Defining the interactome of the pancreas-specific SPCA2 isoform (SPCA2C) identifies unique links to store-operated Ca2+ entry.

Abstract

Calcium (Ca²⁺) is critical for normal cell function, and several protein networks are required for Ca²⁺ signaling. In the pancreas, regulated changes in cytosolic Ca²⁺ allow for the exocytosis of zymogen granules, and altered Ca²⁺ signaling underlies pancreatic pathologies. Previously, our laboratory showed a pancreas-specific isoform of secretory pathway Ca²⁺-ATPase 2 (also known as calcium-transporting ATPase type 2C member 2; SPCA2), termed SPCA2C (gene name Atp2c2c, C-terminally truncated form), affects multiple pathways involved in Ca²⁺ homeostasis. The goal of this study was to define the SPCA2C interactome that contributes to these processes. Using proximity-dependent biotin identification, BioID, we expressed SPCA2C-BirA*^HA in HEK293 cells with constitutive calcium release-activated calcium channel protein 1 (Orai1) expression. A total of 150 high-confidence interacting proteins for SPCA2C were identified. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analyses supported the localization of SPCA2C to the endoplasmic reticulum, and that it functions in Ca²⁺ signaling and vesicular transport. Stromal interaction molecule 1 (STIM1) and coiled-coil domain-containing protein 47 (CCDC47; also known as PAT complex subunit CCDC47) were among several proteins we confirmed as strong interactors through co-immunoprecipitation (co-IP). Co-expression of Atp2c2c and CCDC47 in HEK-Orai1^YFP cells increased store-operated calcium entry (SOCE) and resting cytosolic Ca²⁺ levels compared with the expression of either protein alone. We further teased out the domain determinants of CCDC47 interactions with SPCA2C, STIM1, and Orai1 using co-IP and co-localization experiments. CCDC47 localized with SPCA2C and the key store-operated Ca²⁺ entry mediators STIM1 and Orai1 but did not interact with the long SPCA2 isoform. These interactions were dependent on the presence of the CCDC47 coiled-coil or accessible transmembrane domains. Overall, we define several previously unknown protein interactions for SPCA2C, and suggest that CCDC47 may be involved in the coiled-coil interplay that underlies STIM1 and Orai1-mediated Ca²⁺ entry.