 levels critical to membrane trafficking, cytoskeletal dynamics, and ion transport in receptor-stimulated cells. Mutations in Nir1 are linked to retinal dystrophy and patients with mutations in DGKε suffer from...