Epiregulin Is a Potent Pan-ErbB Ligand That Preferentially Activates Heterodimeric Receptor Complexes

Abstract

The ErbB signaling network consists of four transmembrane receptor tyrosine kinases and more than a dozen ligands sharing an epidermal growth factor (EGF) motif. The multiplicity of ErbB-specific ligands is incompletely understood in terms of signal specificity because all ErbB molecules signal through partially overlapping pathways. Here we addressed the action of epiregulin, a recently isolated ligand of ErbB-1. By employing a set of factor-dependent cell lines engineered to express individual ErbBs or their combinations, we found that epiregulin is the broadest specificity EGF-like ligand so far characterized: not only does it stimulate homodimers of both ErbB-1 and ErbB-4, it also activates all possible heterodimeric ErbB complexes. Consistent with its relaxed selectivity, epiregulin binds the various receptor combinations with an affinity that is approximately 100-fold lower than the affinity of ligands with more stringent selectivity, including EGF. Nevertheless, epiregulin's action upon most receptor combinations transmits a more potent mitogenic signal than does EGF. This remarkable discrepancy between binding affinity and bioactivity is permitted by a mechanism that prevents receptor down-regulation, and results in a weak, but prolonged, state of receptor activation.Various biological processes are controlled by intercellular interactions that are mediated by polypeptide growth factors. Examples include embryonic development, neuronal functions, hematopoiesis, and pathological situations, like wound healing and malignant transformation. The mechanism transmitting extracellular signals ultimately starts with binding of the growth factor to a cell surface receptor, that in many cases carries an intrinsic tyrosine kinase activity (1). These receptors fall into several subgroups sharing structural and functional characteristics. Each subgroup of receptors specifically recognizes a family of structurally homologous growth factors. Perhaps the most striking multiplicity of related growth factors is exemplified by the epidermal growth factor (EGF) 1 family of molecules (2). This six cysteine-containing motif of 45-60 amino acids is shared by all members of the family, and it functions as the receptor binding portion of the molecule. Currently there are four known receptors for EGF-like ligands, constituting the ErbB subgroup of receptor tyrosine kinases (also known as HER, or type I receptor tyrosine kinases (3)). Whereas ErbB-1 binds many ligands, including EGF, transforming growth factor ␣ (TGF␣), and amphiregulin, both ErbB-3 and ErbB-4 bind to a family of isoforms, collectively known as neuregulins (also called Neu differentiation factors, heregulins, glial growth factors, and acetylcholine receptor inducing activity) (4). A related group of molecules, termed NRG2, binds to the same two receptors (5-7), and a third molecule, NRG3, exclusively binds to ErbB-4 (8). Two other ligands, betacellulin (9), and the heparin-binding EGF-like growth factor (10, 11) bind to both ErbB-1 and ErbB-4. Interestingly, the m...

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

“…These observations may suggest the existence of additional, yet undiscovered ErbB proteins, serving as high a nity receptors for these low a nity ligands. Alternatively, low a nity ligands may have a di erent biological function than high a nity growth factors, as they can escape the common rapid endocytic clearance from the extracellular space (Reddy et al, 1996;Shelly et al, 1998). Alternatively, the ligand-less co-receptor of ErbB-4, namely ErbB-2 , may be more e ective in the case of low a nity ligands, such as NRG-3 and NRG-4, thus o ering a mechanism for ®ne-tuning of ErbB signaling.…”

“…This method has been used before to synthesize functionally active derivatives of other EGF-like growth factors (Barbacci et al, 1995;Lin et al, 1988;Shelly et al, 1998). Previously we have established a series of derivatives of the 32D cell line engineered to express di erent ErbB receptors or their combinations (Pinkas-Kramarski et al, 1996;Shelly et al, 1998). The myeloid 32D parental cells require cytokine stimulation, such as interleukin 3 (IL3) for their growth, and were chosen because they lack endogenous ErbB expression.…”

“…The establishment of a series of interleukin 3-(IL-3-) dependent 32D myeloid cells expressing all combinations of ErbB proteins has been described (Alimandi et al, 1997;Pinkas-Kramarski et al, 1996;Shelly et al, 1998). Cells were maintained in RPMI medium with 10% fetal bovine serum (FBS) and dilute IL3-containing conditioned medium.…”

Europium-Labeled Epidermal Growth Factor and Neurotensin: Novel Probes for Receptor-Binding Studies

“…D1 cells expressing EGF receptor (ErbB1, kindly donated by Y. Yarden, Weizmann Institute, Israel) were cultured in suspension as described (14).…”

“…Portions (100 l) of the cell suspension were incubated for 24 h in 96-well plates with the indicated concentrations of Eu-EGF or EGF. Cell survival was determined by MTT assay (14). In short, MTT (0.1 mg/ml) was added to the analyzed cells, which were incubated for 2 h at 37°C.…”