Actin and Keratin are Binding Partners of the 1,25D₃-MARRS Receptor/PDIA3/ERp57

We have shown that the 1,25D₃-MARRS receptor is necessary for the rapid, pre-genomic effects of 1,25(OH)₂D₃ on phosphate and/or calcium absorption in chick intestines. However, a clear understanding of the proteins involved in the signaling mechanisms by which the 1,25D₃-MARRS receptor facilitates 1,25(OH)₂D₃-mediated phosphate or calcium uptake, as well as other cellular effects, is still under investigation. We used co-immunoprecipitation studies and mass spectroscopy to identify actin and keratin as proteins that interact with the 1,25D₃-MARRS receptor. Using confocal microscopy, we visualized 1,25(OH)₂D₃- MARRS receptor localizations relative to actin and/or keratin distribution in chick enterocytes. Cells cultured in media containing phenol red had the 1,25D₃-MARRS receptor and actin localized largely in the nucleus, which was dispersed upon addition of (OH)₂ 1,25(OH)₂D₃. In the absence of phenol red, staining was cytoplasmic. Addition of steroid caused diminished staining at 10 s and 30 s, with a return of intensity between 1 and 5 min. Nuclear staining was observed after 1 min. We found that F-actin concentrations are maximal when 1,25D₃-MARRS receptor localizations within enterocytes are low suggesting that cyclical conversions of F-actin to G-actin are involved in the 1,25(OH)₂D₃-mediated redistribution of the 1,25D₃-MARRS receptor within the cell. We also found that keratin distribution remains constant with 1,25(OH)₂D₃ exposure when Factin depolymerizes into G-actin, which suggests that actin and keratin work in concert to facilitate hormonemediated redistribution of the 1,25D₃-MARRS receptor. We subsequently investigated whether the cyclical redistribution was related to either 1,25(OH)₂D₃-stimulated phosphate or calcium uptake, but no congruent pattern was found.