Expression of fibroblast growth factor receptor 3 by fibroblast growth factor 2 in cultured chick embryo chondrocytes

Although fibroblast growth factor 2 (FGF2) and fibroblast growth factor receptor 3 (FGFR3) both inhibit longitudinal bone growth, little is known about the relationship between FGF2 and FGFR3. Accordingly, the current study examined the expression of FGFR3 mRNA after the administration of FGF2 using cultured chondrocytes from day 17 chick embryos to evaluate the relationship between FGF2 and FGFR3. The chondrocytes were isolated from the caudal one-third portion (LS) of sterna, peripheral regions (USP) and central core regions (USC) of the cephalic portion of the sterna, and lower portion of the proximal tibial growth plate (Ti) of day 17 chick embryo. The expression of FGFR1, FGFR3, and type II and X collagen mRNA in the chondrocytes from the LS, USP, USC, and Ti was determined. FGFR1 was not expressed in the LS and USP chondrocytes, yet strongly expressed in the USC and Ti chondrocytes. With a treatment of FGF2, the expression of FGFR1 slightly increased in the USC chondrocytes and was not related with the concentration of FGF2 in the Ti chondrocytes. FGFR3 was expressed in all the chondrocyte types, yet strongly increased in the LS, USC, USP, and Ti in that order according to the concentration of FGF2. For the LS and USP chondrocytes, the expression of FGFR3 with FGF2 increased in a 4-day culture, yet decreased in a 6-day culture, whereas for the USC chondrocytes, the expression of FGFR3 mRNA with FGF2 increased in a 2-day culture, yet decreased in a 4-day culture, suggesting that the hypertrophic chondrocytes were more numerous and sensitive compared to the proliferative chondrocytes. For all the chondrocyte types, FGF2 appeared to be up-regulated to FGFR3, as the expression of FGFR3 mRNA increased with a higher concentration of FGF2 until a peak level. In conclusion, FGF2 was found to up-regulate to FGFR3 until the peak level of FGFR3 mRNA expression, while in hypertrophic chondrocytes, FGFR3 appeared to cause the differentiaton of chondrocytes, resulting in the inhibition of longitudinal bone growth after the peak level of FGFR3 mRNA expression.     

Cloning and interspecies comparisons of three newt (Notophthalmus viridescens) fibroblast growth factor receptor sequences

We report the nucleotide sequences of two fibroblast growth factor receptor (FGFR) cDNAs, FGFR1 and FGFR3, from the newt species Notophthalmus viridescens. These two cDNA sequences and a previously published newt FGFR cDNA, FGFR2, were used to derive the amino acid sequences which were then compared with their homologues from other species. This comparison shows that the intracellular tyrosine kinase domain is highly conserved across the species examined with the second half of the domain slightly more conserved than the first half. The 3 portion of the carboxyl terminal tail is not very highly conserved. The comparison of the extracellular portion of FGFR2 shows a high degree of conservation among the Ig-like domains and a low degree of conservation in the region that links the third Ig-like domain with the transmembrane domain. (Mol Cell Biochem 175: 11–19, 1997)     

Ligand-induced clathrin-mediated endocytosis of the keratinocyte growth factor receptor occurs independently of either phosphorylation or recruitment of eps15

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells. Following ligand binding, KGFR is rapidly activated and internalized by clathrin-mediated endocytosis. Among the possible receptor substrates which could be involved in the regulation of KGFR endocytosis and down-modulation, we analyzed here the eps15 protein in view of the proposed general role of eps15 in regulating clathrin-mediated endocytosis as well as that of eps15 tyrosine phosphorylation in the control of regulated endocytosis. Immunoprecipitation and Western blot analysis showed that activated KGFR was not able to phosphorylate eps15, suggesting that eps15 is not a receptor substrate. Double immunofluorescence and confocal microscopy revealed that activated KGFR, differently from epidermal growth factor receptor (EGFR), did not induce recruitment of eps15 to the cell plasma membrane. Microinjection of a monoclonal antibody directed against the C-terminal DPF domain which contains the AP2 binding region of eps15 led to inhibition of both pathways of receptor-mediated endocytosis, the EGFR ligand-induced endocytosis and the transferrin constitutive endocytosis, but did not appear to block the KGFR ligand-induced internalization. Taken together our results indicate that the clathrin-mediated uptake of KGFR is not mediated by eps15.     

Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCgamma binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCgamma as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR.     

血小板源生长因子受体与肿瘤

血小板源生长因子(platelet-derived growth factor,PDGF)经由其受体(platelet-derived growth fac tor receptor,PDGFR)表现细胞效应。PDGF和PDGFR涉及多种肿瘤的发病机制并在血管生成中起重要作用。PDGF在肿瘤中的自分泌刺激、PDGFR的过表达或过度活化或者刺激肿瘤内血管生成都会促进肿瘤生长;PDGFR的阻断可以降低实体瘤中组织间质液压而增强药物传送。这些机制可能提示在肿瘤治疗中PDGFR抑制剂单用、与化疗药物或者和其他靶点药物联合用药的可能性和可行性。随着PDGFR拮抗剂,如imatinib的上市,PDGFR作为抗肿瘤药物的靶点备受瞩目。     

Dual functions of the heartless fibroblast growth factor receptor in development of the Drosophila embryonic mesoderm

The Drosophila embryonic mesoderm forms by invagination of the ventral-most blastoderm cells. Subsequent development of this germ layer involves the dorsolateral migration of the internalized cells and expansion by cell division, followed by the specification of particular cell fates through the coordinate actions of both intrinsic and extrinsic regulatory mechanisms. The latter include several intercellular signals that function across germ layers. These processes combine to generate a diversity of mesodermal subtypes, including the cardial and pericardial cells of the heart or dorsal vessel, a complete set of somatic muscle founders each with its unique identity, a population of cells that form the visceral musculature, and other cells that develop into hemocytes and the fat body. Here, we review recent evidence for the involvement of a fibroblast growth factor receptor (FGFR) encoded by the heartless (htl) gene in early directional migration of the Drosophila mesoderm. In addition, we provide new data that 1) demonstrate a second role for Htl in promoting the specification of the precursors to certain cardiac and somatic muscle cells in the Drosophila embryo, independent of its cell migration function, 2) suggest that Ras and at least one other signal transduction pathway act downstream of Htl, and 3) establish a functional relationship between the Ras pathway and Tinman (Tin), a homeodomain factor that is essential for specifying some of the same dorsal mesodermal cells that are dependent on Htl. Finally, parallels between requirements for FGFR signaling in Drosophila and vertebrate mesoderm development are considered. Dev. Genet. 22:212–229, 1998. © 1998 Wiley-Liss, Inc.     

Cortactin involvement in the keratinocyte growth factor and fibroblast growth factor 10 promotion of migration and cortical actin assembly in human keratinocytes

Keratinocyte growth factor (KGF/FGF7) and fibroblast growth factor 10 (FGF10/KGF2) regulate keratinocyte proliferation and differentiation by binding to the tyrosine kinase KGF receptor (KGFR). KGF induces keratinocyte motility and cytoskeletal rearrangement, whereas a direct role of FGF10 on keratinocyte migration is not clearly established. Here we analyzed the motogenic activity of FGF10 and KGF on human keratinocytes. Migration assays and immunofluorescence of actin cytoskeleton revealed that FGF10 is less efficient than KGF in promoting migration and exerts a delayed effect in inducing lamellipodia and ruffles formation. Both growth factors promoted phosphorylation and subsequent membrane translocation of cortactin, an F-actin binding protein involved in cell migration; however, FGF10-induced cortactin phosphorylation was reduced, more transient and delayed with respect to that promoted by KGF. Cortactin phosphorylation induced by both growth factors was Src-dependent, while its membrane translocation and cell migration were blocked by either Src and PI3K inhibitors, suggesting that both pathways are involved in KGF- and FGF10-dependent motility. Furthermore, siRNA-mediated downregulation of cortactin inhibited KGF- and FGF10-induced migration. These results indicate that cortactin is involved in keratinocyte migration promoted by both KGF and FGF10.     

The neural cell adhesion molecule binds to fibroblast growth factor receptor 2

The neural cell adhesion molecule (NCAM) can bind to and activate fibroblast growth factor receptor 1 (FGFR1). However, there are four major FGFR isoforms (FGFR1-FGFR4), and it is not known whether NCAM also interacts directly with the other three FGFR isoforms. In this study, we show by surface plasmon resonance analysis that NCAM can bind to FGFR2 with an affinity similar to that for the NCAM-FGFR1 interaction. However, the kinetic parameters for the NCAM-FGFR2 binding are different from those of the NCAM-FGFR1 binding. Both receptors were shown to cycle relatively fast between the NCAM bound and unbound states, although FGFR2 cycling was clearly faster (13 times) than the FGFR1 cycling. Moreover, ATP was more effective in inhibiting the binding of NCAM to FGFR1 than to FGFR2, indicating that the binding sites in NCAM for the two receptors are similar, but not identical.     

The Xenopus platelet-derived growth factor α receptor: cDNA Cloning and demonstration that mesoderm induction establishes the lineage-specific pattern of ligand and receptor gene expression

We have cloned the Xenopus PDGF α receptor cDNA and have used this clone, along with cDNA encoding PDGF A, to examine their expression pattern in Xenopus embryos and to determine the factors responsible for lineage specificity. Recombinant Xenopus α receptor expressed in COS cells exhibits PDGF-A-dependent tyrosine kinase activity. We find that receptor mRNA is present in cultured marginal zone tissue explants and in animal cap tissue induced to form mesoderm either by grafting to vegetal tissue or by treatment with recombinant activin A. In contrast, PDGF A mRNA is expressed in cultured, untreated animal cap tissue and is suppressed by mesoderm induction. These results suggest that ectodermally produced PDGF A may act on the mesoderm during gastrulation and that mesoderm induction establishes the tissue pattern of ligand and receptor expression. © 1993Wiley-Liss, Inc.     

Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.     

ADP-ribosylation factor 6 regulates mu-opioid receptor trafficking and signaling via activation of phospholipase D2

Endocytosis of the mu-opioid receptor (MOPr) has been shown to play a protective role against the development of tolerance to opioid drugs by facilitating receptor reactivation and recycling. It has been further demonstrated, that the opioid-mediated and ADP-ribosylation factor (ARF)-dependent activation of phospholipase D2 (PLD2) is a prerequisite for MOPr endocytosis. In this study, we investigated which particular ARF protein is involved in opioid-mediated PLD2 activation and what are the mechanisms of ARF function in MOPr trafficking and signaling. By coexpressing the MOPr and dominant negative or constitutively active ARF mutants in human embryonic kidney (HEK) 293 cells and primary cultured cortical neurons as well as by using siRNA technology, we identified the ARF6 protein to be involved in the regulation of MOPr endocytosis. We also found that expression of an effector domain mutant of ARF6, which is incapable of activating PLD, blocked agonist-induced endocytosis suggesting that ARF6 function in MOPr trafficking is PLD2-mediated. Analogously, opioid-mediated activation of PLD2 is blocked in the presence of dominant negative ARF6 mutants. Finally, we also showed that ARF6 protein influences the recycling/reactivation of internalized MOPr and thus modulates agonist-induced MOPr desensitization. Together, these results provide evidence that ARF6 protein regulates MOPr trafficking and signaling via PLD2 activation and hence affects the development of opioid receptor desensitization and tolerance.     

Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells

Hepatocyte growth factor (HGF)-induced migration of endothelial cells is critical for angiogenesis. Sphingosine kinase (SPK) is a key enzyme catalyzing the formation of sphingosine-1-phosphate (S1P), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through both intracellular and extracellular mechanisms. The aim of this study was to investigate whether activation of SPK is involved in the migration of endothelial cells induced by HGF. The biological functions of HGF are mediated through the activation of its high-affinity tyrosine kinase receptor, c-met protooncogene. In the present study, Treatment of ECV304 endothelial cells with HGF resulted in tyrosine phosphorylation of c-Met and activation of SPK in a concentration-dependent manner. Either Ly294002 or PD98059, specific inhibitor of the PI3K and ERK/MAPK pathways, respectively, blocked the HGF-induced activation of SPK. HGF stimulation significantly increased intracellular S1P level, but no detectable secretion of S1P into the cell culture medium was observed. Treatment of ECV304 cells with pertussis toxin (PTX) has no effect on the HGF-induced migration, indicating extracellular S1P is dispensable for this process. Overexpression of wild-type SPK gene in ECV 304 cells increased the intracellular S1P and enhanced the HGF-induced migration, whereas inhibition of cellular SPK activity by N,N-dimethylsphingosine (DMS), a potent inhibitor of SPK, or by expression of a dominant-negative SPK (DN-SK) blocked the HGF-induced migration of ECV 304 cells. It is suggested that PI3K and ERK/MAPK mediated the activation of SPK and would be involved in the HGF-induced migration of endothelial cells. These results elucidate a novel mechanism by which intracellularly generated S1P mediates signaling from HGF/c-Met to the endothelial cell migration.     

Neurotrophin receptor homolog-2 regulates nerve growth factor signaling

The neurotrophin receptor homolog (NRH2) is closely related to the p75 neurotrophin receptor (p75NTR); however, its function and role in neurotrophin signaling are unclear. NRH2 does not bind to nerve growth factor (NGF), however, is able to form a receptor complex with tropomyosin-related kinase receptor A (TrkA) and to generate high-affinity NGF binding sites. Despite this, the mechanisms underpinning the interaction between NRH2 and TrkA remain unknown. Here, we identify that the intracellular domain of NRH2 is required to form an association with TrkA. Our data suggest extensive intracellular interaction between NRH2 and TrkA, as either the juxtamembrane or death domain regions of NRH2 are sufficient for interaction with TrkA. In addition, we demonstrate that TrkA signaling is dramatically influenced by the co-expression of NRH2. Importantly, NRH2 did not influence all downstream TrkA signaling pathways, but rather exerted a specific effect, enhancing src homology 2 domain-containing transforming protein (Shc) activation. Moreover, downstream of Shc, the co-expression of NRH2 resulted in TrkA specifically modulating mitogen-activated protein kinase pathway activation, but not the phosphatidylinositol 3-kinase/Akt pathway. These results indicate that NRH2 utilizes intracellular mechanisms to not only regulate NGF binding to TrkA, but also specifically modulate TrkA receptor signaling, thus adding further layers of complexity and specificity to neurotrophin signaling.     

Caveolin is an inhibitor of platelet-derived growth factor receptor signaling

Caveolin is a major structural component of caveolae and has been implicated in the regulation of the function of several caveolae-associated signaling molecules. Platelet-derived growth factor (PDGF) receptors and caveolin were colocalized in the same subcellular fraction after sucrose density gradient fractionation of fibroblasts. Additionally, we found that the PDGF receptors interacted with caveolin in NIH3T3 fibroblast cells. We then examined whether caveolin directly binds to PDGF receptors and inhibits kinase activity using a recombinant PDGF receptor overexpressed in insect cells and peptides derived from the scaffolding domain of caveolin subtypes. We found the peptide from caveolin-1 and -3, but not -2, inhibited the autophosphorylation of PDGF receptors in a dose-dependent manner. Similarly, caveolin-1 and -3 peptides directly bound to PDGF receptors. Mutational analysis using a series of truncated caveolin-3 peptides (20-, 17-, 14-, and 11-mer peptides) revealed that at least 17 amino acid residues of the peptide were required to inhibit and directly bind to PDGF receptors. Thus, our findings suggest that PDGF receptors directly interact with caveolin subtypes, leading to the inhibition of kinase activity. Caveolin may be another regulating factor of PDGF-mediated tyrosine kinase signaling.     

Overexpression of hepatocyte growth factor receptor in renal carcinoma cells indirectly stimulates tumor growth in vivo

We examined the role of increased expression of HGFR kinase in in vivo growth of renal carcinoma. Human renal carcinoma cell line, ACHN cells, was transfected with plasmid encoding wild-type HGFR gene to generate cell lines with increased HGFR protein. ACHN cells with elevated HGFR expression, denoted clones 8 and 10, respectively, showed higher basal kinase activities of HGFR and PI3-kinase than those of empty-vector (mock)-transfected cells. Clone 8 and 10 cells grew similar to mock cells in culture. In mice, tumors of these clones grew more rapidly than those of mock cells. Microvessel density of clone 8 or 10 tumors was higher than that of mock tumors. Clone 8 and 10 cells secreted vascular endothelial growth factor-A (VEGF-A) more than mock cells and the secretion was PI3-kinase inhibitor, LY294002-sensitive. Anti-VEGF-A neutralizing antibody significantly inhibited tumor growth of clones 8 and 10 in mice. These results indicate for the first time that overexpression of HGFR tyrosine kinase in renal carcinoma cells participates in rapid tumor growth in vivo.     

Mutational identification of fibroblast growth factor receptor 1 and fibroblast growth factor receptor 2 genes in craniosynostosis in Indian population

OBJECTIVE:

The Objective of this study was to identify the association of mutation of fibroblast growth factor receptor 1 (FGFR1), FGFR2 genes with syndromic as well as non-syndromic craniosynostosis in Indian population.

MATERIALS AND METHODS:

Retrospective analysis of our records from January 2008 to December 2012 was done. A total of 41 cases satisfying the inclusion criteria and 51 controls were taken for the study. A total volume of 3 ml blood from the patient as well as parents was taken. Deoxyribonucleic acid extracted using phenol chloroform extraction method followed by polymerase chain reaction-restriction fragment length polymorphism method.

RESULTS:

There were 33 (80.4%) non-syndromic cases of craniosynostosis while 8 (19.5%) were syndromic. Out of these 8 syndromic cases, 4 were Apert syndrome, 3 were Crouzon syndrome and 1 Pfeiffer syndrome. Phenotypically the most common non-syndromic craniosynostosis was scaphocephaly (19, 57.7%) followed by plagiocephaly in (14, 42.3%). FGFR1 mutation (Pro252Arg) was seen in 1 (2.4%) case of non-syndromic craniosynostosis while no association was noted either with FGFR1 or with FGFR2 mutation in syndromic cases. None of the control group showed any mutation.

CONCLUSION:

Our study proposed that FGFR1, FGFR2 mutation, which confers predisposition to craniosynostosis does not exist in Indian population when compared to the western world.     

Exploitation of phage display for the development of anti-cancer agents targeting fibroblast growth factor signaling pathways: New strategies to tackle an old challenge

A tumor is defined as a group of cancer cells and ‘surrounding’ stromal bio-entities. Alongside the extracellular matrix (ECM) in the tumor microenvironment (TME), the stromal cells play key roles in cancer affliction and progression. Carcinoma-associated fibroblasts (CAFs) in the area of the tumor, whether activated or not, dictate the future of tumor cells. The CAFs and corresponding secreted growth factors (GFs), which mediate the crosstalk within the TME, can be targeted in therapies directed at the stroma. The impact of the fibroblast growth factor-fibroblast growth factor receptor (FGF-FGFR) signaling pathway in different kinds of tumors has been explored. Several tyrosine kinase inhibitors (TKIs), monoclonal antibodies (mAbs), and ligand traps targeting the formation of FGF-FGFR complex are in preclinical or early development phases. Moreover, there are numerous studies in the literature reporting the application of phage display technology for the development of peptides and proteins capable of functioning as FGF mimetics or traps, which are able to modulate FGF-related signaling pathways. In this review, prominent research in relation to phage display-assisted ligand identification for the FGF/FGFR system is discussed.     

Keratinocyte growth factor receptor ligands target the receptor to different intracellular pathways

The keratinocyte growth factor receptor (KGFR)/fibroblast growth factor receptor 2b is activated by high-affinity-specific interaction with two different ligands, keratinocyte growth factor (KGF)/fibroblast growth factor (FGF)7 and FGF10/KGF2, which are characterized by an opposite requirement of heparan sulfate proteoglycans and heparin for binding to the receptor. We investigated here the possible different endocytic trafficking of KGFR, induced by the two ligands. Immunofluorescence and immunoelectron microscopy analysis showed that KGFR internalization triggered by either KGF or FGF10 occurs through clathrin-coated pits. Immunofluorescence confocal microscopy using endocytic markers as well as tumor susceptibility gene 101 (TSG101) silencing demonstrated that KGF drives KGFR to the degradative pathway, while FGF10 targets the receptor to the recycling endosomes. Biochemical analysis showed that KGFR is ubiquitinated and degraded after KGF treatment but not after FGF10 treatment, and that the alternative fate of KGFR might depend on the different ability of the receptor to phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2) substrate and to recruit the ubiquitin ligase c-Cbl. The recycling endocytic pathway followed by KGFR upon FGF10 stimulation correlates with the higher mitogenic activity exerted by this ligand on epithelial cells compared with KGF, suggesting that the two ligands may play different functional roles through the regulation of the receptor endocytic transport.     

Molecular Cloning of a Rat Brain cDNA,with Homology to a Tyrosine Kinase Substrate,that Induces Galactosylceramide Expression in COS-7 Cells

Abstract: A rat brain cDNA clone has been isolated, using a eukaryotic cell transient expression system in conjunction with an anti-galactosylceramide (anti-GalCer) monoclonal antibody that induces GalCer expression in COS-7 cells. The protein was designated as GalCer expression factor-1 (GEF-1). A good correlation between GalCer expression and the level of the enzyme activity of UDP-galactose:ceramide galactosyltransferase (CGT) was demonstrated. The cDNA insert encoded a polypeptide of 771 amino acids with a calculated molecular mass of 85,787 Da. The cDNA hybridized to a single mRNA of 3.1 kb in all rat organs examined, including brain, testis, and skeletal muscle. The cDNA product was determined to be a tyrosine-phosphorylated protein with a molecular mass of 110 kDa in transfected COS-7 cells and adult rat brain. COS-7 cells transfected with the cDNA clone showed dramatic morphological changes: The transfected cells appeared to be fibroblast-like cells, whereas the parent COS-7 cells were typical epithelial-like cells. The deduced amino acid sequences revealed a strikingly high homology to a mouse hepatocyte growth factor-regulated tyrosine kinase substrate but no homology to CGT. Taking these results together, it is suggested that GEF-1 may play an important role in regulating GalCer expression in the brain.