The major hypothesis of this revised proposal is that membrane p32 regulates cellular secretory activity by modulating agonist induced intracellular calcium signaling. The proposed studies are designed to understand the modulatory effect of SP-A on cellular signaling and to characterize this effect in vitro as well as in vivo. Studies on cultured cells will examine how the inhibitory signal for p32 affects calcium and inositol phosphate-dependent cell activation signals. Agonists, such as ATP, which induce phospholipase C and D activation and release intracellular second messengers such as inositol phosphates and diacylglycerol, will be studied. Further effects of p32 and SP-A on calmodulin activation and calmodulin-dependent protein kinase activation will be studied, as well. Since these signaling systems often activate transcription, studies are as well designed to look at specific transcriptional activity. These studies will be carried out in cultured cells and in specific p32 knockout mice and p32 overexpressing mice. p32 protein and transcript will be localized in tissue during ontogeny by immunohistochemistry and in situ hybridization and Northern analysis. Mice to be used in these studies are 1) transgenic mice whose p32 expression is controlled by the powerful uteroglobin promoter, and 2) p32 knockout mice. The combination of p32 overexpression and knockout will allow not only the study of the role of p32 in vivo, but also to detect its ability to regulate signal transduction and calcium transients. These studies will define a mechanism of receptor mediated inhibition of signal transduction that may help to understand other types of signal inhibition.
