Francoise Bellot

Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.

The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin‐like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2‐15) x 10(‐11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor‐receptor interactions.

Egf binding to its receptor triggers a rapid tyrosine phosphorylation of the erbB-2 protein in the mammary tumor cell line SK-BR-3.

The epidermal growth factor receptor (EGF‐R) and the erbB‐2 proto‐oncogene product protein are closely related by their structural homology and their shared enzymatic activity as autophosphorylating tyrosine kinases. We show that in mammary tumor cells (SK‐BR‐3) EGF causes a rapid increase in tyrosine phosphorylation of the erbB‐2 protein. Phosphorylation of erbB‐2 does not occur in cells lacking the EGF‐R (MDA‐MB‐453). Phosphorylation of erbB‐2 in SK‐BR‐3 cells is blocked if EGF is prevented from interacting with its receptor by specific monoclonal antibodies. While EGF induces the down‐regulation of its receptor in SK‐BR‐3 cells, EGF has no effect on the stability of the erbB‐2 protein. This result suggests that the erbB‐2 protein is a substrate of the EGF‐R and indicates the possibility of communication between these two proteins early in the signal transduction process.

Ligand-induced transphosphorylation between different FGF receptors.

Recent evidence shows that different fibroblast growth factors (FGF) bind with similar high affinities to two FGF receptors (FGFR) called flg and bek. In order to explore the mechanism of FGFR tyrosine autophosphorylation, we have generated cell lines which co‐express a kinase‐negative mutant of FGFR and an active form of FGFR. The following transfected NIH 3T3 cells were generated: (i) cells which express a shorter truncated form of bek (two Ig domains) together with a kinase‐negative mutant of full length bek (bek K517A), (ii) cells which express wild‐type bek together with kinase‐negative flg (flg K514A) and (iii) cells co‐expressing wild‐type flg together with bek K517A. Immunoprecipitations with either bek‐or flg‐specific antisera followed by immunoblotting indicated that the double transfectants express the desired receptor species. The addition of acidic FGF (aFGF) to the various cell lines followed by immunoprecipitation with anti‐FGFR antibodies and immunoblotting with anti‐phosphotyrosine specific antibodies indicated that aFGF induces tyrosine phosphorylation of the kinase‐negative FGFR mutants. These results show that tyrosine autophosphorylation of the kinase‐negative FGFR is mediated by a transphosphorylation mechanism and that both homologous (bek‐‐‐‐bek) and heterologous (bek‐‐‐‐flg and flg‐‐‐‐bek) transphosphorylation occurs in living cells. Recent evidence shows that tyrosine autophosphorylation of receptors with tyrosine kinase activities is essential for mediating interactions with signaling molecules. Therefore, heterologous transphosphorylation could amplify the response of cells to various forms of FGFs and their cognate receptors.

Functional analysis of the ligand binding site of EGF-receptor utilizing chimeric chicken/human receptor molecules.

The epidermal growth factor (EGF)‐receptor is composed of an extracellular ligand‐binding region connected by a single transmembrane region to the cytoplasmic kinase domain. In spite of its importance for understanding signal transduction, the ligand‐binding domain of the EGF‐receptor is not yet defined. We describe the identification of a major ligand‐binding domain of the EGF‐receptor by utilizing chimeras between the human EGF‐receptor and the chicken EGF‐receptor. This approach is based on the fact that murine EGF binds to the chicken EGF‐receptor with 100‐fold lower affinity as compared to the human EGF‐receptor. Hence, the substitution of various domains of the chicken EGF‐receptor by domains of the human EGF‐receptor may restore the higher binding affinity towards EGF, characteristic of the human receptor. We show that chimeric chicken/human EGF‐receptor, which contains domain III of the extracellular region of the human receptor, behaves like the human EGF‐receptor with respect to EGF binding affinity and biological responsiveness. However, a chimeric chicken/human EGF‐receptor containing domains I and II of the human receptor behaves like the chicken rather than the human EGF‐receptor. Moreover, two different monoclonal antibodies which compete for the binding of EGF to EGF‐receptor recognize specifically domain III of the human EGF‐receptor. It is concluded that domain III which is flanked by the two cysteine‐rich domains is a major ligand‐binding domain of the EGF‐receptor.

Chicken epidermal growth factor (EGF) receptor: cDNA cloning, expression in mouse cells, and differential binding of EGF and transforming growth factor alpha.

The primary structure of the chicken epidermal growth factor (EGF) receptor was deduced from the sequence of a cDNA clone containing the complete coding sequence and shown to be highly homologous to the human EGF receptor. NIH-3T3 cells devoid of endogenous EGF receptor were transfected with the appropriate cDNA constructs and shown to express either chicken or human EGF receptors. Like the human EGF receptor, the chicken EGF receptor is a glycoprotein with an apparent molecular weight of 170,000. Murine EGF bound to the chicken receptor with approximately 100-fold lower affinity than to the human receptor molecule. Surprisingly, human transforming growth factor alpha (TGF-alpha) bound equally well or even better to the chicken EGF receptor than to the human EGF receptor. Moreover, TGF-alpha stimulated DNA synthesis 100-fold better than did EGF in NIH 3T3 cells that expressed the chicken EGF receptor. The differential binding and potency of mammalian EGF and TGF-alpha by the avian EGF receptor contrasts with the similar affinities of the mammalian receptor for the two growth factors.

Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor.

Epidermal growth factor (EGF) treatment of NIH 3T3 cells transfected with wild-type EGF receptor induced tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma). The EGF receptor and PLC-gamma were found to be physically associated such that antibodies directed against PLC-gamma or the EGF receptor coimmunoprecipitated both proteins. The association between PLC-gamma and wild-type EGF receptor was dependent on the concentration of EGF, but EGF did not enhance the association between PLC-gamma and a kinase-negative mutant of the EGF receptor. Oligomerization of the EGF receptor was not sufficient to induce association of the EGF receptor with PLC-gamma, since the kinase-negative mutant receptor underwent normal dimerization in response to EGF yet did not associate with PLC-gamma. The form of PLC-gamma associated with the EGF receptor appeared to be primarily the non-tyrosine-phosphorylated form. It is concluded that the kinase activity of the EGF receptor is essential for association of PLC-gamma with the EGF receptor, possibly by stimulating receptor autophosphorylation.

Localization of a major receptor-binding domain for epidermal growth factor by affinity labeling

Epidermal growth factor (EGF) receptor was affinity labeled with 125I-labeled EGF, using bifunctional covalent cross-linking agents. The affinity-labeled receptor was isolated and cleaved with CNBr to yield a single-labeled fragment, which was unequivocally identified by site-specific antibodies and other methods to encompass residues 294 to 543 of the EGF receptor. On the basis of amino acid sequence conservation, the extracellular portion of EGF receptor can be divided into four domains. The labeled CNBr fragment contains the entire sequence which is flanked by the two cysteine-rich domains of extracellular portion of the EGF receptor denoted as domain III. On the basis of these and other results, we propose that domain III contributes most of the interactions that define ligand-binding specificity of the EGF receptor.

Biological activities of EGF-receptor mutants with individually altered autophosphorylation sites.

In vitro site‐directed mutagenesis was used to replace individually the three known autophosphorylation sites of the epidermal growth factor (EGF)‐receptor (i.e. Tyr1173, Tyr1148 and Tyr1068) by phenylalanine, a residue which cannot serve as a phosphate acceptor site. In another mutant, Tyr1173 was substituted by a serine residue. The cDNA constructs encoding either mutant or wild‐type EGF‐receptors were transfected into NIH‐3T3 cells devoid of endogenous EGF‐receptors. The mutant receptors were expressed on the cell surface and displayed typical high‐ and low‐affinity binding sites for [125I]EGF. Phorbol ester (PMA) modulated the binding affinity of wild‐type and mutant receptors in a similar manner. Mutant EGF‐receptors exhibited EGF‐dependent tyrosine kinase activity leading to self‐phosphorylation and phosphorylation of exogenous substrates both in vitro and in living cells. The internalization and degradation of EGF‐receptors were not affected by the mutations. Cells expressing mutant EGF‐receptors became mitogenically responsive to EGF, indicating that none of the vital functions of the EGF‐receptor were critically impaired by the loss of individual autophosphorylation sites. Maximal mitogenic stimulation correlated with the number of wild‐type or mutant receptors per cell, highly expressing cells showing higher maximal stimulation. However, the dose‐response curves of cells expressing mutant receptors were slightly shifted to lower concentrations of EGF, rendering the cells mitogenically responsive to lower doses of EGF than cells expressing normal EGF‐receptor at similar expression levels. Basal [3H]thymidine incorporation in the presence of 0.5% calf serum was consistently higher for cells expressing mutant receptors, while the response to stimulation with 10% calf serum was not affected.

Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek.

Heparin potentiates the mitogenic activity of acidic fibroblast growth factor (aFGF) by 20-100 fold but mechanisms detailing this potentiation have not yet been fully elucidated. We report that heparin increases the binding affinity of aFGF for the two cloned and overexpressed human FGF receptors, flg and bek, by 2-3 fold. This increase in binding affinity, together with previous data demonstrating a 3-5 fold increase in the stability of aFGF, are likely to account for a significant portion of heparins potentiation of aFGF activity observed in biological assay systems.