RESUMEN
Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.
Asunto(s)
Sustancias de Crecimiento/metabolismo , Células 3T3 , Animales , Sitios de Unión , Línea Celular , Dicroismo Circular , Medio de Cultivo Libre de Suero , Relación Dosis-Respuesta a Droga , Escherichia coli/metabolismo , Factor 1 de Crecimiento de Fibroblastos , Factor 10 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/química , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 3 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/química , Factores de Crecimiento de Fibroblastos/metabolismo , Sustancias de Crecimiento/química , Sustancias de Crecimiento/genética , Humanos , Concentración 50 Inhibidora , Ligandos , Ratones , Ratones Endogámicos BALB C , Mutagénesis Sitio-Dirigida , Mutación Puntual , Unión Proteica , Isoformas de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento/química , Receptores de Factores de Crecimiento/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
The fibroblast growth factor (FGF) family plays a key role in a multitude of physiological and pathological processes. The activities of FGFs are mediated by a family of tyrosine kinase receptors, designated FGFRs. The mechanism by which FGFs induce receptor activation is controversial. Despite their structural similarity, FGFs display distinct receptor binding characteristics and cell type specificity. Previous studies with FGF-2 identified a low affinity receptor binding site that is located within a loop connecting its 9th and 10th beta-strands. The corresponding residues in the other family members are highly variable, and it was proposed that the variability might confer on FGFs unique receptor binding characteristics. We studied the role of this loop in FGF-7 by both site-directed mutagenesis and loop replacement. Unlike the other members of the FGF family, FGF-7 recognizes only one FGFR isoform and is, therefore, ideal for studies of how the specificity in the FGF-FGFR interaction is conferred at the structural level. Point mutations in the loop of FGF-7 did not change receptor binding affinity but resulted in reduced mitogenic potency and reduced ability to induce receptor-mediated phosphorylation events. These results suggest that the loop of FGF-7 fulfills the role of low affinity binding site required for receptor activation. The observation that it is possible to uncouple FGF-7 receptor binding and biological activity favors a bivalent model for FGFR dimerization, and it may be clinically relevant to the design of FGF-7 antagonists. Reciprocal loop replacement between FGF-7 and FGF-2 had no effect on their known receptor binding affinities nor did it alter their known specificity in eliciting a mitogenic response. In conclusion, these results suggest that, despite the diversity in the loop structure of FGF-2 and FGF-7, the loop has a similar function in both growth factors.
Asunto(s)
Factores de Crecimiento de Fibroblastos , Sustancias de Crecimiento/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Células 3T3 , Animales , Sitios de Unión , Línea Celular , Relación Dosis-Respuesta a Droga , Factor 10 de Crecimiento de Fibroblastos , Factor 2 de Crecimiento de Fibroblastos/genética , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/genética , Humanos , Cinética , Ratones , Modelos Moleculares , Mutagénesis , Fosforilación , Mutación Puntual , Unión Proteica/genética , Isoformas de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Temperatura , Tirosina/metabolismoRESUMEN
A plasmid shuttle vector for Escherichia coli and mycobacteria was constructed from an E. coli plasmid containing the ColE1 origin, a 2.6-kb PstI fragment from bacteriophage D29 that grows in numerous mycobacterial species, and the kanamycin resistance gene either of Tn903 or of Tn5. The resultant plasmid is 7.63 kb and can be introduced via transformation into Mycobacterium smegmatis with high efficiency. In M. smegmatis the plasmid is stable and apparently present in multiple copies. Bioluminescence (luxA and luxB of Vibrio harveyi and fischeri) has been expressed in M. smegmatis from the aminoglycoside transferase promoter of Tn5. The D29 fragment should carry an origin of replication and some associated genes that act on it since various mutations destroy the ability of this fragment to replicate in M. smegmatis. The fragment was localized on the D29 genome map.
Asunto(s)
Escherichia coli/genética , Mycobacterium/genética , Plásmidos , Elementos Transponibles de ADN , Genes Bacterianos , Vectores Genéticos , Resistencia a la Kanamicina/genética , Mediciones Luminiscentes , Mapeo Restrictivo , Transformación Genética , Vibrio/genéticaRESUMEN
A Mycobacterium smegmatis PstI library was constructed by cloning these fragments downstream from the lac promoter of the expression vector pHG171. Three identically sized clones were isolated by complementation of an Escherichia coli strain (chi 2338) deficient in citrate synthase. One insert (pBL265) was used in hybridization experiments with DNA from E. coli and M. smegmatis and it was demonstrated that the clones were indeed from M. smegmatis. The transcription of the M. smegmatis citrate synthase gene in E. coli relied upon the lac promoter. In translation experiments performed in vitro pBL265 gave rise to a novel protein of about 42 kDa. This band was not seen in 'opposite-orientation' subclones. Various subclones in which the 5'-end was shortened nevertheless complement E. coli chi 2338 and produce the 42 kDa protein. This demonstrates that the M. smegmatis citrate synthase gene uses its own ribosome-binding site in E. coli. The relevant 1.8 kb of the 2.8 kb insert was sequenced. A consensus E. coli ribosome-binding site was found centred precisely 10 bp upstream of the methionine codon. Other interesting features revealed by the sequence are discussed. Citrate synthase activity was assayed in vitro and the mycobacterial enzyme was found to be similar to those of the Gram-positive bacteria.
Asunto(s)
Citrato (si)-Sintasa/genética , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Mycobacterium/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Citrato (si)-Sintasa/química , ADN Bacteriano/genética , Escherichia coli/enzimología , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , Mapeo Restrictivo , Ribosomas/metabolismo , Transcripción Genética , Transformación BacterianaRESUMEN
During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.
Asunto(s)
Vectores Genéticos , Plásmidos , Proteínas Bacterianas/biosíntesis , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/genética , Escherichia coli/genética , Proteínas Recombinantes/biosíntesis , beta-Galactosidasa/biosíntesisRESUMEN
The bacteriophage lambda early promoter PR has been used to direct the synthesis of lac alpha in a plasmid containing the multiple cloning site of pUC8. The copy number of the plasmid is controlled by the rop(rom) gene and the plasmid incorporates the cI857 gene for temperature regulation of lac alpha expression. Isolation of recombinant derivatives with DNA inserts in the multiple cloning region can be identified by insertional inactivation of lac alpha and consequently, a Lac- phenotype in a host carrying the M15 deletion of lac. The potential of pHG276 as a fully regulated expression vector is examined.
Asunto(s)
Bacteriófago lambda/genética , Vectores Genéticos , Plásmidos , Clonación Molecular , Enzimas de Restricción del ADN , ADN Bacteriano/análisis , ADN Recombinante/análisis , Genes Virales , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Temperatura , beta-Galactosidasa/biosíntesisRESUMEN
Insolubilized lipopolysaccharides (LPS) were prepared by covalently coupling LPS from polysaccharide-deficient S. minnesota R595 and polysaccharide-rich E. coli 055:B5 to carboxylated latex particles. The stability of these LPS-latex complexes was determined using several assays to detect soluble LPS following incubation at ambient and elevated temperatures. Resident and thioglycollate-elicited macrophages from both LPS-responder C3HeB/FeJ and LPS nonresponder C3H/HeJ mice were examined for their capacity to phagocytose the LPS particles following in vitro culture for various time periods. Uptake was demonstrated by an increase in the number of particles within the macrophages with increasing time of incubation. Rough polysaccharide-deficient LPS-latex particles were found to be more readily phagocytosed than control particles, whereas smooth polysaccharide-rich LPS particles were phagocytosed less readily than the controls. Qualitatively similar results were found in the relative rate of uptake of particles by the macrophages from the endotoxin-responsive and -unresponsive mouse strains used in this study.