Dierential expression of NDF/neuregulin receptors ErbB-3 and ErbB-4 and involvement in inhibition of neuronal dierentiation Ronit Pinkas-Kramarski 1 , Raya Eilam 2 , Iris Alroy 1 , Gil Levkowitz 1 , Peter Lonai 3 and Yosef Yarden 1 Departments of 1 Molecular Cell Biology, 2 Neurobiology and 3 Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel Two receptor tyrosine kinases, ErB-3 and ErbB-4, mediate signaling by Neu dierentiation factors (NDFs, also called neuregulins), while ErbB-1 and ErbB-2 serve as co-receptors. We show that the two NDF/neuregulin receptors dier in spatial and temporal expression patterns: The kinase-defective receptor, ErbB-3, is expressed primarily in epithelial layers of various organs, in the peripheral nervous system, and in adult brain, whereas ErbB-4 is restricted to the developing central nervous system and to the embryonic heart. An example of alternating expression of the two receptors is provided by the developing cerebellum: During postnatal cerebellar development, ErbB-4 expression slightly decreases along with a decline in NDF transcription, whereas ErbB-3 expression commences after the peak of neurogenesis. To study functional dierences, we established primary brain cultures and found that ErbB-3 was expressed only in oligodendrocytes, whereas ErbB-4 expression was shared by oligodendrocytes, astrocytes and neurons. Blocking the action of endogenous NDF in vitro, by using a soluble form of ErbB-4, accelerated neurite outgrowth in both primary cultures and in neuronal-type cultures of the P19 teratocarcinoma, suggesting an inhibitory eect of NDF on neural dierentiation. Apparently, ErbB-3 is associated with proliferation of P19 cells, whereas ErbB- 4 correlates with a dierentiated phenotype. We conclude that the two NDF receptors play distinct, rather than redundant, developmental and physiological roles. Keywords: EGF family; signal transduction; tyrosine kinase; NDF/neuregulin; nervous system Introduction The NDF/neuregulin family includes more than a dozen growth and dierentiation factors that share an epidermal growth factor- (EGF-) like motif, serving as the receptor binding domain (Peles and Yarden, 1993; Carraway and Burden, 1995). Various neuregulin isoforms were discovered by dierent bioassays that re¯ect the multiple physiological functions of this polypeptide factor family. Thus, the ability of NDFs to stimulate tyrosine phosphorylation of a breast tumor protein, ErbB-2, and control dierentiation or proliferation of mammary cells, led to the isolation of the Neu dierentiation factor (NDF) (Peles et al., 1992; Wen et al., 1992) and heregulin (Holmes et al., 1992). A strong mitogenic activity on Schwann cells enabled isolation of the glial growth factors (GGFs) (March- ionni et al., 1993), whereas the ability of another neuregulin isoform to elevate expression of the nicotinic acetylcholine receptor in muscle cells was utilized in isolating ARIA (Falls et al., 1993). The availability of recombinant forms of NDF/neuregulin opened the way for more detailed in vitro and in vivo studies, collectively characterizing two major groups of biological roles for NDFs/neuregulins. First, the factors appear to act as potent inducers of epithelial morphogenesis and dierentiation. An example is provided by the mammary gland, where NDF acts as as inducer of dierentiation in vitro (Bacus et al., 1993) and aects lobulo-alveolar budding and milk produc- tion in isolated glands (Yang et al., 1995). Likewise, dierent isoforms of NDF can extend cell survival or act as potent mitogens for cultured keratinocytes (Marikovsky et al., 1995), while a speci®c isoform mediates directional migration and epidermal differ- entiation during cutaneous wound repair in vivo (Danilenko et al., 1995). Apart from their action as short-range paracrine mediators of mesenchyme-epithel inductive processes (Pinkas-Kramarski et al., 1997), NDFs play a pivotal role in the nervous system, primarily in communicating nerve cells to non-neuronal cells. Neuregulins act as the most potent mitogens for precursor Schwann cells in vitro (Dong et al., 1995), and they reduce apoptosis of mature Schwann cells upon axotomy (Grinspan et al., 1996), probably re¯ecting a role in attaining the appropriate ratio of neurons to Schwann cells in adults (Syroid et al., 1996). An in vivo eect on synaptic Schwann cell survival after denervation has been recently demonstrated (Trachtenberg and Thomp- son, 1996). Myelin-forming cells of the central nervous system, the oligodendrocytes, are also targets of NDFs/ neuregulins (Vartanian et al., 1994; Canoll et al., 1996), as are embryonic astroglia, whose maturation and survival are aected, but not their proliferation (Pinkas-Kramarski et al., 1994). An instructive signal of neuregulin for glial dierentiation, on the expense of a neuronal fate, was demonstrated by using neural crest stem cells (Shah et al., 1994). Part of the physiological complexity of the NDF/ neuregulin family may be explained by its unique combinatorial signaling. Two distinct receptor tyrosine kinases, ErbB-3 and ErbB-4, mediate NDF actions by acting as a low and a high anity receptor, respectively (Tzahar et al., 1994). Two other members of the ErbB family, ErbB-1 (also called EGF-receptor) and ErbB-2, function as co-receptors, whose recruitment into heterodimers is hierarchically controlled by NDF Correspondence: Y Yarden Received 4 June 1997; revised 1 August 1997; accepted 1 August 1997 Oncogene (1997) 15, 2803±2815  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00