Engineered Soluble Monomeric IgG1 CH3 Domain: GENERATION,MECHANISMS OF FUNCTION,AND IMPLICATIONS FOR DESIGN OF BIOLOGICAL THERAPEUTICS*

Most of the therapeutic antibodies approved for clinical use are full-size IgG1 molecules. The interaction of the IgG1 Fc with the neonatal Fc receptor (FcRn) plays a critical role in maintaining their long half-life. We have hypothesized that isolated Fc domains could be engineered to functionally mimic full-size IgG1 (nanoantibodies) but with decreased (10-fold) size. Here, we report for the first time the successful generation of a soluble, monomeric CH3 domain (mCH3). In contrast to the wild-type dimeric CH3, the mCH3 exhibited pH-dependent binding to FcRn similar to that of Fc. The binding free energy of mCH3 to FcRn was higher than that of isolated CH2 but lower than that of Fc. Therefore, CH3 may contribute a larger portion of the free energy of binding to FcRn than CH2. A fusion protein of mCH3 with an engineered antibody domain (m36.4) also bound to FcRn in a pH-dependent fashion and exhibited significantly higher neutralizing activity against HIV-1 than m36.4-Fc fusion proteins. The m36.4-mCH3 fusion protein was monomeric, stable, soluble, and expressed at a high level in Escherichia coli. We also found that engineering an additional disulfide bond in mCH3 remarkably increased its thermal stability, whereas the FcRn binding was not affected. These data suggest that mCH3 could not only help in the exploration of the dual mechanisms of the CH3 contribution to Fc functions (dimerization and FcRn interactions) but could also be used for the development of candidate therapeutics with optimized half-life, enhanced tissue penetration, access to sterically restricted binding sites, and increased therapeutic efficacy.     

The IgG Fc contains distinct Fc receptor (FcR) binding sites: the leukocyte receptors Fc gamma RI and Fc gamma RIIa bind to a region in the Fc distinct from that recognized by neonatal FcR and protein A

The CH2-CH3 interface of the IgG Fc domain contains the binding sites for a number of Fc receptors including Staphylococcal protein A and the neonatal Fc receptor (FcRn). It has recently been proposed that the CH2-CH3 interface also contains the principal binding site for an isoform of the low affinity IgG Fc receptor II (Fc gamma RIIb). The Fc gamma RI and Fc gamma RII binding sites have previously been mapped to the lower hinge and the adjacent surface of the CH2 domain although contributions of the CH2-CH3 interface to binding have been suggested. This study addresses the question whether the CH2-CH3 interface plays a role in the interaction of IgG with Fc gamma RI and Fc gamma RIIa. We demonstrate that recombinant soluble murine Fc gamma RI and human Fc gamma RIIa did not compete with protein A and FcRn for binding to IgG, and that the CH2-CH3 interface therefore appears not to be involved in Fc gamma RI and Fc gamma RIIa binding. The importance of the lower hinge was confirmed by introducing mutations in the proposed binding site (LL234,235AA) which abrogated binding of recombinant soluble Fc gamma RIIa to human IgG1. We conclude that the lower hinge and the adjacent region of the CH2 domain of IgG Fc is critical for the interaction between Fc gamma RIIa and human IgG, whereas contributions of the CH2-CH3 interface appear to be insignificant.     

Engineering a Monomeric Fc Domain Modality by N-Glycosylation for the Half-life Extension of Biotherapeutics

Human IgG is a bivalent molecule that has two identical Fab domains connected by a dimeric Fc domain. For therapeutic purposes, however, the bivalency of IgG and Fc fusion proteins could cause undesired properties. We therefore engineered the conversion of the natural dimeric Fc domain to a highly soluble monomer by introducing two Asn-linked glycans onto the hydrophobic C_H3-C_H3 dimer interface. The monomeric Fc (monoFc) maintained the binding affinity for neonatal Fc receptor (FcRn) in a pH-dependent manner. We solved the crystal structure of monoFc, which explains how the carbohydrates can stabilize the protein surface and provides the rationale for molecular recognition between monoFc and FcRn. The monoFc prolonged the in vivo half-life of an antibody Fab domain, and a tandem repeat of the monoFc further prolonged the half-life. This monoFc modality can be used to improve the pharmacokinetics of monomeric therapeutic proteins with an option to modulate the degree of half-life extension.     

X-ray Crystal Structures of Monomeric and Dimeric Peptide Inhibitors in Complex with the Human Neonatal Fc Receptor,FcRn

The neonatal Fc receptor, FcRn, is responsible for the long half-life of IgG molecules in vivo and is a potential therapeutic target for the treatment of autoimmune diseases. A family of peptides comprising the consensus motif GHFGGXY, where X is preferably a hydrophobic amino acid, was shown previously to inhibit the human IgG:human FcRn protein-protein interaction (Mezo, A. R., McDonnell, K. A., Tan Hehir, C. A., Low, S. C., Palombella, V. J., Stattel, J. M., Kamphaus, G. D., Fraley, C., Zhang, Y., Dumont, J. A., and Bitonti, A. J. (2008) Proc. Natl. Acad. Sci. U.S.A., 105, 2337–2342). Herein, the x-ray crystal structure of a representative monomeric peptide in complex with human FcRn was solved to 2.6 Å resolution. The structure shows that the peptide binds to human FcRn at the same general binding site as does the Fc domain of IgG. The data correlate well with structure-activity relationship data relating to how the peptide family binds to human FcRn. In addition, the x-ray crystal structure of a representative dimeric peptide in complex with human FcRn shows how the bivalent ligand can bridge two FcRn molecules, which may be relevant to the mechanism by which the dimeric peptides inhibit FcRn and increase IgG catabolism in vivo. Modeling of the peptide:FcRn structure as compared with available structural data on Fc and FcRn suggest that the His-6 and Phe-7 (peptide) partially mimic the interaction of His-310 and Ile-253 (Fc) in binding to FcRn, but using a different backbone topology.     

Design, expression and characterization of a soluble single-chain functional human neonatal Fc receptor

The neonatal Fc receptor (FcRn) is responsible for transporting maternal IgGs to fetus/newborns and maintaining the homeostasis of IgGs in adults. FcRn resembles class I major histocompatibility complex in structure, and is composed of a transmembrane heavy chain and an invariant beta 2 microglobulin. Changes in the affinity of IgGs to FcRn lead to changes in the half-life of engineered IgGs and Fc fusion proteins. Longer half-life of therapeutic antibodies means lower dose and longer interval between administering. For some diagnostic agents including imaging or radio-labeled agents a shorter half life in circulation results in lower non-specific binding and decreased side effects. Therefore, studying the interaction of FcRn and therapeutic antibodies has direct clinical implications. A reliable method to prepare soluble and functional FcRn protein is essential for such studies. In this study, we describe a new method to express in mammalian cells soluble human FcRn (sFcRn) as a single-chain soluble fusion protein. The highly hydrophilic beta 2 microglobulin was joined with the hydrophobic heavy chain via a 15 amino acid linker. The single-chain fusion protein format not only improved the expression level of the heavy chain but also simplified the purification process. The sFcRn maintained its pH-dependent binding to IgG. This method typically yielded ～1 mg/100ml culture without optimization, and is easy to scale up for production of large quantities.     

Soluble monomeric IgG1 Fc

Antibody fragments are emerging as promising biopharmaceuticals because of their relatively small size and other unique properties. However, compared with full-size antibodies, these antibody fragments lack the ability to bind the neonatal Fc receptor (FcRn) and have reduced half-lives. Fc engineered to bind antigens but preserve interactions with FcRn and Fc fused with monomeric proteins currently are being developed as candidate therapeutics with prolonged half-lives; in these and other cases, Fc is a dimer of two CH2-CH3 chains. To further reduce the size of Fc but preserve FcRn binding, we generated three human soluble monomeric IgG1 Fcs (mFcs) by using a combination of structure-based rational protein design combined with multiple screening strategies. These mFcs were highly soluble and retained binding to human FcRn comparable with that of Fc. These results provide direct experimental evidence that efficient binding to human FcRn does not require human Fc dimerization. The newly identified mFcs are promising for the development of mFc fusion proteins and for novel types of mFc-based therapeutic antibodies of small size and long half-lives.     

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen^TM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling^® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.     

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen^TM) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling^® platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.     

Interaction with Both Domain I and III of Albumin Is Required for Optimal pH-dependent Binding to the Neonatal Fc Receptor (FcRn)

Albumin is an abundant blood protein that acts as a transporter of a plethora of small molecules like fatty acids, hormones, toxins, and drugs. In addition, it has an unusual long serum half-life in humans of nearly 3 weeks, which is attributed to its interaction with the neonatal Fc receptor (FcRn). FcRn protects albumin from intracellular degradation via a pH-dependent cellular recycling mechanism. To understand how FcRn impacts the role of albumin as a distributor, it is of importance to unravel the structural mechanism that determines pH-dependent binding. Here, we show that although the C-terminal domain III (DIII) of human serum albumin (HSA) contains the principal binding site, the N-terminal domain I (DI) is important for optimal FcRn binding. Specifically, structural inspection of human FcRn (hFcRn) in complex with HSA revealed that two exposed loops of DI were in proximity with the receptor. To investigate to what extent these contacts affected hFcRn binding, we targeted selected amino acid residues of the loops by mutagenesis. Screening by in vitro interaction assays revealed that several of the engineered HSA variants showed decreased binding to hFcRn, which was also the case for two missense variants with mutations within these loops. In addition, four of the variants showed improved binding. Our findings demonstrate that both DI and DIII are required for optimal binding to FcRn, which has implications for our understanding of the FcRn-albumin relationship and how albumin acts as a distributor. Such knowledge may inspire development of novel HSA-based diagnostics and therapeutics.     

Discovery and structure–activity relationships of small molecules that block the human immunoglobulin G–human neonatal Fc receptor (hIgG–hFcRn) protein–protein interaction

The neonatal Fc receptor, FcRn, prolongs the half-life of IgG in the serum and represents a potential therapeutic target for the treatment of autoimmune disease. Small molecules that block the protein–protein interactions of human IgG–human FcRn may lower pathogenic autoantibodies and provide effective treatment. A novel class of quinoxalines has been discovered as antagonists of the IgG:FcRn protein–protein interaction through optimization of a hit derived from a virtual ligand-based screen.     

Structural Insights into Neonatal Fc Receptor-based Recycling Mechanisms

We report the three-dimensional structure of human neonatal Fc receptor (FcRn) bound concurrently to its two known ligands. More particularly, we solved the crystal structure of the complex between human FcRn, wild-type human serum albumin (HSA), and a human Fc engineered for improved pharmacokinetics properties (Fc-YTE). The crystal structure of human FcRn bound to wild-type HSA alone is also presented. HSA domain III exhibits an extensive interface of contact with FcRn, whereas domain I plays a lesser role. A molecular explanation for the HSA recycling mechanism is provided with the identification of FcRn His¹⁶¹ as the only potential direct contributor to the corresponding pH-dependent process. At last, this study also allows an accurate structural definition of residues considered for decades as important to the human IgG/FcRn interaction and reveals Fc His³¹⁰ as a significant contributor to pH-dependent binding. Finally, we explain various structural mechanisms by which several Fc mutations (including YTE) result in increased human IgG binding to FcRn. Our study provides an unprecedented relevant understanding of the molecular basis of human Fc interaction with human FcRn.     

Direct detection of the interaction between recombinant soluble extracellular regions in the heterodimeric metabotropic gamma-aminobutyric acid receptor

The gamma-aminobutyric acid, type B (GABAB) receptor is a heterodimeric receptor consisting of two complementary subunits, GABAB1 receptor (GBR1) and GABAB2 receptor (GBR2). GBR1 is responsible for GABA binding, whereas GBR2 is considered to perform a critical role in signal transduction toward downstream targets. Therefore, precise communication between GBR1 and GBR2 is thought to be essential for the proper signal transduction process. However, biochemical data describing the interaction of the two subunits, especially for the extracellular regions, are not sufficient. Thus we began by developing a protein expression system of the soluble extracellular regions. One of the soluble recombinant GBR1 proteins exhibited a ligand binding ability, which is similar to that of the full-length GBR1, and thus the ligand-binding domain was determined. Direct interaction between GBR1 and GBR2 extracellular soluble fragments was confirmed by co-expression followed by affinity column chromatography and a sucrose density gradient sedimentation. In addition, we also found homo-oligomeric states of these soluble extracellular regions. The interaction between the two soluble extracellular regions caused the enhancement of the agonist affinity for GBR1 as previously reported in a cell-based assay. These results not only open the way to future structural studies but also highlight the role of the interaction between the extracellular regions, which controls agonist affinity to the heterodimeric receptor.     

Participation of the extracellular domain in (pro)renin receptor dimerization

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin–angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD participates in dimerization.     

Stoichiometry of the interaction between the major histocompatibility complex-related Fc receptor and its Fc ligand.

The neonatal Fc receptor (FcRn) transports immunoglobulin G (IgG) across epithelia, providing passive immunity and protecting serum IgG from degradation. For both functions, FcRn binds to IgG at the acidic pH of intracellular vesicles (pH 相似文献   

Albumin binding to FcRn: distinct from the FcRn-IgG interaction

The MHC-related Fc receptor for IgG (FcRn) protects albumin and IgG from degradation by binding both proteins with high affinity at low pH in the acid endosome and diverting both from a lysosomal pathway, returning them to the extracellular compartment. Immunoblotting and surface plasmon resonance studies show that both IgG and albumin bind noncooperatively to distinct sites on FcRn, that the affinity of FcRn for albumin decreases approximately 200-fold from acidic to neutral pH, and that the FcRn-albumin interaction shows rapid association and dissociation kinetics. Isothermal titration calorimetry shows that albumin binds FcRn with a 1:1 stoichiometry and the interaction has hydrophobic features as evidenced by a large positive change in entropy upon binding. Our results suggest that the FcRn-albumin interaction has unique features distinct from FcRn-IgG binding despite the overall similarity in the pH-dependent binding mechanism by which both ligands are protected from degradation.     

Crystal structure and immunoglobulin G binding properties of the human major histocompatibility complex-related Fc receptor(,)

The neonatal Fc receptor (FcRn) performs two distinct but related functions: transport of maternal immunoglobulin G (IgG) to pre- or neonatal mammals, thus providing passive immunity, and protection of IgG from normal serum protein catabolism. FcRn is related to class I MHC proteins but lacks a functional peptide binding groove. The crystal structure of human FcRn has been determined at 2.7 A resolution and compared to the previously described structure of rat FcRn [Burmeister et al. (1994) Nature 372, 336-343] and to the structures of MHC and MHC-related proteins. Human FcRn is structurally similar to the rat receptor but does not form receptor dimers in the crystals as observed in crystals of rat FcRn. The interaction between human FcRn and IgG was characterized by determining the binding stoichiometry using equilibrium gel filtration and by deriving binding affinities for the different human IgG subclasses using a surface plasmon resonance assay. Like rat and mouse FcRn, human FcRn interacts with IgG with a 2:1 receptor:ligand stoichiometry. The binding of human FcRn to the four human IgG subclasses shows subclass and allotype variations but no clear subclass affinity differences that correlate with serum half-lives. The structure of human FcRn and studies of its ligand binding are relevant to current efforts to use FcRn-mediated regulation of IgG half-life in serum to increase the lifetimes of antibody-based therapeutics.     

Conformational Destabilization of Immunoglobulin G Increases the Low pH Binding Affinity with the Neonatal Fc Receptor

Crystallographic evidence suggests that the pH-dependent affinity of IgG molecules for the neonatal Fc receptor (FcRn) receptor primarily arises from salt bridges involving IgG histidine residues, resulting in moderate affinity at mildly acidic conditions. However, this view does not explain the diversity in affinity found in IgG variants, such as the YTE mutant (M252Y,S254T,T256E), which increases affinity to FcRn by up to 10×. Here we compare hydrogen exchange measurements at pH 7.0 and pH 5.5 with and without FcRn bound with surface plasmon resonance estimates of dissociation constants and FcRn affinity chromatography. The combination of experimental results demonstrates that differences between an IgG and its cognate YTE mutant vary with their pH-sensitive dynamics prior to binding FcRn. The conformational dynamics of these two molecules are nearly indistinguishable upon binding FcRn. We present evidence that pH-induced destabilization in the CH2/3 domain interface of IgG increases binding affinity by breaking intramolecular H-bonds and increases side-chain adaptability in sites that form intermolecular contacts with FcRn. Our results provide new insights into the mechanism of pH-dependent affinity in IgG-FcRn interactions and exemplify the important and often ignored role of intrinsic conformational dynamics in a protein ligand, to dictate affinity for biologically important receptors.     

Anti-carcinoembryonic antigen single-chain variable fragment antibody variants bind mouse and human neonatal Fc receptor with different affinities that reveal distinct cross-species differences in serum half-life

Serum half-life of IgG is controlled by the neonatal Fc receptor (FcRn) that interacts with the IgG Fc region and may be increased or decreased as a function of altered FcRn binding. Preclinical evaluations of modified IgGs are frequently carried out in mice, but such IgGs may bind differently to mouse and human FcRn (mFcRn and hFcRn). Here, we report a detailed characterization of a matched set of mouse-human chimeric T84.66 scFv-Fc variants with specificity for the tumor carcinoembryonic antigen and mutations in the FcRn-binding site. Binding to soluble mFcRn and hFcRn was measured using in vitro assays, and the results were compared with blood clearance in vivo in normal (mFcRn bearing) and hFcRn transgenic mice. All variants bound better to mFcRn than to hFcRn. The loss of affinity varied among the mutants, however, and also the hierarchy of binding differed depending on the receptor. The mutations had no major impact on binding to the classical Fcγ receptors. Importantly, the trend of blood clearance in both strains of mice correlated with the hierarchy of binding obtained using soluble FcRn. Consequently, in vitro interaction analysis of engineered IgGs regarding their cross-species FcRn binding ability provides information for prediction of in vivo pharmacokinetics.     

pH-dependent Binding Engineering Reveals an FcRn Affinity Threshold That Governs IgG Recycling

The Fc domain of IgG has been the target of multiple mutational studies aimed at altering the pH-dependent IgG/FcRn interaction to modulate IgG pharmacokinetics. These studies have yielded antibody variants with disparate pharmacokinetic characteristics, ranging from extended in vivo half-life to those exhibiting extremely rapid clearance. To better understand pH-dependent binding parameters that govern these outcomes and limit FcRn-mediated half-life extension, we generated a panel of novel Fc variants with high affinity binding at acidic pH that vary in pH 7.4 affinities and assessed pharmacokinetic outcomes. Pharmacokinetic studies in human FcRn transgenic mice and cynomolgus monkeys showed that multiple variants with increased FcRn affinities at acidic pH exhibited extended serum half-lives relative to the parental IgG. Importantly, the results reveal an underappreciated affinity threshold of neutral pH binding that determines IgG recycling efficiency. Variants with pH 7.4 FcRn affinities below this threshold recycle efficiently and can exhibit increased serum persistence. Increasing neutral pH FcRn affinity beyond this threshold reduced serum persistence by offsetting the benefits of increased pH 6.0 binding. Ultra-high affinity binding to FcRn at both acidic and neutral pH leads to rapid serum clearance.     

Soluble N-cadherin stimulates fibroblast growth factor receptor dependent neurite outgrowth and N-cadherin and the fibroblast growth factor receptor co-cluster in cells

A chimeric molecule consisting of the extracellular domain of the adhesion molecule, N-cadherin, fused to the Fc region of human IgG (NCAD-Fc) supports calcium-dependent cell adhesion and promotes neurite outgrowth following affinity-capture to a tissue culture substrate. When presented to cerebellar neurons as a soluble molecule, the NCAD-Fc stimulated neurite outgrowth in a manner equivalent to that seen for N-cadherin expressed as a cell surface glycoprotein. Neurons expressing a dominant-negative version of the fibroblast growth factor (FGF) receptor did not respond to soluble NCAD-Fc. In cells transfected with full-length N-cadherin and the FGF receptor, antibody-clustering of N-cadherin resulted in a co-clustering of the FGF receptor to discrete patches in the cell membrane. The data demonstrate that the ability of N-cadherin to stimulate neurite outgrowth can be dissociated from its ability to function as a substrate associated adhesion molecule. The N-cadherin and the FGF receptor co-clustering in cells provides a basis for the neurite outgrowth response stimulated by N-cadherin being dependent on FGF receptor function.