Efficient coupling with phosphatidylinositol 3-kinase, but not phospholipase C gamma or GTPase-activating protein, distinguishes ErbB-3 signaling from that of other ErbB/EGFR family members.

Abstract

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

c-erbB3 and c-erbB4 expression is a feature of the endocrine responsive phenotype in clinical breast cancer

“…Indeed, high concentrations of c-erbB3/c-erbB4 ligands, may even result in a parallel inhibition of cellular proliferation (Bacus et al, 1992;Grunt et al, 1995). Although there is clearly substantial overlap of the expression of individual erbB family members within the breast cancer population, the di erential e ects of c-erbB3/cerbB4 and the EGFR may result from the ability of cerbB3 to e ciently recruit PI3 Kinase (Solto et al, 1994;Fedi et al, 1994;Prigent and Gullick, 1994) and hence pro®ciently activate additional signal transduction pathways to those employed by EGFR. Relative availabilities of ligands, receptors and secondary pathways would appear to critically alter the proliferation/di erentiation status of breast tumours.…”

Effects of oncogenic ErbB2 on G1 cell cycle regulators in breast tumour cells

“…However, the phosphotyrosine content of ErbB3 decreased in parallel with the loss of ErbB2 from the plasma membrane ( Figure 2b, lower panel). Compared with the other ErbB receptors, the cytoplasmic tail of ErbB3 contains more potential docking sites for the p85 subunit of PI3K (Fedi et al, 1994;Prigent and Gullick, 1994), allowing it to couple e ciently to this kinase . A GST pull-down assay using the SH2 domain of p85, showed that signi®cantly less ErbB3 was precipitated after 72 h scFv-5R induction ( Figure 2c, lane 3).…”

Immune responses to all ErbB family receptors detectable in serum of cancer patients

“…These transfectants have previously been characterized and are known to express individual ErbB receptors in the range of 0.6 ± 3610 6 molecules/cell (Di Fiore et al, 1987b;Kraus et al, 1993;Fedi et al, 1994;. A recombinant model system expressing high receptor levels was utilized, to facilitate sensitive and speci®c detection of single ErbB family members, as frequent co-expression of ErbB receptors in human tumor cell lines may confound de®nition of a receptorspeci®c response.…”

“…A recombinant model system expressing high receptor levels was utilized, to facilitate sensitive and speci®c detection of single ErbB family members, as frequent co-expression of ErbB receptors in human tumor cell lines may confound de®nition of a receptorspeci®c response. Figure 1 illustrates detection of EGFR, ErbB2, ErbB3 or ErbB4 in the 170-185 kDa region of the corresponding transfectant by receptorspeci®c polyclonal rabbit antisera raised against carboxyl-terminal epitopes of single ErbB receptors (Fedi et al, 1994;Alimandi et al, 1995;Kraus et al, 1993;Baulida et al, 1996). Overexpression of the respective recombinant human ErbB receptor was validated in LTR-EGFR, LTR-ErbB2, LTR-ErbB3, or LTR-ErbB4 when compared with NIH3T3 controls (Figure 1).…”

“…NIH3T3 cells transfected with expression vectors for human coding sequences of ErbB family receptors have previously been described including LTR-EGFR (Di Fiore et al, 1987a;Fedi et al, 1994), LTRErbB2 (Di Fiore et al, 1987b), LTR-ErbB3 (Kraus et al, 1993) and LTR-ErbB4 .…”