The polarized nature of acinar cell function 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: a) the role of RGS proteins in Ca2+ signaling; and b) the regulation of ICRAC by IP3R we will test the hypothesis of organization of Ca2+ signaling proteins into complexes by 1. Study the role of RGS proteins in conferring signaling specificity and assembly of Ca2+ signaling complexes. This will be achieved by finding the N-terminal sequence responsible for receptor recognition. Mapping and isolation of signaling complexes with RGS box and the N-terminus. Study the role of native pancreatic RGS proteins in Ca2+ oscillations. These studies will reveal the role of RGS proteins in Ca2+ signaling and determine complexes composition by identifying known and novel proteins in the isolated complexes. 2. Explore Ca2+ signaling complexes at the PM/ER junction - Coupling through ICRAC. For that we will determine if the newly discovered miniature Ca2+ channel Imin is ICRAC and use deletion mutations to identify the domain in IP3R that regulate Imin and the ICRAC analogue Htrp3. This domain will be used as a bait to identify its binding partner in Htrp3 and maybe to Imin. These studies will assess the validity of the coupling hypothesis to capacitative Ca2+ entry and the role of PM/ER junction in assembly of Ca2+ signaling complexes. 3. SERCA3 minus/minus mice and Ca2+ signaling complexes. An alternative approach to organization of Ca2+ signaling complexes is to probe the role of SERCA3 in Ca2+ signaling. To determine the mechanism of receptor-selective impaired Ca2+ signaling in SERCA3 minus/minus cells we will compare a) the biochemical, transport and spatiotemporal properties of Ca2+ signaling b) the coupling of m3 receptors in signaling complexes and c) storage and behavior of ER Ca2+ in WT and SERCA3 minus/minus cells. These studies will provide the ultimate proof for the autonomous functioning of Ca2+ signaling complexes. 4. Prob cellular microdomains with targeted cameleons. ER-cameleon and cameleons fused to the N and C termini of VAMP2 and Syncollin will be packaged into adenoviruses and infected into acinar cells. These constructs should probe dynamics of ER Ca2+ and the [Ca2+] next to signaling complexes to determine their role in local control of [Ca2+]. I believe that the tools developed in the previous grant period, the expertise of the personnel in my lab and the collaborations we established will allow us to achieve our goals and provide a new level of understanding of Ca2+ signaling of general relevance to cell signaling and to the function of exocrine cells.
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