The polarized nature of salivary gland functions requires polarized organization and function of signaling complexes. Understanding assembly of signaling proteins into complexes within cellular microdomains is the central theme of this proposal. Based on our preliminary findings of polarized expression of Ca2+ signaling proteins in secretory cells, expression of multiple P2Rs in a membrane limited manner in salivary gland cells, regulation of GPCR signaling by RGS proteins and the regulation of Icrac channels by IP3R we will test the central hypothesis by 1. Studying the spatial features of Ca2+ signaling by P2Rs and GPCR. Imaging of Fura2, BTC and Mg-Fura2 will be used to capture Ca2+ waves and gradients in SMG cells and correlate them with expression patterns of P2Rs and Ca2 about signaling complexes. Function of individual P2Rs will be probed further by characterizing ionic current activated by ATP and by partial P2R agonist and the sensitivity of these currents to P2Rs inhibitory Abs. 2. Determine interaction of RGS proteins with GPCR. This will be achieved by measuring the potency of several RGS proteins to inhibit Ca2+ signaling evoked by GPCR in SMG cells. RGS proteins specific antibodies and dominant negatives will be used to identify the active RGS proteins in SMG cells and their physiological role. Known constitutively active mutants of the alfa-1BAR will be used in an attempt to identify the site of interaction of RGS proteins with GPCR. 3. Explore Ca2+ signaling complexes at the PM/ER junction: icrac-IP3R complexes. Determine if Imin is Icrac and characterize single channel properties of Icrac in excised patches from native SMG cells. Determine whether Icrac in SMG cells is gated by the newly discovered conformational coupling mode. Study regulation of Icrac by the Homer family of scaffolding proteins in the context of their role in assembly of 1crac-IP3R and signaling complexes. I believe that the tools available in my lab for the proposed projects, the expertise of the personnel in my lab and the collaborations we established will allow us to achieve our goals and provide insights of general relevance to cell signaling and to the function of salivary gland cells.
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