Recombinant expression of a chimeric EGFR/ErbB-3 receptor in NIH 3T3 fibroblasts allowed us to investigate cytoplasmic events associated with ErbB-3 signal transduction upon ligand activation. An EGFR/ErbB-3 chimera was expressed on the surface of NIH 3T3 transfectants as two classes of receptors possessing epidermal growth factor (EGF) binding affinities comparable to those of the wild-type EGF receptor (EGFR). EGF induced autophosphorylation in vivo of the chimeric receptor and DNA synthesis of EGFRI ErbB-3 transfectants with a dose response similar to that of EGFR transfectants. However, the ErbB-3 and EGFR cytoplasmic domains exhibited striking differences in their interactions with several known tyrosine kinase substrates. We demonstrated strong association of phosphatidylinositol 3-kinase activity with the chimeric receptor upon ligand activation comparable in efficiency with that of the platelet-derived growth factor receptor, while the EGFR exhibited a 10-to 20-fold-lower efficiency in phosphatidylinositol 3-kinase recruitment. By contrast, both phospholipase Cy and GTPase-activating protein failed to associate with or be phosphorylated by the ErbB-3 cytoplasmic domain under conditions in which they coupled with the EGFR. In addition, though certain signal transmitters, including Shc and GRB2, were recruited by both kinases, EGFR and ErbB-3 elicited tyrosine phosphorylation of distinct sets of intracellular substrates. Thus, our findings show that ligand activation of the ErbB-3 kinase triggers a cytoplasmic signaling pathway that hitherto is unique within this receptor subfamily.Signal transduction by receptor tyrosine kinases (RTKs) involves tyrosine phosphorylation and/or physical association of a number of cytosolic enzymes by the activated receptor. Initially identified as immediate targets of the platelet-derived growth factor (PDGF) receptor (PDGFR) and the epidermal growth factor (EGF) receptor (EGFR), these substrates include phospholipase C-y (PLCy), GTPaseactivating protein (GAP), and phosphatidylinositol 3-kinase (PtdIns 3-kinase) (2,11,13,24, 36,39,41,58,64). Their coding sequences share regions of homology with the noncatalytic portion of c-src, termed SH2 and SH3, that are involved in the assembly of signaling complexes with the activated receptor. Thereby, SH2 domains bind to discrete phosphotyrosine (P-Tyr)-containing peptide sequences present in RTKs, whereas SH3 domains appear to associate with proline-rich motifs in proteins regulating the activity of low-molecular-mass G proteins (5,14,26,43,49,52,69

In an effort to isolate novel growth factors, we identified a human protein, designated Sk, that co-eluted with Neuregulin during chromatographic separation of conditioned medium from the SK-LMS-1 human leiomyosarcoma cell line. Degenerate oligonucleotides based on amino-terminal sequence analysis of the purified protein were used to isolate the corresponding cDNA from a library generated from this cell line. Sk is a novel 266-amino acid protein that contains a signal peptide sequence and two cysteine-rich domains with no similarity to other known growth factors. A single major 2-kilobase transcript was expressed in several embryonic tissues. Transfection of mammalian cells demonstrated that the protein was secreted and expressed as a doublet of approximately 35 kDa. In vitro translation and endoglycosylase analysis indicated that this doublet, which was also observed in cells expressing the endogenous protein, arises from posttranslational modification. A search of the GenBank TM data base revealed a match of Sk with Dkk-1, which is a novel secreted protein required for head induction in amphibian embryos and a potent Wnt inhibitor. When coexpressed with Wnt-2 in NIH3T3 cells, human Sk/Dkk-1 caused reversion of Wnt-2 induced morphological alterations and inhibited the Wnt-2 induced increase in uncomplexed ␤-catenin levels. These results provide biochemical evidence that human Sk/Dkk-1 antagonizes Wnt signaling upstream of its effect on ␤-catenin regulation.

The predicted human erbB-3 gene product is closely related to epidermal growth factor receptor (EGFR) and erbB-2, which have been implicated as oncogenes in model systems and human neoplasia. We expressed the erbB-3 coding sequence in NIH 3T3 fibroblasts and identified its product as a 180-kDa glycoprotein, gplsOerbB-3. Tunicamycin and pulsechase experiments revealed that the mature protein was processed by N-linked glycosylation of a 145-kDa erbB-3 core polypeptide. The intrinsic catalytic function of gpl8OerbB3 was shown by its ability to autophosphorylate in vitro. Liganddependent signaling of its cytoplasmic domain was established employing transfectants that express a chimeric EGFR/erbB-3 protein, gplSOI FR/erbB-3* EGF induced tyrosine phosphorylation of the chimera and promoted soft agar colony formation of such transfectants. These fimdings combined with the detection of constitutive tyrosine phosphorylation of gpl8OerbB3 in 4 of 12 human mammary tumor cell lines implicate the activated erbB-3 product in the pathogenesis of some human malignancies.

Interleukin-3 (IL-3)-dependent murine 32D cells do not detectably express epidermal growth factor receptors (EGFRs) and do not proliferate in response to EGF, heregulin (HRG) or other known EGF-like ligands. Here, we report that EGF specifically binds to and can be crosslinked to 32D transfectants co-expressing ErbB2 and ErbB3 (32D.E2/E3), but not to transfectants expressing either ErbB2 or ErbB3 individually. [125I]EGF-crosslinked species detected in 32D. E2/E3 cells were displaced by HRG and betacellulin (BTC) but not by other EGF-like ligands that were analyzed. EGF, BTC and HRG also induced receptor tyrosine phosphorylation, activation of downstream signaling molecules and proliferation of 32D.E2/E3 cells. 32D transfectants were also generated which expressed an ErbB3-EGFR chimera alone (32D.E3-E1) or in combination with ErbB2 (32D. E2/E3-E1). While HRG stimulation of 32D.E3-E1 cells resulted in DNA synthesis and receptor phosphorylation, EGF and BTC were inactive. However, EGF and BTC were as effective as HRG in mediating signaling when ErbB2 was co-expressed with the chimera in the 32D.E2/E3-E1 transfectant. These results provide evidence that ErbB2/ErbB3 binding sites for EGF and BTC are formed by a previously undescribed mechanism that requires co-expression of two distinct receptors. Additional data utilizing MDA MB134 human breast carcinoma cells, which naturally express ErbB2 and ErbB3 in the absence of EGFRs, supported the results obtained employing 32D cells and suggest that EGF and BTC may contribute to the progression of carcinomas that co-express ErbB2 and ErbB3.

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