Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells.
In addition to the mature 50 amino acid transforming growth factor alpha (TGF alpha), some transformed cells appear to produce multiple higher molecular weight forms. The structure and derivation of most of these larger soluble TGF alpha species remain to be established. We previously reported that a chemically induced rat hepatocellular carcinoma cell line, JM1, secreted acid-stable proteins which bind to epidermal growth factor receptors and stimulate DNA synthesis in primary cultures of normal adult rat hepatocytes. Purification and characterization of these hepatoma-derived growth factors have indicated their relationship to TGF alpha. Two EGF-competing activities of apparent Mr 30K and 10K were separated by gel filtration of concentrated JM1-conditioned medium and further purified by ion-exchange chromatography and reverse-phase HPLC. Both growth factors were detected by a radioimmunoassay specific for TGF alpha. Western blotting with antibodies to the 50 amino acid TGF alpha revealed that the lower molecular weight factor comigrated with the synthetic 6-kDa rat TGF alpha. The higher molecular weight TGF alpha appeared on immunoblots as a diffuse band of 18-21 kDa, which converted to the mature 6-kDa form upon digestion with elastase, confirming a precursor-product relationship. However, the 18-21-kDa proteins did not react with antibodies directed against the carboxy-terminal cytoplasmic segment of the transmembrane TGF alpha precursor. Enzymatic deglycosylation of the 18-21-kDa TGF alpha species by sequential removal of sialic acids and O- and N-linked carbohydrate reduced the molecular weight to 11K. The size and soluble nature of this polypeptide suggest that it represents the extracellular domain of the transmembrane TGF alpha precursor.(ABSTRACT TRUNCATED AT 250 WORDS)
Luetteke
N C
NC
Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710.
Michalopoulos
G K
GK
Teixidó
J
J
Gilmore
R
R
Massagué
J
J
Lee
D C
DC
D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D008099:Liver / Q000378:metabolism*; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011916:Rats, Inbred F344; D015291:Transforming Growth Factors / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D012333:RNA, Messenger; D015291:Transforming Growth Factors