Neuregulin-4: a novel growth factor that acts through the ErbB-4 receptor tyrosine kinase

Abstract

The ErbB/HER family of receptor tyrosine kinases consists of four receptors that bind a large number of growth factor ligands sharing an epidermal growth factor-(EGF)-like motif. Whereas ErbB-1 binds seven di erent ligands whose prototype is EGF, the three families of neuregulins (NRGs) activate ErbB-3 and/or ErbB-4. Here we characterize a fourth neuregulin, NRG-4, that acts through ErbB-4. The predicted pro-NRG-4 is a transmembrane protein carrying a unique EGF-like motif and a short cytoplasmic domain. A synthetic peptide encompassing the full-length EGF-like domain can induce growth of interleukin-dependent cells ectopically expressing ErbB-4, but not cells expressing the other three ErbB proteins or their combinations. Consistent with speci®city to ErbB-4, NRG-4 can displace an ErbB-4-bound NRG-1 and can activate signaling downstream of this receptor. Expression of NRG-4 mRNA was detected in the adult pancreas and weakly in muscle; other tissues displayed no detectable NRG-4 mRNA. The primary structure and the pattern of expression of NRG-4, together with the strict speci®city of this growth factor to ErbB-4, suggest a physiological role distinct from that of the known ErbB ligands.

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

“…In previous work on NRG4 expression in breast cancer, we observed using reverse transcription (RT)-PCR additional NRG4 mRNA products (Dunn et al, 2004). Here we characterize two of these variants which both posses transmembrane sequences; the previously described NRG4 variant described by Harari et al (1999) which we designate NRG4A1 and a novel 270 bp transcript designated as NRG4A2.…”

“…It is unclear therefore how NRG4 is inserted into the membrane and how the ectodomain is shed (as is the case for some NRG1 isoforms, Wakatsuki et al, 2004) to produce an ErbB4 receptoractivating ligand. Harari et al (1999) who reported the first sequence of NRG4 showed that a refolded peptide representing the EGF domain of mouse NRG4 stimulated the phosphorylation of HER4 and the downstream second messenger protein mitogen-activated protein kinase.Extensive alternative mRNA splicing and the use of at least six sites of initiation of transcription in NRG1 allows the production of more than 15 variants (Harrison and Law, 2006). NRG2 is also alternatively spliced to produce a and b variants (Yamada et al, 2000), and recently isoforms of NRG3 have been described (Carteron et al, 2006).…”

“…It is unclear therefore how NRG4 is inserted into the membrane and how the ectodomain is shed (as is the case for some NRG1 isoforms, Wakatsuki et al, 2004) to produce an ErbB4 receptoractivating ligand. Harari et al (1999) who reported the first sequence of NRG4 showed that a refolded peptide representing the EGF domain of mouse NRG4 stimulated the phosphorylation of HER4 and the downstream second messenger protein mitogen-activated protein kinase.…”

“…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 proteolytic site.…”