Purification and characterization of a novel growth factor from human breast cancer cells.
We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.
Lupu
R
R
Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, Washington, DC 20007.
Wellstein
A
A
Sheridan
J
J
Ennis
B W
BW
Zugmaier
G
G
Katz
D
D
Lippman
M E
ME
Dickson
R B
RB
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001943:Breast Neoplasms; D002448:Cell Adhesion; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D006023:Glycoproteins / Q000302:isolation & purification* / Q000494:pharmacology; D006133:Growth Substances / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010449:Peptide Mapping; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011863:Radioimmunoassay; D011869:Radioligand Assay; D011960:Receptors, Estrogen / Q000032:analysis; D016211:Transforming Growth Factor alpha / Q000302:isolation & purification* / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D006133:Growth Substances; D011960:Receptors, Estrogen; C092466:TGF-alpha-like 30-kDa glycoprotein, human; D016211:Transforming Growth Factor alpha; D014415:Tunicamycin; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases