OUR SPECIFIC OBJECTIVES FOR THIS PAST YEAR:[unreadable] [unreadable] 1) With respect to PLD, our earlier studies revealed that PLD2 co-localized with components of "lipid rafts" and that phosphorylation of PLD2 by Src kinases was dependent on integrity of these "lipid rafts". In the current period we have investigated whether PLD itself is required for the functional integrity of "lipid rafts" because localized production of phosphatidic acid and diacylglycerol through PLD could have a substantial impact on membrane dynamics.[unreadable] [unreadable] 2) We have also investigated the role of PLD in the activation of sphingosine kinase (SK) because PLD products are potential activators of SK and SK has been implicated in the generation of a calcium signal.[unreadable] [unreadable] 3) As noted in the Introduction, we hypothesized that Ca2+-specific CRAC was not the sole mechanism for the entry of Ca2+ because Sr2+ and other divalent cations also permeate and support degranulation in stimulated mast cells. The transient receptor potential cannonical (TRPC) channels were originally considered as candidates for CRAC but their electrophysiological properties did not fit with those of CRAC and some TRPCs were known to conduct divalent metal ions such as Sr2+. Therefore, we examined the possibility that TRPCs interact with the recently identified CRAC components, Orai1 and its Ca2+ sensor STIM1.[unreadable] [unreadable] RESULTS: [unreadable] [unreadable] 1) PLD AND LIPID RAFT FUNCTION: Suppression of PLD function, either with primary alcohols or siRNAs, results in dispersal of lipid raft components including LAT, Thy1, and GPI. Once mast cells are activated, the translocation of the IgE receptor (FceRI) and its associated tyrosine kinases, Lyn and Syk, into lipid rafts as well as downstream phosphorylation events are also blocked. Consistent results were obtained whether cells were examined by use of fluorescent tagged molecules and confocal microscopy or by classical membrane fractionation techniques. These techniques indicated that tagged PLD2 also colocalizes with lipid raft constituents, a process that is prevented by the lipid raft dispersing agents. These observations suggest that not only is PLD2 activation dependent on lipid raft integrity but that this activation contributes to the functional organization of lipid rafts (submitted for publication).[unreadable] [unreadable] [unreadable] 2) REGULATION OF SPHINGOSINE KINASE (SK), CALCIUM MOBILIZATION, AND DEGRANULATION BY PLD: In addition to being the primary source diacylglycerides for the activation of diglyceride-dependent isoforms of PKC(Peng and Beaven, J. Immunol. 174:5201,2005), PLD1 is reported to regulate activation of SK1. The SK product, sphingosine 1-phosphate (S1P), acting in conjunction with IP3 was said to promote release of Ca2+ from intracellular stores and thus indirectly influx of external Ca2+. However, contrary to previous reports, we find that knockdown of PLD2 and SK2, and not PLD1 or SK1, blocks entry of Ca2+ with minimal effect on release of Ca2+ from intracellular stores in stimulated mast cells. Degranulation is also impaired, indicating that Ca2+ influx is critical for complete degranulation (ref. 1). We have confirmed the PLD2/SK2/Ca2+ influx pathway by knockdown of PLD1 or PLD2 in bone marrow-derived cells (BMMC) from SK1 and SK2 knockout mice. Also contrary to previous reports, we find that the regulation of Ca2+ homeostasis by SK in mast cells is not unique to antigen stimulation, but is intrinsic to the calcium signaling process rather than depending on stimulant. For example, Ca2+-influx is impaired in PLD2 or SK2 deficient BMMC by merely depleting the intracellular Ca2+-stores with thapsigargin or Ca2+-ionophore, both of which activate SOCE by store depletion (see next section). From these and earlier studies we believe that SK may be essential for proper translocation of either the Ca2+-sensor, STIM1, or the ion channel proteins, Orai1 and TRPC5, to their proper destinations for Ca2+-influx (see next section). [unreadable] [unreadable] 3) IDENTIFICATION OF CALCIUM CHANNELS AND REGULATORY PROTEINS FOR CALCIUM ENTRY: As noted in the Introduction, depletion of intracellular stores of Ca2+ in the endoplasmic reticulum activates influx of external Ca2+ (SOCE) via the well characterized Ca2+-specifc current CRAC. CRAC has now been attributed to the interaction of a Ca2+-sensor, STIM1, in the endoplasmic reticulum and the Ca2+-specific channel protein Orai1. We find that overexpression of STIM1 and Orai1 allows entry of Ca2+ but not Sr2+ in stimulated mast cells. However, primary and tumor mast cell lines also express various members of the TRPC channels some of which can conduct Sr2+ as well as Ca2+. Knock down of individual TRPCs with siRNAs indicate that entry of Ca2+ and Sr2+ as well as degranulation was dependent on TRPC5 and not on any other TRPC channel protein. However, knockdown of STIM1 and Orai1 also impaired entry of both ions and the response could only be reconstituted by overexpression of TRPC5, Orai1 and STIM1. These and similar experiments suggest that TRPC5 associates with STIM1 and Orai1 in a stoichiometric manner to enhance entry of Ca2+ or Sr+ to generate a signal for degranulation. This scenario bridges seemingly paradoxical observations of non-selective uptake of divalent metal ions by stimulated mast cells (our studies) and highly selective uptake of Ca2+ through Orai1 (studies by others). Our studies also add a new facet in our understanding of the mechanisms underlying SOCE (ref. 2). Preliminary studies with overexpressed tagged molecules of TRPC5, Orai1, and STIM1 also reveal that SK (see previous section) may be essential for the interaction of STIM1 with either TRPC5 or Orai1 as monitored by confocal microscopy.