Regulation of acinar cell function

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Acinar cells produce, store and secrete enzymes through a process known as regulated exocytosis, which involves spatial and temporal Ca2+ release in response to stimulation. Control of Ca2+ concentrations ([Ca2+]) is also essential for gene transcription and protein processing. Improper [Ca2+] alters gene expression, affects protein processing and promotes apoptosis. Recently, we identified a novel isoform of Secretory Pathway Ca2+ATPase 2 (SPCA2) termed SPCA2C, which aligns with the carboxy terminus of the longer SPCA2 protein, but lacks the ATPase domain. Over-expression of SPCA2C increases ER and cytosolic [Ca2+] and affects G protein coupled receptor (GPCR) signalling and Store-Operated Ca2+ Entry (SOCE). SOCE is activated in response to low [Ca2+] in the ER, which activates STIM and ORAI channels to transport Ca2+ into the ER from outside the cell. The short term goals for the next cycle of this research program are to (1) determine mechanisms by which SPCA2C affects Ca2+ homeostasis in acinar and non-acinar cells, and (2) identify proteins that interact with SPCA2C to regulate its function.***Governing hypothesis: SPCA2C is an important regulator of Ca2+ homeostasis essential for acinar cell function.***AIMS:***(1) Identify Ca2+-signaling pathways affected by SPCA2C. Tagged SPCA2C (SPCA2CFLAG) will be expressed in HEK293 or HEK293 cells expressing YFP-tagged Orai1 (HEK293-Orai1YFP). GPCR signalling, SOCE and Store Independent Ca2+ Entry (SICE) will be examined. Fura2 imaging, co-IF, and confocal microscopy will assess cytosolic free Ca2+ concentration ([Ca2+]i) and protein localization. Pharmacological inhibitors of SOCE and GPCR signaling will determine SPCA2C's role in these pathways.***(2) Identify the role of SPCA2C on acinar cell biology. We have developed adenoviral vectors for expression of epitope-tagged SPCA2C in acinar cells cultures or in vivo through intraductal injection. We will assess acinar cell morphology, [Ca2+]i, gene expression and exocytosis ability following SPCA2C over-expression to determine its importance to these functions in acinar cells.***(3) Define the protein interactors for SPCA2C. We will perform an unbiased screen using BioID followed by mass spectrometry to identify interacting proteins for SPCA2C. We will use co-IP and Lumiere assays to validate interacting partners for SPCA2C in HEK293-Orai1YFP cells and acinar cells. Identified proteins will be characterized as described in Aims 1 and 2, These experiments will define SPCA2C interacting proteins which may also mediate [Ca2+]i.***Characterizing SPCA2C will address our Long Term Goals to (a) delineate mechanisms by which diverse Ca2+-regulating signaling pathways are coordinated to maintain acinar function and respond to environmental cues, and (b) define how changes to these pathways affect critical cell activities including transcription, protein folding and exocytosis.