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1.
Nature ; 618(7966): 862-870, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37286607

RESUMO

α/ßKlotho coreceptors simultaneously engage fibroblast growth factor (FGF) hormones (FGF19, FGF21 and FGF23)1,2 and their cognate cell-surface FGF receptors (FGFR1-4) thereby stabilizing the endocrine FGF-FGFR complex3-6. However, these hormones still require heparan sulfate (HS) proteoglycan as an additional coreceptor to induce FGFR dimerization/activation and hence elicit their essential metabolic activities6. To reveal the molecular mechanism underpinning the coreceptor role of HS, we solved cryo-electron microscopy structures of three distinct 1:2:1:1 FGF23-FGFR-αKlotho-HS quaternary complexes featuring the 'c' splice isoforms of FGFR1 (FGFR1c), FGFR3 (FGFR3c) or FGFR4 as the receptor component. These structures, supported by cell-based receptor complementation and heterodimerization experiments, reveal that a single HS chain enables FGF23 and its primary FGFR within a 1:1:1 FGF23-FGFR-αKlotho ternary complex to jointly recruit a lone secondary FGFR molecule leading to asymmetric receptor dimerization and activation. However, αKlotho does not directly participate in recruiting the secondary receptor/dimerization. We also show that the asymmetric mode of receptor dimerization is applicable to paracrine FGFs that signal solely in an HS-dependent fashion. Our structural and biochemical data overturn the current symmetric FGFR dimerization paradigm and provide blueprints for rational discovery of modulators of FGF signalling2 as therapeutics for human metabolic diseases and cancer.


Assuntos
Fator de Crescimento de Fibroblastos 23 , Proteoglicanas de Heparan Sulfato , Hormônios , Receptores de Fatores de Crescimento de Fibroblastos , Transdução de Sinais , Humanos , Microscopia Crioeletrônica , Fator de Crescimento de Fibroblastos 23/química , Fator de Crescimento de Fibroblastos 23/metabolismo , Fator de Crescimento de Fibroblastos 23/ultraestrutura , Proteoglicanas de Heparan Sulfato/química , Proteoglicanas de Heparan Sulfato/metabolismo , Hormônios/química , Hormônios/metabolismo , Proteínas Klotho/química , Proteínas Klotho/metabolismo , Proteínas Klotho/ultraestrutura , Multimerização Proteica , Receptores de Fatores de Crescimento de Fibroblastos/química , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura
2.
Cell Tissue Res ; 319(2): 267-78, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15654655

RESUMO

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) and its selective agonist fluprostenol increase basic fibroblast growth factor (FGF-2) mRNA and protein production in osteoblastic Py1a cells. The present report extends our previous studies by showing that Py1a cells express FGF receptor-2 (FGFR2) and that treatment with PGF(2alpha) or fluprostenol decreases FGFR2 mRNA. We have used confocal and electron microscopy to show that, under PGF(2alpha) stimulation, FGF-2 and FGFR2 proteins accumulate near the nuclear envelope and colocalize in the nucleus of Py1a cells. Pre-treatment with cycloheximide blocks nuclear labelling for FGF-2 in response to PGF(2alpha). Treatment with SU5402 does not block prostaglandin-mediated nuclear internalization of FGF-2 or FGFR2. Various effectors have been used to investigate the signal transduction pathway. In particular, pre-treatment with phorbol 12-myristate 13-acetate (PMA) prevents the nuclear accumulation of FGF-2 and FGFR2 in response to PGF(2alpha). Similar results are obtained by pre-treatment with the protein kinase C (PKC) inhibitor H-7. In addition, cells treated with PGF(2alpha) exhibit increased nuclear labelling for the mitogen-activated protein kinase (MAPK), p44/ERK2. Pre-treatment with PMA blocks prostaglandin-induced ERK2 nuclear labelling, as confirmed by Western blot analysis. We conclude that PGF(2alpha) stimulates nuclear translocation of FGF-2 and FGFR2 by a PKC-dependent pathway; we also suggest an involvement of MAPK/ERK2 in this process.


Assuntos
Núcleo Celular/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Osteoblastos/efeitos dos fármacos , Osteoblastos/enzimologia , Prostaglandinas/farmacologia , Receptores Proteína Tirosina Quinases/efeitos dos fármacos , Receptores de Fatores de Crescimento de Fibroblastos/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular Transformada , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Dinoprosta/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fator 2 de Crescimento de Fibroblastos/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Imuno-Histoquímica , Microscopia Confocal , Microscopia Imunoeletrônica , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Prostaglandinas F Sintéticas/farmacologia , RNA Mensageiro/efeitos dos fármacos , Ratos , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/ultraestrutura , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura
3.
J Cell Physiol ; 200(1): 31-44, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15137055

RESUMO

The expression of the keratinocyte growth factor receptor (KGFR) has been analyzed on intestinal epithelial Caco-2 cells upon confluence-induced spontaneous differentiation. Western blot and immunofluorescence analysis showed that the expression of functional KGFRs, differently from that of epidermal growth factor receptor (EGFR), was up-modulated in post-confluent differentiated cultures compared with the pre-confluent cells. Confocal microscopy and immunoelectron microscopy revealed that the up-regulated KGFRs displayed a basolateral polarized distribution on the cell surfaces in the monolayer. In vivo immunohistochemical analysis on normal human colon tissue sections showed that KGFRs, differently from EGFRs, were mostly distributed on the more differentiated cells located on the upper portion of the intestinal crypt. Bromodeoxyuridine incorporation assay and Ki67 labeling indicated that the differentiated cells were able to proliferate in response to the two ligands of KGFR, KGF and FGF-10, whereas they were not stimulated by the EGFR ligands TGFalpha and EGF. Western blot and quantitative immunofluorescence analysis of the expression of carcinoembryonic antigen (CEA) in post-confluent cells revealed that incubation with KGF induced an increase of cell differentiation. Taken together these results indicate that up-modulation of KGFR may be required to promote proliferation and differentiation in differentiating cells and that, among the cells componing the intestinal epithelial monolayer, the target cells for KGFR ligands appear to be different during differentiation from those responsive to EGFR ligands.


Assuntos
Diferenciação Celular , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Queratinócitos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Anticorpos Monoclonais/metabolismo , Western Blotting , Células CACO-2 , Antígeno Carcinoembrionário/metabolismo , Divisão Celular , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Receptores ErbB/ultraestrutura , Fator 10 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/farmacologia , Técnica Indireta de Fluorescência para Anticorpo , Substâncias de Crescimento/farmacologia , Células HT29 , Humanos , Intestinos/citologia , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/ultraestrutura , Antígeno Ki-67/metabolismo , Ligantes , Microscopia Confocal , Microscopia Imunoeletrônica , Modelos Biológicos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura , Regulação para Cima/efeitos dos fármacos
4.
Arch Pathol Lab Med ; 120(5): 490-6, 1996 May.
Artigo em Inglês | MEDLINE | ID: mdl-8639054

RESUMO

OBJECTIVE: To characterize the distribution of the receptor for the fibroblast growth factor in human normal tissues and tumors using a new monoclonal antibody. DESIGN: Monoclonal antibodies for a kinase-insert region of fibroblast growth factor receptor-1 (FGFR-1) were generated. We conducted an immunohistological analysis of FGFR-1 using a highly specific antibody we generated, and we examined the distribution of this receptor in normal human tissues and in tumors. RESULTS: Intense positivity of FGFR-1 was observed in astrocytes in the brain, smooth muscles in the uterus, cardiac myocytes, respiratory epithelium in the lung, tubular epithelium in the kidney, acinar cells in the pancreas, follicular cells in the thyroid, and ductal and lobular epithelium in the breast. We also observed FGFR-1 expression in the fibroblasts and the tissue microvasculature. In addition to some nonepithelial tumors, some epithelial tumors expressed FGFR-1, including pancreatic adenocarcinomas, thyroid papillary carcinomas, invasive ductal carcinomas of the breast, lung adenocarcinomas, renal cell carcinomas, and colonic adenocarcinomas. Although FGFR-1 was expressed in colonic adenocarcinomas, which have invasive potential, tubular adenomas, which are noninvasive, did not express FGFR-1. CONCLUSION: We have been able to define the distribution of FGFR-1 in human normal tissues and tumors. Especially in colonic tumors, FGFR-1 expression may lead adenoma cells to invade and grow in the surrounding tissue.


Assuntos
Anticorpos Monoclonais , Biomarcadores Tumorais/análise , Proteínas Tirosina Quinases , Receptores Proteína Tirosina Quinases , Receptores de Fatores de Crescimento de Fibroblastos/análise , Receptores de Fatores de Crescimento de Fibroblastos/imunologia , Células Tumorais Cultivadas/química , Células Tumorais Cultivadas/patologia , Sequência de Aminoácidos , Especificidade de Anticorpos , Biomarcadores Tumorais/imunologia , Western Blotting , Fixadores , Formaldeído , Humanos , Imunoglobulina M/imunologia , Dados de Sequência Molecular , Inclusão em Parafina , RNA Mensageiro/biossíntese , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura , Células Tumorais Cultivadas/ultraestrutura
5.
Nat Struct Biol ; 2(12): 1068-74, 1995 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-8846218

RESUMO

Fibroblast growth factor receptors (FGFRs) have three extracellular domains that belong to the immunoglobulin superfamily. We have determined the outline structures for these domains on the basis of their homology to the I set molecule telokin. The outline structures describe the relative positions of residues in each domain; their major secondary structures, and the extent to which residues are accessible to the solvent. They also provide the basis of a coherent description of the change in recognition properties that occur when the IIIb and IIIc exons are switched and of the effects of mutations in FGFRs that cause genetic diseases.


Assuntos
Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura , Sequência de Aminoácidos , Sítios de Ligação , Dados de Sequência Molecular , Conformação Proteica , Receptores de Fatores de Crescimento de Fibroblastos/química , Homologia de Sequência de Aminoácidos
6.
Cell ; 83(4): 621-30, 1995 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-7585965

RESUMO

We have developed a functional screen in yeast to identify ligands for receptor tyrosine kinases. Using this method, we cloned two Xenopus genes that activate the fibroblast growth factor (FGF) receptor. These encode novel secreted proteins, designated FRL1 and FRL2, distantly related to the epidermal growth factor and angiogenin/ribonuclease families, respectively. Both genes activate the FGF receptor in Xenopus oocytes as well as in yeast. Overexpression induces mesoderm and neural-specific genes in Xenopus explants; induction is blocked by a dominant negative inhibitor of the FGF receptor. FRL1 is broadly expressed during gastrulation and neurulation, while FRL2 is expressed principally in the axial mesoderm and brain at later stages. Our results indicate that despite their lack of similarity with FGF, FRL1 and FRL2 are ligands for the FGF receptor that play distinct roles in development.


Assuntos
Proteínas de Membrana/genética , Receptores de Superfície Celular , Receptores de Fatores de Crescimento de Fibroblastos/genética , Saccharomyces cerevisiae/genética , Proteínas de Xenopus , Xenopus/embriologia , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/análise , Indução Embrionária/genética , Biblioteca Gênica , Testes Genéticos , Immunoblotting , Ligantes , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Mesoderma/fisiologia , Dados de Sequência Molecular , Oócitos/fisiologia , Ligação Proteica/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura , Fatores de Tempo , Transformação Genética
7.
Eur J Pediatr ; 154(3): 215-9, 1995 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-7758520

RESUMO

Achondroplasia, the most common form of chondrodysplasia, has been associated with mutations in the gene of the fibroblast growth factor receptor-3 (FGFR-3) on chromosome 4p. All 39 achondroplasia alleles studied so far carried point mutations which caused the same amino acid exchange, a substitution of glycine by arginine at position 380 (G380R) in the transmembrane domain of the receptor. We report on a newborn with achondroplasia who does not carry a G380R mutation but has a mutation causing substitution of a nearby glycine with a cysteine (G375C). This observation indicates allelic heterogeneity and confirms the role of mutations in the transmembrane domain of FGFR-3 in the pathogenesis of achondroplasia.


Assuntos
Acondroplasia/genética , Cromossomos Humanos Par 4 , Cisteína , Glicina , Mutação Puntual , Receptores de Fatores de Crescimento de Fibroblastos/genética , Acondroplasia/diagnóstico por imagem , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/patologia , Humanos , Recém-Nascido , Masculino , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Radiografia , Receptores de Fatores de Crescimento de Fibroblastos/ultraestrutura
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