Grant: Discovery Grants Program - Individual

Exocrine cells possess signaling networks allowing external cues to rapidly generate functional outcomes. These networks typically rely on 2nd messengers such as calcium (Ca2+). Proper Ca2+ localization is required for such diverse functions as transcription, protein folding and exocytosis, but high Ca2+ levels promote cell death. Therefore, Ca2+ levels and locations must be tightly regulated. The long-term goal of my NSERC- funded program is to identify and characterize proteins regulating processes involved in exocrine cell function. With NSERC funding we identified and characterized SPCA2C, an exocrine-specific isoform of secretory pathway Ca2+ATPase (SPCA2) transcribed from the last 4 exons of Atp2c2, the gene encoding SPCA2. We showed SPCA2C over-expression increased store-operated and store-independent Ca2+ entry (SOCE and SICE) and recently defined the SPCA2C interactome, identifying >150 putative binding partners. The short-term goals for the next five years will be to determine how these interactions affect acinar/exocrine cell function. We hypothesize that SPCA2C interacts with proteins that directly affect both Ca2+ and non-Ca2+ signaling pathways to promote acinar cell function. To address this hypothesis, we have 3 objectives: OBJECTIVE 1: Determine how SPCA2C-CCDC47 interactions affect Ca2+ homeostasis. CCDC47 (coiled-coiled domain containing 47) was identified as a SPCA2C-interacting protein. We mutated the CCDC47 cytosolic domain and showed SPCA2C interaction occurs through the transmembrane domain. We will now characterize CCDC47's luminal domain and the importance of Ca2+ for these interactions. We will target residues within the EF hand domains (required for Ca2+ binding) and determine how this affects CCDC47 localization and interactions with SPCA2C, ORAI1 and STIM1. OBJECTIVE 2: Characterization of Atp2c2?24 mice. We generated a targeted deletion of exon 24 of Atp2c2 in mouse embryos (Atp2c2?24) which abolishes SPCA2C expression and greatly reduced full length SPCA2 expression as well. To examine SPCA2C's role in exocrine cells, we will analyze acinar cell morphology, SPCA2C interacting proteins and Ca2+ signaling pathways in Atp2c2?24 pancreatic tissue. OBJECTIVE 3: Characterize exocytosis and enzyme secretion in the absence of SPCA2C. Preliminary data suggests interactions between SPCA2C and RAB1A, RAB8A and STX5, and STRING analysis of the SPCA2C interactome identified several functional pathways with roles in exocrine cell biology such as vesicle-mediated trafficking. Using similar strategies as described for CCDC47, we will define how SPCA2C interacts with these proteins and whether exocytosis is altered in Atp2c2?24 mice. These experiments will provide novel insight into the fundamental mechanisms regulating exocytosis in non-excitatory cells. While the initial experiments focus on pancreatic acinar cells, Atp2c2?24 mice provide an excellent opportunity to understand its function in other secretory cells as well.