Cell specific processing and release of the pro‐hormone candidate tumor suppressor, Ecrg4, from the epithelial cell surface

Abstract

ObjectiveTo study the processing of human Ecrg4 in epithelial cells.MethodsPlasmids encoding human Ecrg4, Ecrg4 mutants and Ecrg4‐derived peptides fused with green fluorescent protein were transiently expressed in the prostate cancer (PC‐3) or human embryonic kidney (HEK) epithelial cells. The expression of Ecrg4 was analyzed by immunoblotting, immunohistochemistry and fluorescence and confocal microscopy. Cell surface‐bound Ecrg4 was detected by cell surface fractionation.ResultsEcrg4 can be detected in the conditioned media of PC‐3 but not HEK cells 30 hours after transient transfection of pcDNA3‐hEcrg4. In both HEK and PC3 cells however, a “secreted” 14 kDa Ecrg4 product was found tethered to the outer cell surface and could not be released by washing cells with neutral (PBS), high salt (2M NaCl), acidic (Glycine, pH 2.8) or basic (Na2CO3, pH 11) buffers. Fusion of leader sequences with GFP established that Ecrg4 retention was mediated by amino acids 1–15 of the 30 amino acid leader sequence while Ecrg4(16–30) promoted retention. Mutagenesis studies further established that processing occurred at predicted furin and thrombin consensus sites at the cell surface.ConclusionEcrg4 is cell membrane protein that can be shed into media by cell stimulation. This surface bound ECRG4 can be further processed at the furin site to release the ligand‐like peptides in conditioned media.