Characterization of the cell membrane-associated products of the Neuregulin 4 gene.
The NRG4 gene is a member of a family of four genes that encode a class of epidermal growth factors. This gene has been reported to express a protein designated here as NRG4A1. We describe here a novel splice variant of the NRG4 gene, NRG4A2, which encodes a C-terminal region containing a predicted type I PDZ-binding peptide. Both NRG4A1 and NRG4A2 were shown to be expressed on the cell surface, as expected by the presence of a predicted transmembrane sequence, and were modified at a single N-linked glycosylation site in the extracellular domain. Significant stabilization of expression of both proteins was seen in the presence of the proteosome inhibitor MG-132 suggesting that they are normally degraded by this system. N-terminal cleavage was inhibited in both isotypes by the broad-spectrum matrix metalloproteinase inhibitor, galardin (GM 6001). A glycosylated, secreted form of NRG4A1 was detected in the cell medium which showed biological activity in two assays, phosphorylation of the HER4 receptor and stimulation of neurite formation in PC-12 cells stably expressing HER4. Transfection and expression of green fluorescent protein-tagged proteins and immunofluorescent staining with specific anti-peptide antibodies showed that NRG4A1 is localized to membrane ruffles, while NRG4A2 has a more punctate membrane distribution.
Cancer Biology Laboratory, Research School of Biosciences, University of Kent, Canterbury, UK.
N V L
NV
Hayes
R J
RJ
Newsam
A J
AJ
Baines
W J
WJ
Gullick
D001483:Base Sequence; D001943:Breast Neoplasms / Q000473:pathology; D002462:Cell Membrane / Q000737:chemistry; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D020880:Neuregulins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D066247:Receptor, ErbB-4; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D014407:Tumor Cells, Cultured
D004151:Dipeptides; D004791:Enzyme Inhibitors; D007976:Leupeptins; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D020880:Neuregulins; D020033:Protein Isoforms; C467747:neuregulin-4; C587689:ERBB4 protein, human; D066246:ErbB Receptors; D066247:Receptor, ErbB-4; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde