title: Cell-specific processing and release of the hormone-like precursor and candidate tumor suppressor gene product, Ecrg4.
abstract: The human open reading frame C2orf40 encodes esophageal cancer-related gene-4 (Ecrg4), a newly recognized neuropeptide-like precursor protein whose gene expression by cells in vitro, over-expression in mice in vivo, and knock-down in zebrafish affects cell proliferation, migration and senescence, progenitor cell survival and differentiation, and inflammatory function. Unlike traditionally secreted neuropeptide precursors, however, we find that Ecrg4 localizes to the epithelial cell surface and remains tethered after secretion. Here, we used cell surface biotinylation to establish that 14-kDa Ecrg4 localizes to the cell surface of prostate (PC3) or kidney (HEK) epithelial cells after transfection. Accordingly, this Ecrg4 is resistant to washing cells with neutral, high salt (2 M NaCl), acidic (50 mM glycine, pH 2.8), or basic (100 mM Na(2)CO(3), pH 11) buffers. Mutagenesis of Ecrg4 established that cell tethering was mediated by an NH(2)-terminus hydrophobic leader sequence that enabled both trafficking to the surface and tethering. Immunoblotting analyses, however, showed that different cells process Ecrg4 differently. Whereas PC3 cells release cell surface Ecrg4 to generate soluble Ecrg4 peptides of 6-14 kDa, HEK cells do neither, and the 14-kDa precursor resembles a sentinel attached to the cell surface. Because a phorbol ester treatment of PC3 cells stimulated Ecrg4 release from, and processing at, the cell surface, these data are consistent with a multifunctional role for Ecrg4 that is dependent on its cell of origin and the molecular form produced.
authors.affiliation: Department of Surgery, School of Medicine University of California, San Diego, San Diego, CA 92103, USA.
authors.forename: Xitong
authors.initials: X
authors.lastname: Dang
authors.forename: Sonia
authors.initials: S
authors.lastname: Podvin
authors.forename: Raul
authors.initials: R
authors.lastname: Coimbra
authors.forename: Brian
authors.initials: B
authors.lastname: Eliceiri
authors.forename: Andrew
authors.initials: A
authors.lastname: Baird
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D003470:Culture Media / Q000494:pharmacology; D057809:HEK293 Cells; D006728:Hormones / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008970:Molecular Weight; D016296:Mutagenesis / Q000187:drug effects / Q000235:genetics; D009363:Neoplasm Proteins / Q000737:chemistry / Q000378:metabolism*; D009928:Organ Specificity / Q000187:drug effects; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003470:Culture Media; C476999:ECRG4 protein, human; D006728:Hormones; D009363:Neoplasm Proteins; D011498:Protein Precursors; D025521:Tumor Suppressor Proteins; D013755:Tetradecanoylphorbol Acetate