Identification and biological characterization of an epidermal growth factor-related protein: cripto-1

• Published in: The Journal of biological chemistry 1994-06, Vol.269 (25), p.17320-17328
• Main Authors: BRANDT, R, NORMANNO, N, KIM, N, SALOMON, D. S, GULLICK, W. J, JIING-HUEY LIN, HARKINS, R, SCHNEIDER, D, JONES, B.-W, CIARDIELLO, F, PERSICO, M. G, ARMENANTE, F
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Blotting, Western; Cell Division; CHO Cells; Cloning, Molecular; Cricetinae; DNA, Complementary - genetics; Epidermal Growth Factor; Fundamental and applied biological sciences. Psychology; Gene Expression; GPI-Linked Proteins; Growth Substances; Humans; Intercellular Signaling Peptides and Proteins; Membrane Glycoproteins; Mice; Molecular Sequence Data; Molecular Weight; Neoplasm Proteins - chemistry; Peptides - chemistry; Precipitin Tests; Protein hormones. Growth factors. Cytokines; Proteins; Radioligand Assay; Recombinant Proteins; RNA, Messenger - genetics
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)32557-7

The human and mouse cripto-1 (CR-1) genes can code for proteins related in structure to epidermal growth factor (EGF). A specific 36-kDa immunoreactive protein was detected by Western blot analysis in human cell lines that express CR-1 mRNA but not in cell lines that fail to express this transcript. Immunoprecipitation of GEO colon carcinoma or mouse embryonal carcinoma cells detected 27-29-kDa and 24-kDa proteins, respectively. Cell lysates and conditioned medium that were prepared from several CHO clones and were expressing either a recombinant human or mouse CR-1 cDNA contained immunospecific 27-29-kDa and 24-kDa proteins, respectively. Monensin or tunicamycin treatment resulted in a shift of the 27-29-kDa human CR-1 protein to 24 kDa and 20 kDa, respectively. The 20-kDa protein was also observed after digestion of the 27-29-kDa human CR-1 protein with N-glycosidase F. Using two CR-1 synthetic refolded peptides that correspond to the EGF-like domain of the human CR-1 sequence or conditioned medium obtained from human CR-1 expressing CHO cells, growth stimulatory activity could be detected on non-transformed human mammary epithelial cells and on two human breast cancer cell lines. EGF receptor-blocking antibody did not inhibit the growth stimulatory action of the CR-1 protein. Likewise, the CR-1 refolded peptides or conditioned medium from the human CR-1-expressing CHO cells failed to inhibit the binding of 125I-EGF in an EGF-radioreceptor assay. These data demonstrate that the CR-1 is a glycoprotein that can function as a growth factor through an EGF receptor-independent pathway.