Heregulin-mediated activation of particular erbB receptor combinations was used as a model system to investigate the interaction of erbB3 and erbB4 with the adaptor protein growth factor receptor-bound (Grb)7. In human breast cancer cell lines, co-immunoprecipitation of Grb7 with both receptors was detected upon heregulin stimulation. This association was direct and mediated by the Grb7 Src homology (SH)2 domain. Coexpression of erbB2 with erbB3 point mutants was used to map Grb7 binding sites. This demonstrated that tyrosine 1180 and 1243 represent the major and minor sites of Grb7 interaction, respectively. Although these recognition sequences possess an Asn residue at ؉2 relative to the phosphotyrosine and therefore represent potential Grb2 binding sites, phosphopeptide competition and "pull-down" experiments demonstrated that they interact preferentially with the Grb7 versus the Grb2 SH2 domain. Substitution analysis indicated that an Arg residue at ؉3 could act as a selectivity determinant, but the effect was context-dependent. Consequently, the Grb2 and Grb7 SH2 domains possess overlapping, but distinct, specificities. These studies therefore identify Grb7 as an in vivo target of erbB3 and erbB4 and provide an underlying mechanism for the ability of erbB3 to recruit Grb7 and not Grb2, a property unique among erbB receptors.Recently it has become evident that a complex series of interactions governs signaling by the erbB family of receptor tyrosine kinases. This family currently contains four members, the epidermal growth factor receptor (EGFR) 1 or erbB1, erbB2, erbB3, and erbB4, which differ both qualitatively and quantitatively in their signaling potential (1-3) and biological activities (4 -7). A variety of ligands exhibit distinct specificities for one or more of these receptors. For example, the EGFR possesses multiple ligands, including EGF, transforming growth factor-␣, amphiregulin, and betacellulin, but betacellulin also binds erbB4 (8), and both erbB3 and erbB4 provide receptors for the heregulin/neuregulin 1 and neuregulin 2 families of ligands (9 -16). Furthermore, ligand-induced erbB receptor heterodimerization, first detected between the EGFR and erbB2 (17,18), diversifies the signals that can be generated by particular ligands (6,19,20). Importantly, this occurs in a hierarchical fashion and also with directionality (20). In particular, erbB2-containing heterodimers are preferred, and this receptor favors interaction with erbB3. Moreover, heterodimerization is critical for the activity of the erbB3 receptor because it is kinase-impaired and hence reliant on transphosphorylation by other receptors for signal generation (2, 21-23).Dimerization of erbB receptors leads to kinase activation and phosphorylation of their cytoplasmic domains on specific tyrosine residues, thus creating binding sites for signaling molecules containing phosphotyrosine binding or Src homology (SH) 2 domains (24). The latter are conserved noncatalytic regions of approximately 100 amino acids which, along with other modu...
Members of the erbB family of receptor tyrosine kinases are commonly overexpressed in human breast cancer. However, the relative contribution of particular signalling pathways activated downstream of these receptors to the mitogenic response of transformed breast epithelial cells remains poorly characterized. Administration of heregulin-b2 (HRG), a ligand for erbB3 and erbB4, to growth arrested T-47D human breast cancer cells leads to activation of both the PI3-kinase and MAP kinase signalling pathways and potent stimulation of cell cycle progression. Speci®c inhibitors were used to assess the role of these pathways in HRG-induced mitogenesis and to identify underlying mechanisms in terms of regulation of gene expression. Treatment with the MEK inhibitor PD98059 led to a complete block of HRG-induced entry into S-phase, whilst administration of the PI3-kinase inhibitor wortmannin resulted in only modest inhibition. In addition, administration of PD98059 8 h after HRG was equipotent with simultaneous administration in inhibiting entry into S-phase. However, delaying addition for 14 ± 16 h after HRG, when the cells were entering Sphase, was without e ect. HRG stimulation led to sequential induction of c-myc, cyclin D1, cyclin E and cyclin A gene expression and hyperphosphorylation of the retinoblastoma protein pRB. p21 (WAF1/CIP1/ SDI1) gene expression was rapidly induced by HRG, but signi®cant changes in p27 (KIP1) protein levels were not detected. Preincubation with PD98059 blocked the HRG-dependent induction of cyclin D1 mRNA, p21 and c-Myc protein and pRB phosphorylation. These ®ndings demonstrate that MEK activation is critical to HRGinduced S-phase entry in these cells whilst PI3-kinase plays a minor role. Moreover, these data are compatible with HRG-induced activation of MEK being critical for a mid-G1 transition point and implicate c-myc and cyclin D1 as key targets of the MAP kinase pathway involved in this response.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.