Conflicting results concerning the ability of the epidermal growth factor (EGF) receptor to associate with and/or activate phosphatidylinositol (Ptdlns) 3-kinase have been published. Despite the ability of EGF to stimulate the production of Ptdlns 3-kinase products and to cause the appearance of PtdIns 3-kinase activity in antiphosphotyrosine immunoprecipitates in several cell lines, we did not detect EGF-stimulated Ptdlns 3-kinase activity in anti-EGF receptor immunoprecipitates. This result is consistent with the lack of a phosphorylated Tyr-X-X-Met motif, the p85 Src homology 2 (SH2) domain recognition sequence, in this receptor sequence. The EGF receptor homolog, ErbB2 protein, also lacks this motif. However, the ErbB3 protein has seven repeats of the Tyr-X-X-Met motif in the carboxy-terminal unique domain. Here we show that in A431 cells, which express both the EGF receptor and ErbB3, Ptdlns 3-kinase coprecipitates with the ErbB3 protein (pl80eR3) in response to EGF. p180B3 is also shown to be tyrosine phosphorylated in response to EGF. In contrast, a different mechanism for the activation of Ptdlns 3-kinase in response to EGF occurs in certain cells (PC12 and A549 cells). Thus, we show for the first time that ErbB3 can mediate EGF responses in cells expressing both ErbB3 and the EGF receptor.

c‐erbB‐3 is a member of the type I (EGF receptor‐related) family of growth factor receptors for which no ligand has been identified. To facilitate ligand stimulation we have constructed a chimeric receptor which possesses an activatable kinase and promotes the growth of NIH 3T3 fibroblasts. In this study we have shown that SHC and phosphatidylinositol 3′‐kinase bind to the activated EGF receptor/c‐erbB‐3 chimera. Whereas p85 is not phosphorylated to a significant extent, SHC appears to be a major substrate for phosphorylation on tyrosine. In contrast to EGF receptor and c‐erbB‐2, we were unable to detect binding of activated c‐erbB‐3 to GRB2. Using synthetic peptides corresponding to each of 13 potential phosphorylation sites on c‐erbB‐3, we have shown that tyrosine 1309 is responsible for SHC binding. Peptides containing the motif YXXM inhibit p85 association. By comparison with recently reported SHC binding sites on Middle T antigen and Trk we have identified a SHC binding motif, NPXY.

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Summary Abnormalities of the EGF receptor and/or the related ERBB2 receptor occur in a significant proportion of cases of human breast cancer and are important influences in the behaviour of this tumour type.In this report we demonstrate by nucleic acid analysis and immunohistochemistry that the recently recognised third member of this gene Costa et al., 1988). Overexpression of the ERBB2 protein is also a marker of poor prognosis for node-positive disease (Slamon et al., 1987) and probably also for node-negative disease (Perren, 1991). More exciting, however, is the accumulating evidence that the ERBB2 receptor protein has potential as a target for antibody-directed therapy (Shepard et al., 1991) and other approaches such as inhibition of receptor dimerisation and tyrosine kinase function (Gullick 1990b Thirty six biopsy samples of non-neoplastic breast tissue

A mutant epidermal growth factor receptor (⌬EGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG.⌬EGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated ⌬EGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTPbound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.⌬EGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of ⌬EGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

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Thyroid-stimulating hormone stimulates proliferation through both the cAMP-dependent protein kinase and Ras but not through Raf-1 and mitogen-activated and extracellular signal-related kinase kinase. We now report that thyroid-stimulating hormone represses mitogen-activated protein kinase activity and that microinjection of an effector domain mutant Ha-Ras protein, Ras(12V,37G), defective in Raf-1 binding and mitogenactivated protein kinase activation, stimulates DNA synthesis in quiescent and thyroid-stimulating hormonetreated thyrocytes. A yeast two-hybrid screen identified RalGDS as a Ras(12V,37G) binding protein and therefore a potential effector of Ras in these cells. Associations between Ras and RalGDS were observed in extracts prepared from thyroid cells. Microinjection of a mutant RalA(28N) protein thought to sequester RalGDS family members reduced DNA synthesis stimulated by Ras as well as cAMP-mediated DNA synthesis in two cell lines which respond to cAMP with mitogenesis. These results support the idea that RalGDS may be an effector of Ras in cAMP-mediated growth stimulation.The elucidation of the critical role played by Ras in growth control initiated an intensive effort to identify Ras binding effector molecules. The first Ras binding protein identified was p120GAP (1), which functions both as an effector and downregulator of Ras. The best characterized Ras effector is the cytoplasmic serine/threonine protein kinase Raf-1 (2, 3). Direct interaction between the N-terminal domain of Raf-1 and the effector loop of Ras was first demonstrated using the yeast two-hybrid system (4, 5), while later studies reported the coprecipitation of Ras and Raf (6, 7). A number of other proteins bind directly to GTP-bound Ras, including the p110 catalytic subunit of phosphatidylinositol 3-kinase (8), AF6 (9), Rin-1 (10), NF1 (11), MEKK1 (12), protein kinase C (13), a herpesvirus-encoded protein (14), and a guanine nucleotide dissociation stimulator for the Ras-related protein Ral (RalGDS) and a closely related protein (RGL) (15-18). While interaction of Ras with Raf-1, p110 (8), and RalGDS (19) results in increased enzyme activity, the biological outcomes of these other Rasprotein interactions are less understood. TSH 1 -stimulated DNA synthesis requires both the cAMP-dependent protein kinase and Ras (20). Although Ras-dependent, mitogenic signaling induced by TSH does not require Raf-1 or MEK (21), suggesting that Ras utilizes alternate effectors in the presence of cAMP. To identify such effectors, an extensive two-hybrid screen of a thyroid cell cDNA library was performed with an effector domain mutant of Ras which does not bind Raf-1 (22) but stimulates DNA synthesis in thyrocytes. This screen repeatedly identified RalGDS as a potential Ras effector. Consistent with such a role, interaction between Ras and RalGDS was detected in extracts prepared from thyroid cells. Further, microinjection of a mutant RalA(28N) protein thought to sequester RalGDS (19, 23) specifically reduced cAMP-mediated DNA synthesis in both rat ...

Using recombinant talin polypeptides and an SDS/PAGE-blot overlay assay, we have previously identified three regions of talin that are involved in binding to vinculin [Gilmore, Wood, Ohanian, Jackson, Patel, Rees, Hynes and Critchley (1993) J. Cell Biol. 122, 337-347]. We have confirmed these observations by using a yeast two-hybrid assay and shown that talin residues 498-656, 852-950 and 1929-2029 are each capable of binding to vinculin residues 1-258. We have further defined the three vinculin-binding sites in talin to residues 607-636, 852-876 and 1944-1969; alignment of these sequences shows 59% similarity, although there are only two identical residues. Predictions of secondary structure indicate that this vinculin-binding motif forms an amphipathic alpha-helix. The hydrophobic face of helix 607-636 contains three aligned leucines (residues 608, 615 and 622), which show conservative substitutions in the other two sites. To test the possibility that this might constitute a leucine zipper involved in vinculin binding, we mutated each leucine residue to an alanine. The results showed that this leucine repeat is not essential to the interaction between talin and vinculin. We also used the yeast two-hybrid system to define further the talin-binding site within vinculin residues 1-258. C-terminal deletions made in accordance with exon boundaries showed that vinculin residues 1-167 are capable of interacting with each of the three vinculin-binding sites in talin. However, all N-terminal deletions abolished binding. The results suggest that the talin-binding site in vinculin has a relatively complex fold, whereas the vinculin-binding motif in talin is contained within a short linear peptide sequence that is repeated three times in the talin rod domain.