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Biosynthetic processing of neu differentiation factor. Glycosylation trafficking, and regulated cleavage from the
T L Burgess1, S L Ross, Y X Qian
1Department of Mammalian Cell Molecular Biology, Amgen Inc., Thousand Oaks, California 91320-1789, USA.
Abstract:
new differentiation factor (NDF), also known as heregulin, is structurally related to the epidermal growth factor family of growth factors; it stimulates tyrosine phosphorylation of the neu/HER-2 oncogene and causes differentiation of certain human breast cancer cell lines. Alternative splicing of a single gene gives rise to multiple isoforms of NDF/heregulin, as well as the neuronal homologues, designated ARIA (acetylcholine receptor inducing activity) and GGF (glial growth factor); at least 15 structural variants are known. All but two of the NDF/heregulin cDNAs are predicted to encode transmembrane, glycosylated precursors of soluble NDF. In this report we characterized the biosynthetic processing of different NDF isoforms in stably transfected Chinese hamster ovary cells expressing individual NDF isoforms, and in the native cell line Rat 1-EJ, which expresses at least six different NDF isoforms. We found that the precursors for NDF undergo typical glycosylation and trafficking. A portion of the molecules are proteolytically cleaved intracellularly leading to the constitutive secretion of soluble, mature NDF into the culture media. However, a significant portion of the newly synthesized NDF precursor molecules escape intracellular cleavage and are transported to the cell surface of both transfected and native cells, where they reside as full-length, transmembrane proteins. Finally we show that these full-length, transmembrane NDF molecules can undergo phorbol ester regulated cleavage from the membrane, releasing the soluble growth factor into the medium.
Insights
New differentiation factor (NDF), also known as heregulin, is processed into soluble forms and also remains as transmembrane proteins. Phorbol ester treatment releases the membrane-bound NDF, indicating regulated secretion. This impacts breast cancer cell differentiation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- New differentiation factor (NDF), or heregulin, is structurally related to epidermal growth factor family.
- NDF stimulates tyrosine phosphorylation of the neu/HER-2 oncogene and induces differentiation in human breast cancer cell lines.
- Alternative splicing generates multiple NDF/heregulin isoforms and neuronal homologues (ARIA, GGF), with at least 15 known variants.
Purpose of the Study:
- To characterize the biosynthetic processing of different NDF isoforms.
- To investigate NDF processing in stably transfected Chinese hamster ovary cells and the native Rat 1-EJ cell line.
- To determine the fate of NDF precursors, including secretion and cell surface expression.
Main Methods:
- Stable transfection of Chinese hamster ovary cells with individual NDF isoforms.
- Analysis of NDF processing in the native Rat 1-EJ cell line.
- Characterization of glycosylation, trafficking, and proteolytic cleavage of NDF precursors.
Main Results:
- NDF precursors undergo typical glycosylation and trafficking.
- Intracellular cleavage results in constitutive secretion of soluble, mature NDF.
- A significant portion of NDF precursors remain as full-length transmembrane proteins on the cell surface.
- Phorbol ester treatment induces regulated cleavage of transmembrane NDF, releasing soluble growth factor.
Conclusions:
- NDF exists in both secreted and transmembrane forms due to differential processing.
- Transmembrane NDF can be released as a soluble factor via regulated cleavage.
- These findings provide insights into the complex regulation of NDF/heregulin signaling in breast cancer and neuronal systems.