Análise da localização nuclear do receptor de inositol 1,4,5-trifosfato isoforma III (ITPR3) em células tumorais e não tumorais

Abstract

The cell nucleus has the same calcium signaling machinery as the cytoplasm, and Ca2+ in the nucleus regulates distinct processes such as gene expression and cell proliferation. In the nucleus, we found inositol 1,4,5-triphosphate receptor isoform 3 (ITPR3), but its nuclear localization and how this receptor reaches the nucleus has never been demonstrated in detail. The aim of this study was to demonstrate the nuclear expression of ITPR3 in tumor and non-tumor cells and to investigate the role of the C-terminal domain in its nuclear localization. Initially, we used the GEPIA2 database to verify the overexpression of ITPR3 in different tumor types, mainly in the gastrointestinal tract. To investigate the location of the receptor in cells, proteomics and Western Blot assays were performed, showing that the receptor is expressed in both the cytoplasm and nucleus of MDCK, U2OS, and MzChA-1 cells. Immunofluorescence assays were performed on these three cell lines, confirming the results of previous studies. Immunofluorescence quantification demonstrated that the three cell lines showed no difference in receptor expression in the cytoplasm, but the tumor cell lines showed higher receptor expression in the nucleus than the non-tumor line. To understand the mechanism by which ITPR3 is localized in the nucleus, we performed in silico prediction of a possible nuclear localization signal (NLS) in the C-terminal region. Three constructs were made with the last two transmembrane domains: (1) the complete C-terminal portion, (2) the C-terminal portion without the last 38 amino acids, and (3) the C-terminal portion modified with four alanine residues. U2OS cells were transfected with these constructs and visualized by immunofluorescence. The mean intensity of ITPR3 in the nucleus and contact points between ITPR3 and lamina B or Hoechst labeling were quantified. All constructs were expressed in the nucleus. However, removal of the last 38 amino acids from the C-terminal portion increased the expression of the construct in the nucleoplasm. Finally, to verify the possibility of the construct forming tetramers as the complete receptor, prediction was carried out using AlphaFold2, suggesting that hydrogen bonds occur between Glu291 and Lys293 in the C-terminal portions of the construct. In conclusion, this is the first work that confirms, using different techniques, the nuclear localization of ITPR3, both in the nuclear envelope and in the nucleoplasm. In tumor cells, ITPR3 is more highly expressed in the nucleus than in the cytoplasm, enabling new ways to understand the molecular dynamics of this receptor and its relationship with cancer development. Furthermore, we demonstrated that the C-terminal portion of the receptor is expressed in the nucleus, and that the last 38 amino acids do not seem to be decisive in guaranteeing the expression of the receptor in this cellular compartment.