Characterization of the cell membrane-associated products of the Neuregulin 4 gene

Abstract

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.Oncogene ( The neuregulins (NRGs) are a family of growth factors that possess a wide range of possible activities including promoting cell growth, differentiation, migration, apoptosis and adhesion (Falls, 2003). There are four mammalian NRG genes (NRG1-4) each of which can encode a homologous epidermal growth factor (EGF)-like domain which is the minimum requirement for the stimulation of ErbB receptor tyrosine kinases. NRGs 1 and 2 interact with ErbB3 and ErbB4, while NRG3 and NRG4 are reported to bind only ErbB4 (Hobbs et al., 2002). Each NRG gene has a characteristic pattern of expression in normal tissues, NRG1-3 are all expressed in the nervous system, while NRG4 was detected in a limited number of adult tissues but not the brain (Harari et al., 1999). To date, only one product has been reported for the NRG4 gene which encodes a 115 amino acid transmembrane protein containing an EGF domain homologous to the other NRGs, but outside this region shares little sequence homology with the other NRGs (Buonanno and Fishbach, 2001). It is probable that NRG4 is synthesized as a pro-form, similar to other ErbB ligands, with which it shares structural similarity. Hydropathy plot analysis predicts a single transmembrane region between amino acids 64 and 83 and sequence analysis predicts an EGF domain at the immediate N terminus of the protein (Harari et al., 1999) which contains a single N-linked glycosylation site (NYT) located at residue 39 between the fifth and sixth cysteine, a feature found at the N terminus of the EGF domain of NRG2 (Higashiyama et al., 1997) but not in any of the other EGF receptor ligands. This is followed by a relatively short serine-rich region, representing a putative proteoly...

Brown and white adipose tissues: intrinsic differences in gene expression and response to cold exposure in mice

“…8 C ). To test whether NRG4 was secreted into the culture media and whether this had any effect in promoting neurite outgrowth, conditioned medium of mature brown adipocytes was used to treat PC12 cells stably expressing ErbB4 (25). NRG4 was detected by ELISA in conditioned medium from differentiated brown adipocytes and was undetectable in unconditioned medium and medium from preadipocytes (Fig.…”

Association between circulating neuregulin4 levels and diabetes mellitus: A meta-analysis of observational studies

“…They concluded that the theory of "Nrg4 resistance" contributes to this correlation. Besides, although Nrg4 preserves glucose homeostasis in mice hepatocytes by activation of ErBb3/ErBb4 signaling, the universal distribution characteristic of ErBb3/ErBb4 signaling may lead to a totally different biological role of Nrg4 in other sites of human body [9,32,33]. On the contrary, Wang and Zhang [9,26] argued that circulating Nrg4 levels were negatively correlated with glucose parameters.…”

Neurological dysfunctions associated with altered BACE1‐dependent Neuregulin‐1 signaling

“…The broad‐spectrum matrix metalloproteinase inhibitor, galardin (GM 6001), is capable of inhibiting Nrg4 cleavage (Hayes et al . ), although the details remain to be determined. A stretch of eleven residues spanning the BACE1 cleavage site, but not the ADAM cleavage site, is identical between Nrg1 and Nrg3; altered Nrg3 cleavage has been detected in BACE1‐null mouse brains (Hu et al .…”