Sensitization of cholangiocarcinoma cells to chemotherapy through BCRP inhibition with ß-caryophyllene oxide.

Cholangiocarcinomas (CCAs) are cancers originated in the biliary tree, which are characterized by their high mortality and marked chemoresistance, partly due to the activity of ATP-binding cassette (ABC) export pumps, whose inhibition has been proposed as a strategy for enhancing the response to chemotherapy. We have previously shown that ß-caryophyllene oxide (CRYO) acts as a chemosensitizer in hepatocellular carcinoma by inhibiting ABCB1, MRP1, and MRP2. Here, we have evaluated the usefulness of CRYO in inhibiting BCRP and improving the response of CCA to antitumor drugs. The TCGA-CHOL cohort (n = 36) was used for in silico analysis. BCRP expression (mRNA and protein) was assayed in samples from intrahepatic (iCCA) and extrahepatic (eCCA) tumors (n = 50) and CCA-derived cells (EGI-1 and TFK-1). In these cells, BCRP-dependent mitoxantrone transport was determined by flow cytometry. At non-toxic concentrations, CRYO inhibited BCRP function, which enhanced the cytostatic effect of drugs used in the treatment of CCA. The BCRP ability to confer resistance to a panel of antitumor drugs was determined in Chinese hamster ovary (CHO) cells with stable BCRP expression. At non-toxic concentrations, CRYO markedly reduced BCRP-induced resistance to known substrate drugs (mitoxantrone and SN-38) and cisplatin, gemcitabine, sorafenib, and 5-FU but not oxaliplatin. Neither CRYO nor cisplatin alone significantly affected the growth of BCRP-expressing tumors subcutaneously implanted in immunodeficient mice. In contrast, intratumor drug content was enhanced when administered together, and tumor growth was inhibited. In sum, the combined treatment of drugs exported by BCRP with CRYO can improve the response to chemotherapy in CCA patients.

3D Spheroid Primary Human Hepatocytes for Prediction of Cytochrome P450 and Drug Transporter Induction.

Primary human hepatocytes (PHHs) have been the gold standard in vitro model for the human liver and are crucial to predict hepatic drug-drug interactions. The aim of this work was to assess the utility of 3D spheroid PHHs to study induction of important cytochrome P450 (CYP) enzymes and drug transporters. The 3D spheroid PHHs from three different donors were treated for 4 days with rifampicin, dicloxacillin, flucloxacillin, phenobarbital, carbamazepine, efavirenz, omeprazole, or ß-naphthoflavone. Induction of CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, and transporters P-glycoprotein (P-gp)/ABCB1, multidrug resistance-associated protein 2 (MRP2)/ABCC2, ABCG2, organic cation transporter 1 (OCT1)/SLC22A1, SLC22A7, SLCO1B1, and SLCO1B3 were evaluated at mRNA and protein levels. Enzyme activity of CYP3A4, CYP2B6, CYP2C19, and CYP2D6 were also assessed. Induction of CYP3A4 protein and mRNA correlated well for all donors and compounds and had a maximal induction of five- to sixfold for rifampicin, which closely correlates to induction observed in clinical studies. Rifampicin induced the mRNA of CYP2B6 and CYP2C8 by 9- and 12-fold, whereas the protein levels of these CYPs reached 2- and 3-fold induction, respectively. Rifampicin induced CYP2C9 protein by 1.4-fold, whereas the induction of CYP2C9 mRNA was over 2-fold in all donors. Rifampicin induced ABCB1, ABCC2, and ABCG2 by 2-fold. In conclusion, 3D spheroid PHHs is a valid model to investigate mRNA and protein induction of hepatic drug-metabolizing enzymes and transporters, and this model provides a solid basis to study induction of CYPs and transporters, which translates to clinical relevance.

Organic Cation Transporter I and Na+ /taurocholate Co-Transporting Polypeptide are Involved in Retrorsine- and Senecionine-Induced Hepatotoxicity in HepaRG cells.

SCOPE: 1,2-unsaturated pyrrolizidine alkaloids (PAs) are secondary plant metabolites that are found in many plant species throughout the world. They are of concern for risk assessment as consumption of contaminated foodstuff can cause severe liver damage. Of late, transporter-mediated uptake and transport has advanced as a vital determinant of PA toxicity. In this study, the authors investigate a transporter-mediated uptake of PAs and its implications in PA toxicity. METHODS AND RESULTS: We show that transporter expression levels are significantly affected by treatment with the PAs senecionine (Sc) and retrorsine (Re) in the human hepatoma cell line HepaRG. Furthermore, the specific contribution to PA uptake of the two transporters Na+ /taurocholate co-transporting polypeptide (SLC10A1) and organic cation transporter I (SLC22A1), both belonging to the heterogeneous solute carrier super family, is investigated by means of a siRNA-mediated knockdown approach. Knockdown of both uptake transporters result in reduced uptake of Re and Sc in a time-dependent manner and attenuated PA-mediated cytotoxic effects in HepaRG cells. CONCLUSION: Our results confirm previous findings of active transport mechanisms of PAs into hepatocytes and highlight the importance of toxicokinetic studies for the risk assessment of PAs.

A multiplexed, automated immuno-matrix assisted laser desorption/ionization mass spectrometry assay for simultaneous and precise quantitation of PTEN and p110α in cell lines and tumor tissues.

The PI3-kinase/AKT/mTOR pathway plays a central role in cancer signaling. While p110α is the catalytic α-subunit of PI3-kinase and a major drug target, PTEN is the main negative regulator of the PI3-kinase/AKT/mTOR pathway. PTEN is often down-regulated in cancer, and there are conflicting data on PTEN's role as breast cancer biomarker. PTEN and p110α protein expression in tumors is commonly analyzed by immunohistochemistry, which suffers from poor multiplexing capacity, poor standardization, and antibody crossreactivity, and which provides only semi-quantitative data. Here, we present an automated, and standardized immuno-matrix-assisted laser desorption/ionization mass spectrometry (iMALDI) assay that allows precise and multiplexed quantitation of PTEN and p110α concentrations, without the limitations of immunohistochemistry. Our iMALDI assay only requires a low-cost benchtop MALDI-TOF mass spectrometer, which simplifies clinical translation. We validated our assay's precision and accuracy, with simultaneous enrichment of both target proteins not significantly affecting the precision and accuracy of the quantitation when compared to the PTEN- and p110α-singleplex iMALDI assays (<15% difference). The multiplexed assay's linear range is from 0.6-20 fmol with accuracies of 90-112% for both target proteins, and the assay is free of matrix-related interferences. The inter-day reproducibility over 5-days was high, with an overall CV of 9%. PTEN and p110α protein concentrations can be quantified down to 1.4 fmol and 0.6 fmol per 10 µg of total tumor protein, respectively, in various tumor tissue samples, including fresh-frozen breast tumors and colorectal cancer liver metastases, and patient-derived xenograft (PDX) tumors.

An eight-compound mixture but not corresponding concentrations of individual chemicals induces triglyceride accumulation in human liver cells.

In real life, organisms are exposed to complex mixtures of chemicals at low concentration levels, whereas research on toxicological effects is mostly focused on single compounds at comparably high doses. Mixture effects deviating from the assumption of additivity, especially synergistic effects, are of concern. In an adverse outcome pathway (AOP)-guided manner, we analyzed the accumulation of triglycerides in human HepaRG liver cells by a mixture of eight steatotic chemicals (amiodarone, benzoic acid, cyproconazole, flusilazole, imazalil, prochloraz, propiconazole and tebuconazole), each present below its individual effect concentration at 1-3 µM. Pronounced and significantly enhanced triglyceride accumulation was observed with the mixture, and similar effects were seen at the level of pregnane-X-receptor activation, a molecular initiating event leading to hepatic steatosis. Transcript pattern analysis indicated subtle pro-steatotic changes at low compound concentrations, which did not exert measurable effects on cellular triglycerides. Mathematical modeling of mixture effects indicated potentially more than additive behavior using a model for compounds with similar modes of action. The present data underline the usefulness of AOP-guided in vitro testing for the identification of mixture effects and highlight the need for further research on chemical mixtures and harmonization of data interpretation of mixture effects.

Cross-species analysis of hepatic cytochrome P450 and transport protein expression.

Most drugs and xenobiotics are metabolized in the liver. Amongst others, different cytochrome P450 (CYP) enzymes catalyze the metabolic conversion of foreign compounds, and various transport proteins are engaged in the excretion of metabolites from the hepatocytes. Inter-species and inter-individual differences in the hepatic levels and activities of drug-metabolizing enzymes and transporters result from genetic as well as from environmental factors, and play a decisive role in determining the pharmacokinetic properties of a compound in a given test system. To allow for a meaningful comparison of results from metabolism studies, it is, therefore, of utmost importance to know about the specific metabolic properties of the test systems, especially about the levels of metabolic enzymes such as the CYPs. Using a targeted proteomics approach, we, therefore, compared the hepatic levels of important CYP enzymes and transporters in different experimental systems in vivo and in vitro, namely Wistar rats, C57/Bl6 mice, mice humanized for the two xeno-sensing receptors PXR (pregnane-X-receptor) and CAR (constitutive androstane receptor), mice with human hepatocyte-repopulated livers, human HepaRG hepatocarcinoma cells, primary human hepatocytes, and human liver biopsies. In addition, the effects of xenobiotic inducers of drug metabolism on CYP enzymes and transporters were analyzed in selected systems. This study for the first time presents a comprehensive overview of similarities and differences in important drug metabolism-related proteins among the different experimental models.

Impact of iron oxide nanoparticles on xenobiotic metabolism in HepaRG cells.

Iron oxide nanoparticles are used in various industrial fields, as a tool in biomedicine as well as in food colorants, and can therefore reach human metabolism via oral uptake or injection. However, their effects on the human body, especially the liver as one of the first target organs is still under elucidation. Here, we studied the influence of different representative iron oxide materials on xenobiotic metabolism of HepaRG cells. These included four iron oxide nanoparticles, one commercially available yellow food pigment (E172), and non-particulate ionic control FeSO4. The nanoparticles had different chemical and crystalline structures and differed in size and shape and were used at a concentration of 50 µg Fe/mL. We found that various CYP enzymes were downregulated by some but not all iron oxide nanoparticles, with the Fe3O4-particle, both γ-Fe2O3-particles, and FeSO4 exhibiting the strongest effects, the yellow food pigment E172 showing a minor effect and an α-Fe2O3 nanoparticle leading to almost no inhibition of phase I machinery. The downregulation was seen at the mRNA, protein expression, and activity levels. Thereby, no dependency on the size or chemical structure was found. This underlines the difficulty of the grouping of nanomaterials regarding their physiological impact, suggesting that every iron oxide nanoparticle species needs to be evaluated in a case-by-case approach.

Hepatotoxic effects of cyproconazole and prochloraz in wild-type and hCAR/hPXR mice.

The agricultural fungicides cyproconazole and prochloraz exhibit hepatotoxicity in rodent studies and are tumorigenic following chronic exposure. Both substances are suspected to act via a CAR (constitutive androstane receptor)/PXR (pregnane-X-receptor)-dependent mechanism. Human relevance of these findings is under debate. A 28-day toxicity study was conducted in mice with humanized CAR and PXR (hCAR/hPXR) with two dose levels (50 or 500 ppm) of both substances, using the model CAR activator phenobarbital as a reference. Results were compared to wild-type mice. A treatment-related increase in liver weights was observed for all three substances at least at the high-dose level. Changes in the expression of classic CAR/PXR target genes such as Cyp2b10 were induced by cyproconazole and phenobarbital in both genotypes, while prochloraz treatment resulted in gene expression changes indicative of additional aryl hydrocarbon receptor activation, e.g. by up-regulation of Cyp1a1 expression. Cyproconazole-induced effects on CAR-dependent gene expression, liver weight, and hepatic lipid accumulation were more prominent in wild-type mice, where significant genotype differences were observed at the high-dose level. Moreover, high-dose cyproconazole-treated mice from the wild-type group responded with a marked increase in hepatocellular proliferation, while hCAR/hPXR mice did not. In conclusion, our data demonstrate that cyproconazole and PB induce CAR/PXR downstream effects in hepatocytes in vivo via both, the murine and human receptors. At high doses of cyproconazole, however, the responses were clearly more pronounced in wild-type mice, indicating increased sensitivity of rodents to CAR agonist-induced effects in hepatocytes.

Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition.

Adenosine diphosphate (ADP) enhances platelet activation by virtually any other stimulant to complete aggregation. It binds specifically to the G-protein-coupled membrane receptors P2Y1 and P2Y12, stimulating intracellular signaling cascades, leading to integrin αIIbß3 activation, a process antagonized by endothelial prostacyclin. P2Y12 inhibitors are among the most successful antiplatelet drugs, however, show remarkable variability in efficacy. We reasoned whether a more detailed molecular understanding of ADP-induced protein phosphorylation could identify (1) critical hubs in platelet signaling toward aggregation and (2) novel molecular targets for antiplatelet treatment strategies. We applied quantitative temporal phosphoproteomics to study ADP-mediated signaling at unprecedented molecular resolution. Furthermore, to mimic the antagonistic efficacy of endothelial-derived prostacyclin, we determined how Iloprost reverses ADP-mediated signaling events. We provide temporal profiles of 4797 phosphopeptides, 608 of which showed significant regulation. Regulated proteins are implicated in well-known activating functions such as degranulation and cytoskeletal reorganization, but also in less well-understood pathways, involving ubiquitin ligases and GTPase exchange factors/GTPase-activating proteins (GEF/GAP). Our data demonstrate that ADP-triggered phosphorylation occurs predominantly within the first 10 seconds, with many short rather than sustained changes. For a set of phosphorylation sites (eg, PDE3ASer312, CALDAG-GEFISer587, ENSASer109), we demonstrate an inverse regulation by ADP and Iloprost, suggesting that these are central modulators of platelet homeostasis. This study demonstrates an extensive spectrum of human platelet protein phosphorylation in response to ADP and Iloprost, which inversely overlap and represent major activating and inhibitory pathways.

A Nexus Consisting of Beta-Catenin and Stat3 Attenuates BRAF Inhibitor Efficacy and Mediates Acquired Resistance to Vemurafenib.

Acquired resistance to second generation BRAF inhibitors (BRAFis), like vemurafenib is limiting the benefits of long term targeted therapy for patients with malignant melanomas that harbor BRAF V600 mutations. Since many resistance mechanisms have been described, most of them causing a hyperactivation of the MAPK- or PI3K/AKT signaling pathways, one potential strategy to overcome BRAFi resistance in melanoma cells would be to target important common signaling nodes. Known factors that cause secondary resistance include the overexpression of receptor tyrosine kinases (RTKs), alternative splicing of BRAF or the occurrence of novel mutations in MEK1 or NRAS. In this study we show that ß-catenin is stabilized and translocated to the nucleus in approximately half of the melanomas that were analyzed and which developed secondary resistance towards BRAFi. We further demonstrate that ß-catenin is involved in the mediation of resistance towards vemurafenib in vitro and in vivo. Unexpectedly, ß-catenin acts mainly independent of the TCF/LEF dependent canonical Wnt-signaling pathway in resistance development, which partly explains previous contradictory results about the role of ß-catenin in melanoma progression and therapy resistance. We further demonstrate that ß-catenin interacts with Stat3 after chronic vemurafenib treatment and both together cooperate in the acquisition and maintenance of resistance towards BRAFi.

Innate immune system favors emergency monopoiesis at the expense of DC-differentiation to control systemic bacterial infection in mice.

DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ-signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis.

Release of matrix metalloproteinase-8 during physiological trafficking and induced mobilization of human hematopoietic stem cells.

Previous studies indicate that the release of proteases, including the gelatinase matrix metalloproteinase (MMP)-9, from mature granulocytes plays a crucial role in cytokine-induced hematopoietic stem and progenitor cell (HSPC) mobilization. However, studies with MMP-9-deficient mice revealed that HSPC mobilization was normal in these animals, suggesting that additional proteases must be active at clinically relevant cytokine concentrations. In the present study, we provide evidence that the collagenase MMP-8 is involved in stem cell mobilization. A rapid release of MMP-8 from isolated neutrophil granulocytes can be observed during an in vitro culture. During granulocyte colony-stimulating factor-induced HSPC mobilization, highly elevated serum concentrations of MMP-8 were observed on days 4 to 6 of the mobilization regimen, concomitantly with elevated MMP-9 serum levels and higher numbers of circulating CD34(+) cells. Elevated serum concentrations of both proteases were also found in umbilical cord blood serum. In functional assays, adhesion of HSPC to osteoblasts as an essential component of the endosteal stem cell niche is negatively influenced by MMP-8. The chemokine CXCL12, which is critically involved in stem cell trafficking, can be proteolytically processed by MMP-8 treatment. This degradation has a strong inhibitory influence on HSPC migration. Taken together, our data strongly suggest that MMP-8 can be directly involved in hematopoietic stem cell mobilization and trafficking.

Concurrent epigenetic silencing of wnt/ß-catenin pathway inhibitor genes in B cell chronic lymphocytic leukaemia.

BACKGROUND: The Wnt/ß-catenin signalling is aberrantly activated in primary B cell chronic lymphocytic leukaemia (CLL). Epigenetic silencing of pathway inhibitor genes may be a mechanism for its activation. In this study, we investigated systematically and quantitatively the methylation status of 12 Wnt/ß-catenin pathway inhibitor genes - CDH1, DACT1, DKK1, DKK2, DKK3, DKK4, SFRP1, SFRP2, SFRP3, SFRP4, SFRP5 and WIF1 - in the cell lines EHEB and MEC-1 as well as patient samples. METHODS: Quantification of DNA methylation was performed by means of bisulphite pyrosequencing and confirmed by bisulphite Sanger sequencing. Gene expression was analysed by qPCR using GAPDH as internal control. E-cadherin and ß-catenin protein quantification was carried out by microsphere-based immunoassays. Methylation differences observed between the patient and control groups were tested using generalised least squares models. RESULTS: For 10 genes, a higher methylation level was observed in tumour material. Only DKK4 exhibited similarly high methylation levels in both tumour and normal specimens, while DACT1 was always essentially unmethylated. However, also for these inhibitors, treatment of cells with the demethylating agent 5-aza-2´-deoxycytidine resulted in an induction of their expression, as shown by quantitative PCR, suggesting an indirect epigenetic control of activity. While the degree of demethylation and its transcriptional consequences differed between the genes, there was an overall high correlation of demethylation and increased activity. Protein expression studies revealed that no constitutive Wnt/ß-catenin signalling occurred in the cell lines, which is in discrepancy with results from primary CLL. However, treatment with 5-aza-2´-deoxycytidine caused accumulation of ß-catenin. Simultaneously, E-cadherin expression was strongly induced, leading to the formation of a complex with ß-catenin and thus demonstrating its epigenetically regulated inhibition effect. CONCLUSIONS: The results suggest an epigenetic silencing mechanism of the Wnt/ß-catenin pathway inhibitor genes in CLL. Hypermethylation and silencing of functionally related genes may not be completely stochastic but result from the tumour epigenome reprogramming orchestrated by Polycomb-group repressive complexes. The data are of interest in the context of epigenetic-based therapy.

Combining ultracentrifugation and peptide termini group-specific immunoprecipitation for multiplex plasma protein analysis.

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques.

Receptor tyrosine kinase inhibitor profiling using bead-based multiplex sandwich immunoassays.

Receptor tyrosine kinases (RTK) are important targets in drug discovery processes. Studying the phosphorylation pattern of RTKs enables the determination of their activation and inactivation states. Multiplex bead-based sandwich immunoassays are powerful tools for measuring the phosphorylation state of key regulators within cellular signalling networks. Here, we describe the analysis of the phosphorylation state of receptor tyrosine kinases using the epidermal growth factor receptor (EGFR) as an example. We provide a protocol for a bead-based sandwich immunoassay that enables a relative quantification of the EGFR and its generic tyrosine phosphorylation. We also present data from a kinase inhibitor experiment using 96-well cell-culture plates and a commercially available kit for the analysis of seven receptor tyrosine kinases.

Snapshots of protein dynamics and post-translational modifications in one experiment--beta-catenin and its functions.

ß-catenin plays multiple roles in the canonical Wnt signaling pathway and in cell-cell adhesion complexes. In addition, ß-catenin is a proto-oncogene and activating ß-catenin mutations are relevant in the genesis of colorectal, hepatocellular and other common cancers. Different functions of ß-catenin as transcriptional co-activator or cell adhesion molecule are orchestrated by changes in concentration and phosphorylation as well as its ability to complex with proteins such as cadherins or transcription factors. Detailed quantitative and time-resolved analysis of ß-catenin, based on the evaluation of the changes in the Wnt pathway, enable greater insights into health- and disease-related ß-catenin function. The present paper describes a novel suspension bead array assay panel for ß-catenin, which requires minimal amounts of sample and is able to relatively quantify total ß-catenin, the extent of phosphorylation at multiple sites and the ratio of complexed and free ß-catenin. This is the first study to combine three biochemical methods--sandwich immunoassay, co-immunoprecipitation, and protein-protein interaction assay--in one suspension bead assay panel. The assay was used to measure changes in the concentration of eight different ß-catenin forms in HEK293 cells in a time-resolved manner. In contrast to the general consensus, our study demonstrates an increase in ß-catenin phosphorylated at Ser-45 upon treatment of cells with rWnt3a or a GSK3 inhibition; we also link C-terminal phosphorylation of ß-catenin on Ser-552 and Ser-675 with canonical Wnt signaling.

Analysis of non-canonical fibroblast growth factor receptor 1 (FGFR1) interaction reveals regulatory and activating domains of neurofascin.

Fibroblast growth factor receptors (FGFRs) are important for many different mechanisms, including cell migration, proliferation, differentiation, and survival. Here, we show a new link between FGFR1 and the cell adhesion molecule neurofascin, which is important for neurite outgrowth. After overexpression in HEK293 cells, embryonal neurofascin isoform NF166 was able to associate with FGFR1, whereas the adult isoform NF186, differing from NF166 in additional extracellular sequences, was deficient. Pharmacological inhibitors and overexpression of dominant negative components of the FGFR signaling pathway pointed to the activation of FGFR1 after association with neurofascin in neurite outgrowth assays in chick tectal neurons and rat PC12-E2 cells. Both extra- and intracellular domains of embryonal neurofascin isoform NF166 were able to form complexes with FGFR1 independently. However, the cytosolic domain was both necessary and sufficient for the activation of FGFR1. Cytosolic serine residues 56 and 100 were shown to be essential for the neurite outgrowth-promoting activity of neurofascin, whereas both amino acid residues were dispensable for FGFR1 association. In conclusion, the data suggest a neurofascin intracellular domain, which activates FGFR1 for neurite outgrowth, whereas the extracellular domain functions as an additional, regulatory FGFR1 interaction domain in the course of development.