Oncogene (2000) 19, 5568 ± 5573 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc Ligand discrimination by ErbB receptors: dierential signaling through dierential phosphorylation site usage Colleen Sweeney*,1 and Kermit L Carraway III1 1Department of Cell Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, Massachusetts, MA 02215, USA The four members of the ErbB family of receptor signaling through RTKs features ligand-stimulated tyrosine kinases (RTKs) mediate a variety of cellular receptor dimerization as a mechanism of kinase responses to epidermal growth factor (EGF)-like growth activation (Heldin, 1995; Weiss and Schlessinger, factors, and serve as a model for the generation of both 1998). Binding of growth factor to receptor extra- diversity and speci®city in RTK signaling. Previous cellular domains induces receptor dimerization, studies indicate that receptor ± receptor interactions bringing intracellular kinase domains into proximity. ®gure prominently in signaling through ErbB receptors. Each receptor subunit within the dimeric complex In addition to a role in receptor kinase activation, ligand- then cross-phosphorylates tyrosine residues in the induced ErbB receptor homo- and heterodimerization is activation loop (A-loop) region of the kinase domain thought to account for the diversity of biological of its neighbor, removing a physical constraint and responses stimulated by EGF-like growth factors. Since signi®cantly enhancing kinase activity (Hubbard et each receptor has the potential to couple to dierent al., 1998). Receptor subunits then cross-phosphory- complements of signaling pathways, EGF-like ligands late each other on 3 ± 6 speci®c tyrosine residues specify cellular response by dictating which pairs of responsible for the recruitment and activation of receptors become activated. More recently evidence has intracellular signaling molecules. These signaling been uncovered for ligand discrimination by individual molecules then couple activated receptors to signal ErbB receptor dimers; receptors appear to realize which transduction cascades leading to the nucleus or ligand is binding and dierentially respond through cytoskeleton, such that combinations of signaling autophosphorylation site usage. These observations events emanating from activated receptors culminate indicate that ligand stimulation of RTKs is not generic, in a cellular response. and point to another layer in the ErbB signal The recruitment of intracellular signaling proteins diversi®cation mechanism. The mechanistic implications possessing src homology-2 (SH2) or phosphotyrosine of ligand discrimination are discussed. Oncogene (2000) binding (PTB) domains to speci®c phosphorylated 19, 5568 ± 5573. tyrosine residues within activated RTKs is strictly context dependent; the 5 ± 8 amino acid residues Keywords: ErbB receptor; tyrosine kinase; EGF-like immediately surrounding the phosphotyrosine deter- ligands; ligand discrimination mine which signaling protein becomes recruited (Margolis, 1992; van der Geer and Pawson, 1995) and therefore which signaling pathway is engaged. Introduction Hence a critical determinant of signaling speci®city by RTKs is the identity of the tyrosine residues that Growth factors act on individual cells within tissues to become phosphorylated in response to growth factor signal a variety of cellular processes including pro- binding. liferation, dierentiation, migration, fate, survival or The EGF-like growth factor family, encompassing apoptosis. Many cell types are pluripotent, and can over a dozen dierent growth factor ligands, signals respond dierently depending on the identity of the through the four known RTKs of the ErbB family: growth factor presented to the cell. A particularly well- EGF receptor, ErbB2, ErbB3 and ErbB4. ErbB studied example is the PC12 pheochromacytoma cell receptor signaling is thought to contribute to a variety line, which proliferates in response to EGF but of developmental processes and oncogenic events dierentiates in response to nerve growth factor. While (Burden and Yarden, 1997), and the EGF-like ligands some studies point to a role for signaling strength in exhibit a marked range of activities toward cultured cellular response speci®cation, the mechanisms under- cells. For this reason the ErbB system has served as a lying dierential growth factor activities remain model for the generation of diversity and speci®city in unclear. RTK signaling. Examination of the tyrosine residues of Growth factors bind to and activate the kinase the intracellular domains of the ErbB receptors reveals activities of cell surface RTKs, which propagate the that each is capable of interacting with unique ligand-encoded signal across the plasma membrane complements of signaling proteins (Carraway and and translate it into a cellular response. The most Cantley, 1994; Alroy and Yarden, 1997; Olayioye et widely accepted general model for growth factor al., 2000). This, coupled with observations that the ErbB receptors undergo a variety of ligand-stimulated receptor homo- and heterodimerization events, allows *Correspondence: C Sweeney, Beth Israel Deaconess Medical for the generation of a broad range of intracellular Center, Harvard Institutes of Medicine, Room 1018, 330 Brookline signals and thus a variety of cellular responses (Riese Avenue, Boston, Massachusetts, MA 02215, USA and Stern, 1998). Ligand discrimination by ErbB receptors C Sweeney and KL Carraway III 5569 Ligand selection, heterodimerization and dierential CEM cells stably transfected with ErbB4, we observed signaling that at saturating concentrations the ligands BTC, NRG1, NRG2 and NRG3 all induced similar gross The domain structures of the ErbB receptors are typical levels of ErbB4 receptor dimerization and tyrosine of RTKs in that they possess a large extracellular ligand phosphorylation. Despite this, each of the ligands binding domain, a single transmembrane domain, and an exhibited dierent biological potencies in a cell growth intracellular portion containing tyrosine kinase and and viability assay. More importantly, the dierent autophosphorylation domains. The domain structures ligands induced the preferential recruitment of dierent of the growth factors vary considerably, but each signaling molecules to activated ErbB4, and dieren- possesses an EGF-like domain that is necessary and tially stimulated the activities of signaling kinases sucient for receptor binding and activation. Ligands downstream of activated receptors (Sweeney et al., may be subdivided into three categories based on their 2000). These observations indicate that a single RTK primary receptor binding properties (Riese and Stern, signaling species, an ErbB4 homodimer, is capable of 1998; Olayioye et al., 2000): EGF, transforming growth distinguishing between binding ligands to elicit a range factor-a (TGFa), and amphiregulin are speci®c for EGF of biochemical and biological responses. receptor, neuregulins-1 and -2 can bind to either ErbB3 One obvious mechanism by which ligands might or ErbB4, heparin binding EGF (HB-EGF), betacellulin dierentially signal through a single receptor species is (BTC) and epiregulin can bind to either EGF receptor or by inducing the dierential phosphorylation of the ErbB4, and neuregulins-3 and -4 are speci®c for ErbB4. receptor itself. That is, although each ligand induces a It has been suggested that EGF-like ligands are similar level of overall receptor tyrosine phosphoryla- bivalent, containing separate regions responsible for tion, the speci®c tyrosine residues phosphorylated binding to a primary ErbB receptor with high anity within the receptor dier with each growth factor. and narrow speci®city, and for binding to a dimerizing Phosphopeptide mapping of ErbB4 demonstrated that ErbB receptor with lower anity and broader this was indeed the case (Sweeney et al., 2000). Some speci®city (Tzahar et al., 1997). Such a mechanism phosphopeptides were induced by all four growth may account for the range of receptor dimerization factors while others were speci®c for one, two or three events observed with a single growth factor, as well as of the growth factors. A model illustrating the the preferential heterodimerization of ErbB2 with other relationship between ligand binding, ErbB4 phosphor- ligand-bound ErbB family members (Karunagaran et ylation, signaling protein recruitment and cellular al., 1996; Graus-Porta et al., 1997). On the other hand, response is illustrated in Figure 1. By virtue of their ErbB2 may intrinsically have a higher propensity to ability to potently couple the activated receptor to p85, dimerize, suggested by its stronger overexpression- the 85 kDa regulatory subunit of phosphatidylinositol induced autoactivation relative to the other ErbB 3-kinase, NRG1 and NRG2 may be suited to stimulate receptors (Lonardo et al., 1990). cellular survival or motility (Rameh and Cantley, The preferential activation of ErbB2 in response to 1999). BTC or NRG1 binding to ErbB4 may also several EGF-like growth factors, together with the promote cellular proliferation or dierentiation path- observation that ligands can selectively stimulate some ways through the preferential recruitment of the pairs of ErbB receptors over others (Pinkas-Kramarski adaptor proteins Grb2 and Shc. et al., 1996), point to the existence of a hierarchical Other observations (Crovello et al., 1998; Sweeney network of ligand-stimulated receptor dimerization and