The HER-2/neu receptor tyrosine kinase gene encodes a secreted autoinhibitor

Abstract

HER-2͞neu (erbB-2) encodes an 185-kDa orphan receptor tyrosine kinase that is constitutively active as a dimer and displays potent oncogenic activity when overexpressed. Here we describe a secreted protein of Ϸ68 kDa, designated herstatin, as the product of an alternative HER-2 transcript that retains intron 8. This alternative transcript specifies 340 residues identical to subdomains I and II from the extracellular domain of p185HER-2 followed by a unique C-terminal sequence of 79 aa encoded by intron 8. The recombinant product of the alternative transcript specifically binds to HER-2-transfected cells with a K D of Ϸ14 nM and was chemically crosslinked to p185HER-2, whereas the intron encoded sequence alone also binds with high affinity to transfected cells and associates with p185 solubilized from cell extracts. The herstatin mRNA is expressed in normal human fetal kidney and liver, but is at reduced levels relative to p185HER-2 mRNA in carcinoma cells that contain an amplified HER-2 gene. Herstatin appears to be an inhibitor of p185HER-2, because it disrupts dimers, reduces tyrosine phosphorylation of p185, and inhibits the anchorageindependent growth of transformed cells that overexpress HER-2.

HER2/Neu: mechanisms of dimerization/oligomerization

“…A truncated EGFR seen in human glioblastoma, for example, can dimerize and transform mammalian cells in a ligand-independent fashion (Moscatello et al, 1996) Homologous to mammalian erbB proteins, avian viral v-erbB truncation mutants display ligand-independent e ects, though the signi®cance of the dimerization status of these proteins is unclear (Adelsman et al, 1996). Herstatin, a recently discovered erbB-2 alternate transcript, consisting of 340 amino acids of the erbB-2 extracellular domain and 79 amino acids of novel sequence, binds full-length erbB-2, inhibiting its function (Doherty et al, 1999). Studies with the deletion mutants suggest that the extracellular domains of erbB receptor that small structural components in a subdomain may function not only to mediate dimerization in response to ligand, but also to prevent surreptitious dimerization.…”

Antagonism of the human epidermal growth factor receptor family controls disease severity in murine collagen‐induced arthritis

“…Thereafter, the tPA signal/pro region was amplified using the primers 5Ј-ATTAGAATTCCACCA-TGGAT GCAATGAAGAGAGGG-3Ј containing an Eco RI site, and 5Ј-ATTATCTAGATCTGGCTCCTCTTCTGAATC-G-3Ј containing an Xba I site. The herstatin coding region with 6 histidine N-terminal tags (14,19) was amplified (excluding the signal sequence) using the primers 5Ј-AATTTCTAGAC-AAGTGTGCACCGGCACAGAC-3Ј containing an Xba I site and 5Ј-AAGGAAAAGCGGCCGCTCAGCCTTCATACCG-GGAC-3Ј containing a Not I site. The PCR-amplified products were purified, digested with restriction enzymes, and subcloned into the pcDNA3.1 expression plasmid (Invitrogen, San Diego, CA).…”

“…All of these receptors are encoded by the Her gene family, and all except HER-3 exhibit strong tyrosine kinase activity, which, upon dimerization of the single chains on binding to specific ligand motifs, induces downstream signaling events leading to cell proliferation, division, motility, change in phenotype, production of extracellular matrix, and transcription of specific genes (12). Interestingly, an alternative splice variant of Her2, retaining intron 8, resulted in the formation of an ϳ68-kd protein, herstatin, which disrupts dimerization and thus serves as a natural inhibitor of native p185Her-2, as well as EGFR and HER-3 (14). The existence of regulatory soluble forms of naturally occurring splice variants of cell surface receptors is an interesting finding, since they could potentially serve as therapeutic tools and play a role in homeostatic regulation during health and disease states.…”

Herstatin inhibits heregulin-mediated breast cancer cell growth and overcomes tamoxifen resistance in breast cancer cells that overexpress HER-2

“…The HER-2 gene, in addition to encoding the fulllength receptor, also encodes an alternate product, herstatin (Hst), created by intron retention (Doherty et al, 1999). Hst consists of the first two subdomains of the HER-2 ectodomain and an intron-encoded C-terminus that confers binding to HER-2 itself as well as to HER-1 (Doherty et al, 1999;Azios et al, 2001).…”

“…Hst consists of the first two subdomains of the HER-2 ectodomain and an intron-encoded C-terminus that confers binding to HER-2 itself as well as to HER-1 (Doherty et al, 1999;Azios et al, 2001). The expression of Hst appears to be regulated, since it is relatively low in breast carcinoma cells with HER-2 gene amplification and is expressed at high levels in a tissuespecific manner in fetal kidney and liver (Doherty et al, 1999). Hst has been found to block HRG-mediated HER-2/HER-3 heterodimer formation in a dominantnegative manner in transiently transfected nontumorigenic cells (Azios et al, 2001).…”