title: Identification of potential tumor differentiation factor (TDF) receptor from steroid-responsive and steroid-resistant breast cancer cells.
abstract: Tumor differentiation factor (TDF) is a recently discovered protein, produced by the pituitary gland and secreted into the bloodstream. TDF and TDF-P1, a 20-amino acid peptide selected from the open reading frame of TDF, induce differentiation in human breast and prostate cancer cells but not in other cells. TDF protein has no identified site of action or receptor, and its mechanism of action is unknown. Here, we used TDF-P1 to purify and identify potential TDF receptor (TDF-R) candidates from MCF7 steroid-responsive breast cancer cells and non-breast HeLa cancerous cells using affinity purification chromatography (AP), and mass spectrometry (MS). We identified four candidate proteins from the 70-kDa heat shock protein (HSP70) family in MCF7 cells. Experiments in non-breast HeLa cancerous cells did not identify any TDF-R candidates. AP and MS experiments were validated by AP and Western blotting (WB). We additionally looked for TDF-R in steroid-resistant BT-549 cells and human dermal fibroblasts (HDF-a) using AP and WB. TDF-P1 interacts with potential TDF-R candidates from MCF7 and BT-549 breast cells but not from HeLa or HDF-a cells. Immunofluorescence (IF) experiments identified GRP78, a TDF-R candidate, at the cell surface of MCF7, BT-549 breast cells, and HeLa cells but not HDF-a cells. IF of other HSP70 proteins demonstrated labeling on all four cell types. These results point toward GRP78 and HSP70 proteins as strong TDF-R candidates and suggest that TDF interacts with its receptor, exclusively on breast cells, through a steroid-independent pathway.
authors: Department of Chemistry and Biomolecular Science, Biochemistry and Proteomics Group, Clarkson University, Potsdam, New York 13699-5810, USA.
authors: Izabela
authors: I
authors: Sokolowska
authors: Alisa G
authors: AG
authors: Woods
authors: Mary Ann
authors: MA
authors: Gawinowicz
authors: Urmi
authors: U
authors: Roy
authors: Costel C
authors: CC
authors: Darie
mesh_terms: D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009363:Neoplasm Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D018000:Receptors, Peptide / Q000819:agonists / Q000235:genetics / Q000378:metabolism*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; D018840:HSP70 Heat-Shock Proteins; C000717247:HSPA5 protein, human; D006360:Heat-Shock Proteins; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins; C483315:PDF protein, human; D010455:Peptides; D018000:Receptors, Peptide