The N-terminal Domains of Neuregulin 1 Confer Signal Attenuation

Abstract

Degradation of activated ERBB receptors is an important mechanism for signal attenuation. However, compared with epidermal growth factor (EGF) receptor, the ERBB2/ERBB3 signaling pair is considered to be attenuation-deficient. The ERBB2/ERBB3 ligands of the neuregulin family rely on an EGFlike domain for signaling and are generated from larger membrane-bound precursors. In contrast to EGF, which is processed to yield a 6-kDa peptide ligand, mature neuregulins retain a variety of segments N-terminal to the EGF-like domain. Here we evaluate the role of the N-terminal domain of neuregulin 1 in signaling and turnover of ERBB2/ERBB3. Our data suggest that whereas the EGF-like domain of neuregulin 1 is required and sufficient for the formation of active receptor heterodimers, the presence of the N-terminal Ig-like domain is required for efficient signal attenuation. This manifests itself for both ERBB2 and ERBB3 but is more pronounced and coupled directly to degradation for ERBB3. When stimulated with only the EGF-like domain, ERBB3 shows degradation rates comparable with constitutive turnover, but stimulation with full-length neuregulin 1 resulted in receptor degradation at rates that are comparable with activated EGF receptor. Most of the enhancement in downregulation was maintained after replacing the Ig-like domain with a thioredoxin protein of comparable size but different amino acid composition, suggesting that the physical presence but not specific properties of the Ig-like domain are needed. This sequence-independent effect of the N-terminal domain correlates with an enhanced ability of full-size neuregulin 1 to disrupt higher order oligomers of the ERBB3 extracellular domains in vitro.

Geldanamycin selectively targets the nascent form of ERBB3 for degradation

“…Cells were treated with 3 µM GA or DMSO as a vehicle control for 0-15 h and lysed at indicated time points. Samples were resolved by SDS-PAGE and immunoblotted using antibodies to the C terminus of ERBB3 (C17) or ERBB2 (C18) cycloheximide alone provides an estimate for the half-life of the receptors and confirms previous observations that ERBB3 does have a significantly shorter half-life compared to ERBB2 (Warren et al 2006). However, while the addition of GA significantly shortens the half-life of ERBB2, we again observed a modest increase in half-life for ERBB3.…”

The Growth Factor Receptor ERBB2 Regulates Mitochondrial Activity on a Signaling Time Scale

“…However, in contrast to MAPK inhibition, the comparable rapid increase in UCP2 levels is followed by a steady decline to ϳ20% below controls at 9 h. These data suggest that both ERBB2 and MAPK take part in the regulation of steady state levels of UCP2 protein, with comparable contributions to the short term reduction but additional signals from ERBB2 that are needed to sustain UCP2 levels long term. Consistent with the rebound that becomes apparent after 9 h of NRG treatment, sustained ligand stimulation for 48 h results in an increase in proliferation (42) as well as an increase in UCP2 protein levels (Fig. 3B).…”

“…Commercial reagents were obtained from the following sources: oligomycin and MAPK inhibitors UO126 and PD98059 (Cell Signaling), Canertinib (LC Laboratories), antimycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), dihydroethidium, and the AKT inhibitor LY294002 (Calbiochem), KCN, lipoic acid, and cycloheximide (Sigma), tetramethylrhodamine methyl ester (Invitrogen), Genipin (Wako chemicals), Lipofectamine and Alamar blue reagent (Invitrogen), and control siRNA and UCP2 siRNA (Dharmacon). Recombinant NRG␤1, benchmarked against full-length commercial Ig-NRG␤1, was produced as a thioredoxin fusion protein in Escherichia coli as described previously (42)(43)(44). The constitutively active ⌬MEK1 construct was provided by Dr. Natalie Ahn (University of Colorado) (45).…”

Role of heregulin in human cancer

“…Although the Ig-like domain is not directly involved in receptor binding and activation [3,48], this domain seems to be essential for efficient interaction of the EGF-like domain with ErbB receptors [67] as well as to be important for efficient signal attenuation [68]. Furthermore, the Ig-like domain contributes to the association of HRGs with the extracellular matrix by binding to cell surface heparan sulfate proteoglycans [69], leading to a local enrichment of the growth factor at the site of action [70].…”