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.     

Expression and purification of recombinant human fibroblast growth factor receptor in Escherichia coli

Human fibroblast growth factor receptor (FGFR) is responsible for multifunctional signaling that regulates developmental processes. The three immunoglobulin-like extracellular domains of FGFR (D1, D2, and D3) include the determinants of ligand binding and specificity for fibroblast growth factor and heparan sulfate. D1 and the D1-D2 linker with a contiguous stretch of acidic amino acids are known to be involved in auto-inhibitory regulation. In an effort to gain a better understanding of the role of D1 and the linker in FGFR regulation, we have subcloned, overexpressed, and purified the extracellular fragments, D1-D2 and D1-D3, of FGFR1 in Escherichia coli. The recombinant proteins were produced in an insoluble form and were renatured using a dropwise or on-column refolding method. In addition, D2-D3 was coexpressed with chaperones to test the possibility that the presence of chaperones might enhance refolding efficiencies. A combination of immobilized nickel and heparin affinity chromatography and size-exclusion chromatography resulted in the purification of recombinant ectodomain proteins D1-D2 and D1-D3 of high purity for structural studies.     

Cysteine-rich fibroblast growth factor receptor alters secretion and intracellular routing of fibroblast growth factor 3

Expression of the cysteine-rich fibroblast growth factor (FGF) receptor (CFR) in COS-1 cells strongly inhibits the secretion of co-expressed FGF3. By using a column retention assay and affinity chromatography, we demonstrate that at physiological salt concentrations FGF3 binds with strong affinity to CFR in vivo and in vitro. Furthermore, to show that FGF3 binds to CFR in vivo, truncation mutants of CFR with changed subcellular distributions were shown to cause a similar redistribution of FGF3. Although CFR is a 150-kDa integral membrane glycoprotein that is primarily located in the Golgi apparatus, we show here that in COS-1 cells a substantial proportion of CFR is secreted. This is due to a carboxyl-terminal proteolytic cleavage that releases the intraluminal portion of the protein for secretion. However, the apparent size of the integral membrane and secreted CFR appears similar, since the loss of protein mass is balanced by a gain of complex carbohydrates. The released CFR is associated with the extracellular matrix through its affinity for glycosaminoglycans. These findings show that CFR can modulate the secretion of FGF3 and may control its biological activity by regulating its secretion.     

NCAM-derived peptides function as agonists for the fibroblast growth factor receptor

The neural cell adhesion molecule (NCAM) directly interacts with the fibroblast growth factor receptor (FGFR). Both fibronectin type III (FN3) modules of NCAM are involved in this interaction. One of the NCAM–FGFR contact sites has been localized recently to the upper N-terminal part of the second NCAM FN3 module encompassing the F and G β-strands and the interconnecting loop region. Here, we investigated whether any of the six putative strand-loop-strand regions in the first NCAM FN3 module are involved in FGFR interactions. Peptide sequences encompassing these regions, termed encamins, were synthesized and tested for their ability to bind and activate FGFR. Encamins localized to the N-terminal part of the first FN3 module did not interact with FGFR, whereas encamins localized to the C-terminal part, termed EncaminA, C and E, bound to and activated FGFR. The encamins induced FGFR-dependent neurite outgrowth, and EncaminC and E promoted neuronal survival and enhanced pre-synaptic function. In conclusion, the interaction between NCAM and FGFR probably involves multiple contact sites at an interface formed by the two NCAM FN3 modules and FGFR, and encamins could constitute important pharmacological tools for the study of specific functional aspects of NCAM, including neuroprotection and modulation of plasticity.     

Brain-derived neurotrophic factor induces phosphorylation of fibroblast growth factor receptor substrate 2

Brain-derived neurotrophic factor (BDNF) promotes neuronal survival. Gaining an understanding of how BDNF, via the tropomyosin-related kinase B (TRKB) receptor, elicits specific cellular responses is of contemporary interest. Expression of mutant TrkB in fibroblasts, where tyrosine 484 was changed to phenylalanine, abrogated Shc association with TrkB, but only attenuated and did not block BDNF-induced phosphorylation of mitogen-activated protein kinase (MAPK). This suggests there is another BDNF-induced signaling mechanism for activating MAPK, which compelled a search for other TrkB substrates. BDNF induces phosphorylation of fibroblast growth factor receptor substrate 2 (FRS2) in both fibroblasts engineered to express TrkB and human neuroblastoma (NB) cells that naturally express TrkB. Additionally, BDNF induces phosphorylation of FRS2 in primary cultures of cortical neurons, thus showing that FRS2 is a physiologically relevant substrate of TrkB. Data are presented demonstrating that BDNF induces association of FRS2 with growth factor receptor-binding protein 2 (GRB2) in cortical neurons, fibroblasts, and NB cells, which in turn could activate the RAS/MAPK pathway. This is not dependent on Shc, since BDNF does not induce association of Shc and FRS2. Finally, the experiments suggest that FRS2 and suc-associated neurotrophic factor-induced tyrosine-phosphorylated target are the same protein.     

Fibroblast growth factor/fibroblast growth factor receptor system in angiogenesis

Fibroblast growth factors (FGFs) are a family of heparin-binding growth factors. FGFs exert their pro-angiogenic activity by interacting with various endothelial cell surface receptors, including tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins. Their activity is modulated by a variety of free and extracellular matrix-associated molecules. Also, the cross-talk among FGFs, vascular endothelial growth factors (VEGFs), and inflammatory cytokines/chemokines may play a role in the modulation of blood vessel growth in different pathological conditions, including cancer. Indeed, several experimental evidences point to a role for FGFs in tumor growth and angiogenesis. This review will focus on the relevance of the FGF/FGF receptor system in adult angiogenesis and its contribution to tumor vascularization.     

Epidermal growth factor: the receptor and its function

Epidermal growth factor (EGF) is a small polypeptide hormone with mitogenic properties in vivo and in vitro. EGF elicits biologic responses by binding to a cell surface receptor which is a transmembrane glycoprotein containing a cytoplasmic protein tyrosine kinase. EGF responses are mediated by ligand binding and activation of this intrinsic protein kinase. The receptor can be phosphorylated by other protein kinases, and this may regulate receptor function. Stimulation of the receptor tyrosine kinase activity by ligand binding must regulate the activity of an as yet undefined molecule(s) responsible for transmitting a mitogenic signal to the nucleus.     

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.     

Involvement of fibroblast growth factor receptor 2 isoform switching in mammary oncogenesis

We identified the IIIb C2 epithelial cell-specific splice variant of fibroblast growth factor receptor 2 (FGFR2 IIIb C2) receptor tyrosine kinase in a screen for activated oncogenes expressed in T-47D human breast carcinoma cells. We found FGFR2 IIIb C2 expression in breast carcinoma cell lines and, additionally, expression of the mesenchymal-specific FGFR2 IIIc splice variant in invasive breast carcinomas. FGFR2 IIIc expression was associated with loss of epithelial markers and gain of mesenchymal markers. Although FGFR2 IIIb is expressed in epithelial cells, previous studies on FGFR2 IIIb transformation have focused on NIH 3T3 fibroblasts. Therefore, we compared the transforming activities of FGFR2 IIIb C2 in RIE-1 intestinal cells and several mammary epithelial cells. FGFR2 IIIb C2 caused growth transformation of epithelial cells but morphologic transformation of only NIH 3T3 cells. FGFR2 IIIb C2-transformed NIH 3T3, but not RIE-1 cells, showed persistent activation of Ras and increased cyclin D1 protein expression. NIH 3T3 but not RIE-1 cells express keratinocyte growth factor, a ligand for FGFR2 IIIb C2. Ectopic treatment with keratinocyte growth factor caused FGFR2 IIIb C2-dependent morphologic transformation of RIE-1 cells, as well as cyclin D1 up-regulation, indicating that both ligand-independent and stromal cell-derived, ligand-dependent mechanisms contribute to RIE-1 cell transformation. Our results support cell context distinct mechanisms of FGFR2 IIIb C2 transformation.     

The discovery of basic fibroblast growth factor/fibroblast growth factor-2 and its role in haematological malignancies

Basic fibroblast growth factor/fibroblast growth factor-2 is one of the best characterized of the pro-angiogenic cytokines. This review describes its history, as well as its role in tumor angiogenesis associated with haematological malignancies, as traced by the main contributions to the international medical literature.     

Purification of acidic fibroblast growth factor from bovine heart and its localization in the cardiac myocytes

We found an endogenous growth factor, referred to here as heart-derived growth factor (HDGF), that stimulates the proliferation of vascular endothelial cells. HDGF was purified from bovine myocardium using a procedure that involves denaturation of undesired proteins with methanol and chloroform. Soluble HDGF was purified essentially to homogeneity in a single step by heparin affinity chromatography. The purified HDGF was identified to be acidic fibroblast growth factor based on the following properties: molecular weight of 18,000, isoelectric point of 5.2, amino acid composition and sequence, its dissociation from a heparin affinity column at 0.9 M NaCl, potentiation of activity in the presence of heparin, and antigenicity. Our yield of HDGF was 500 micrograms/kg of tissue. Antiserum raised to HDGF localized HDGF in the cardiac myocytes in culture. These data indicate that a large amount of acidic fibroblast growth factor is present in the heart, and the cardiac myocytes are likely to be a major source of it.     

Purification of an active receptor for acidic and basic fibroblast growth factor from bovine retina.

Acidic and basic fibroblast growth factors (FGFs) influence cell division and differentiation in retina cells. Their effects are thought to be mainly mediated through stimulation of a specific membrane receptor and subsequent generation of an intracellular signal pathway. In this study, we purified a FGF receptor of 130 kDa from bovine neural retina using wheat germ agglutinin affinity chromatography followed by FGF-affinity chromatography. The isolated receptor showed ligand binding activity with dissociation constants of 0.8 nM and 2 nM for aFGF and bFGF, respectively. Furthermore, binding of aFGF and bFGF to purified receptor resulted in self-phosphorylation, demonstrating that the isolated receptor had an unaltered intrinsic kinase activity.