The carboxyl-terminal valine residues of proTGF alpha are required for its efficient maturation and intracellular routing.
Soluble forms of transforming growth factor-alpha (TGF alpha) are derived by proteolytic processing of an integral membrane glycoprotein precursor (pro TGF alpha). Previous studies indicated that phorbol ester-induced cleavage of pro TGF alpha in CHO cells is dependent on the presence of a valine residue located at the carboxyl terminus of the precursor's cytoplasmic domain. We reassessed this requirement with epitope-tagged constructs introduced into transformed rat liver epithelial cells that normally express and process TGF alpha. We found that pro TGF alpha mutants lacking the terminal valine residues showed greatly reduced maturation to the fully glycosylated form. Additionally, they were present at substantially reduced levels on the cell surface and, instead, accumulated in the endoplasmic reticulum. Consistent with these results, enzyme-linked immunosorbent assay (ELISA) and Western blot analyses revealed little or no soluble TGF alpha in medium conditioned by cells expressing the mutant constructs. Finally, a truncated pro TGF alpha mutant lacking most of the cytoplasmic domain but retaining a carboxyl-terminal valine was processed and cleaved in a near-normal manner. These results, some of which were reproduced in CHO cells, indicate that the predominant effect of the carboxyl-terminal valines is to ensure normal maturation and routing of the precursor.
Briley
G P
GP
University of North Carolina, Lineberger Comprehensive Cancer Center 27599-7295, USA.
Hissong
M A
MA
Chiu
M L
ML
Lee
D C
DC
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells / Q000502:physiology; D018929:Cell Culture Techniques; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D004848:Epithelium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008099:Liver / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D016211:Transforming Growth Factor alpha / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D014633:Valine / Q000502:physiology*
D016211:Transforming Growth Factor alpha; D014633:Valine