Common and specific determinants for fibroblast growth factors in the ectodomain of the receptor kinase complex.

The assembly and activation of oligomeric complexes of FGF, the transmembrane receptor kinase (FGFR), and heparan sulfate transmit intracellular signals regulating growth and function of cells. An understanding of the structural relationships between the three subunits and their redundancy and specificity is essential for understanding the ubiquitous FGF signaling system in health and disease. Previously, we reported that a primary heparin or heparan sulfate binding site resides in a distinct sequence in immunoglobulin (Ig)-like module II of the three modules of FGFR. Here we report that in the absence of flanking sequences, isolated Ig module II of FGFR1 supports the binding of FGF-1, FGF-2, and FGF-7 in respective order of affinity. None of the three FGFs detectably bind Ig module I or the IIIb and IIIc splice variants of Ig module III in the absence of flanking sequences. Ig module I and the C-terminus of Ig module III are dispensable for high-affinity binding of FGF-1, FGF-2, and FGF-7. Alterations in highly conserved Ig module II in the heparin binding domain and substitution of individual sequence domains spanning the entire sequence of Ig module II with those from Ig module I obliterated FGF binding. Addition of a specific number of FGFR sequences to the C-terminus of Ig module II resulted in a gain in affinity for FGF-7. Several site-specific alterations in the C-terminus of full-length FGFR1IIIc, an isoform that otherwise absolutely rejects FGF-7, resulted in gain of FGF-7 binding. These results suggest that a complex of Ig module II and heparan sulfate is the base common active core of the FGFR ectodomain and that flanking structural domains modify FGF affinity and determine specificity.

A homeo-interaction sequence in the ectodomain of the fibroblast growth factor receptor.

Interaction of fibroblast growth factor receptors (FGFR) sufficient for a trans-phosphorylation event in which one intracellular domain is substrate for the other is essential for signal transduction. By analysis of the direct interaction of recombinant constructions co-expressed in baculoviral-infected insect cells, we identified a 17-amino acid sequence that is required for the stable interaction between ectodomains of FGFR. The sequence 160ERSPHRPILQAGLPANK176 (Glu160-Lys176) connects immunoglobulin modules II and III. In insect cells, the interaction between Glu160-Lys176 domains occurs independently of intact heparin or FGF binding domains. The sequence is not required for the binding of heparin or FGF-1, but is essential for mitogenic activity of the FGFR kinase in mammalian cells. The results support a model in which the homeo-interaction between Glu160-Lys176 in the ectodomain contributes to the interaction between intracellular domains in mammalian cell membranes (Kan, M., Wang, F., Kan, M., To, B., Gabriel, J. L., and McKeehan, W. L. (1996) J. Biol. Chem. 271, 26143-26148). We propose that the Glu160-Lys176 domain plays a pivotal role in restriction of the interaction between kinases by pericellular matrix heparan sulfate proteoglycan and divalent cations. Restrictions are overcome by FGF or constitutively by diverse gain of function mutations which cause skeletal and craniofacial abnormalities.

Inhibin antagonizes inhibition of liver cell growth by activin by a dominant-negative mechanism.

The beta:beta activin homodimer and alpha:beta inhibin heterodimer are mutual antagonists which share a common beta subunit. Recently, it has been shown that, similar to transforming growth factor-beta 1, activin is an inhibitor of hepatocyte DNA synthesis. The activin receptor appears to be an obligatory complex of genetically distinct type I and II transmembrane serine/threonine kinases. Activin type I receptors, SKR1 and SKR2, were first cloned from well differentiated human hepatoma cells (HepG2). This prompted us to investigate the binding of activin and inhibin to receptors from HepG2 cells and the effect of the two ligands on DNA synthesis. Here we show that beta:beta activin binds to the activin type II receptor kinase (ActRII) which induces activin binding to the type I receptor kinase SKR2 to form ActRII.beta:beta.SKR2 complexes in which an activin beta chain occupies each receptor subunit. Inhibin also binds to ActRII through its beta subunit, competes with the binding of activin to ActRII, but fails to form the ActRII.SKR2 complex. No specific binding site for inhibin could be demonstrated in HepG2 cells. Inhibin, which had no activity of its own, antagonized the inhibitory effect of activin on DNA synthesis. The results suggest that inhibin may be a natural antagonist of assembly of the heterodimeric activin receptor complex through a dominant-negative mechanism.

Genomic structure and cloned cDNAs predict that four variants in the kinase domain of serine/threonine kinase receptors arise by alternative splicing and poly(A) addition.

Heterodimers of types I and II serine/threonine kinase receptor monomers compose the active receptor complex for ligands of the transforming growth factor beta family. Here we show that the genomic organization of coding sequences for the intracellular domain of a widely expressed type I serine/threonine kinase receptor is similar to that of the activin type II receptor gene. The genomic structure and cDNA clones indicate that poly(A) addition to alternative exons at each of three carboxyl-terminal coding exon-intron junctions may be a common feature of both type I and II receptor genes. The predicted products are monomers truncated at kinase subdomains VII, IX, and X which vary in kinase activity and potential serine, threonine, and tyrosine phosphorylation sites. These results suggest that combinations of variants that affect the signal-transducing intracellular kinase domain of both type I and II receptor monomers within the transforming growth factor beta ligand family may add to the heterogeneity of biological effects of individual ligands in the family.

Identification of tyrosines 154 and 307 in the extracellular domain and 653 and 766 in the intracellular domain as phosphorylation sites in the heparin-binding fibroblast growth factor receptor tyrosine kinase (flg).

Four tyrosine residues have been identified as phosphorylation sites in the tyrosine kinase isoform of the heparin-binding fibroblast growth factor receptor flg (FGF-R1). Baculoviral-insect cell-derived recombinant FGF-R1 was phosphorylated and fragmented with trypsin while immobilized on heparin-agarose beads. Phosphotyrosine peptides were purified by chromatography on immobilized anti-phosphotyrosine antibody and analyzed by Edman degradation and electrospray tandem mass spectrometry. Tyrosine residue 653, which is in a homologous spatial position to major autophosphorylation sites in the catalytic domain of the src and insulin receptor kinases, is the major intracellular FGF-R1 phosphorylation site. Residue 766 in the COOH-terminus outside the kinase domain is a secondary site. Tyrosine residues 154 and 307, which are in the extracellular domain of transmembrane receptor isoforms and are in an unusual sequence context for tyrosine phosphorylation, were also phosphorylated.

Gaps and contract: evaluating the diffusion of new information. Part I. A description of the strategy.

Time and fiscal constraints on oncology nurses require that continuing education programs yield demonstrable benefit to the clinician, the institution, and patient outcomes. This article describes "gaps and contract," a strategy that provides nurses with a tool to transfer knowledge from theory to practice, in a measurable form. The framework for this strategy lies within the concept of innovation diffusion. This strategy was used by two universities collaborating to educate 912 oncology health professionals in 38 continuing education programs. Part II of this article will describe the measurement of this strategy.

Gaps and contract: evaluating the diffusion of new information. Part II. The measurement of the strategy.

This article is the second of a two-part series describing "gaps and contract," a strategy that provides nurses with a mechanism to transfer knowledge from theory into practice in a measurable format. Two universities under contract from the Pennsylvania state cancer plan used this strategy to evaluate the effectiveness of a cancer continuing education program. Two hundred seventy-four (274) contracts from one setting and 205 from a second setting are described. Recommendations for using this strategy to measure the efficacy of cancer continuing education programs are included.

Substitution of putative half-cystine residues in heparin-binding fibroblast growth factor receptors. Loss of binding activity in both two and three loop isoforms.

Alternate use of an exon coding for an 89-residue NH2 terminal immunoglobulin-like disulfide loop results in isoforms of the heparin-binding fibroblast growth factor receptor (FGF-R) with three (FGF-R alpha) and two (FGF-R beta) Ig-like loops in the extracellular domain. Both FGF-R alpha and FGF-R beta isoforms exhibit qualitatively similar ligand-binding activities. In this report, we show by site-directed mutagenesis and analysis of ligand-binding activity in transfected cells that substitution of a cysteine that potentially forms an intra-loop disulfide in either juxtamembrane Loop II or III disrupted maturation and formation of the ligand-binding site in both FGF-R alpha and FGF-R beta isoforms. Neither three loop FGF-R alpha constructions coding for intact Loops I and II adjacent to defective Loop III nor intact Loops I and III separated by defective Loop II exhibited ligand-binding activity. In addition, a two-loop molecule of tandem Loops I and III was inactive. The results suggest that single Loops I, II, or III of FGF-R are insufficient to form a ligand-binding site. Loop I does not form an independent ligand-binding site with either Loop II or III, but interacts with a common ligand-binding site formed by Loops II and III (Xu, J., Nakahara, M., Crabb, J. W., Shi, E., Matuo, Y., Fraser, M., Kan, M., Hou, J., and McKeehan, W. L. (1992) J. Biol. Chem. 267, 17792-17803, 1992).

Fibroblast growth factor receptors from liver vary in three structural domains.

Changes in heparin-binding fibroblast growth factor gene expression and receptor phenotype occur during liver regeneration and in hepatoma cells. The nucleotide sequence of complementary DNA predicts that three amino-terminal domain motifs, two juxtamembrane motifs, and two intracellular carboxyl-terminal domain motifs combine to form a minimum of 6 and potentially 12 homologous polypeptides that constitute the growth factor receptor family in a single human liver cell population. Amino-terminal variants consisted of two transmembrane molecules that contained three and two immunoglobulin-like disulfide loops, as well as a potential intracellular form of the receptor. The two intracellular juxtamembrane motifs differed in a potential serine-threonine kinase phosphorylation site. One carboxyl-terminal motif was a putative tyrosine kinase that contained potential tyrosine phosphorylation sites. The second carboxyl-terminal motif was probably not a tyrosine kinase and did not exhibit the same candidate carboxyl-terminal tyrosine phosphorylation sites.

Two apparent human endothelial cell growth factors from human hepatoma cells are tumor-associated proteinase inhibitors.

Two polypeptides from secretory products of human hepatoma cells were isolated and characterized on the basis of their stimulation of maintenance and growth of human endothelial cells in serum-free cell culture. Both factors were purified to homogeneity by a combination of reverse-phase, ion exchange, and molecular filtration high performance liquid chromatography. One factor (endothelial cell growth factor (ECGF-2a) had Mr approximately 6,500 and pI near 6. The second (ECGF-2b) had Mr = 27,000 and a pI below 4.0. Both ECGF-2a and ECGF-2b exhibited single NH2-terminal sequences. The first 25 NH2-terminal residues of ECGF-2a and the first 49 residues of ECGF-2b were determined by gas-phase microsequencing. All clearly determined residues of ECGF-2a were identical with human pancreatic secretory trypsin inhibitor. All assignable residues of ECGF-2b were identical with urinary glycoprotein proteinase inhibitor (HI-30/EDC1). Both proteins are absent or at low levels in normal plasma and urine, but appear during acute inflammatory disease and cancer. Amino acid composition of ECGF-2a and ECGF-2b was also similar to human pancreatic secretory inhibitor and HI-30/EDC1, respectively. Both ECGF-2a and ECGF-2b inhibited bovine pancreatic trypsin (2 micrograms/ml) by 50% at 750 ng/ml. ECGF-2a and ECGF-2b stimulated endothelial cell number at a half-maximal dose of 50 ng/ml (8 nM) and 80 to 130 ng/ml (5 to 9 nM) protein, respectively. When assayed under identical conditions, no effect of either factor on human smooth muscle cells, human hepatoma cells, or human, rat, and mouse fibroblasts could be detected.

Changes in NAD(P)+-dependent malic enzyme and malate dehydrogenase activities during fibroblast proliferation.

A sensitive isotope exchange method was developed to assess the requirements for and compartmentation of pyruvate and oxalacetate production from malate in proliferating and nonproliferating human fibroblasts. Malatedependent pyruvate production (malic enzyme activity) in the particulate fraction containing the mitochondria was dependent on either NAD+ or NADP+. The production of pyruvate from malate in the soluble, cytosolic fraction was strictly dependent on NADP+. Oxalacetate production from malate (malate dehydrogenase, EC 1.1.1.37) in both the particulate and soluble fraction was strictly dependent on NAD+. Relative to nonproliferating cells, NAD+-linked malic enzyme activity was slightly reduced and the NADP+-linked activity was unchanged in the particulate fraction of serum-stimulated, exponentially proliferating cells. However, a reduced activity of particulate malate dehydrogenase resulted in a two-fold increase in the ratio of NAD(P)+-linked malic enzyme to NAD+-linked malate dehydrogenase activity in the particulate fraction of proliferating fibroblasts. An increase in soluble NADP+-dependent malic enzyme activity and a decrease in NAD+-linked malate dehydrogenase indicated an increase in the ratio of pyruvate-producing to oxalacetate-producing malate oxidase activity in the cytosol of proliferating cells. These coordinate changes may affect the relative amount of malate that is oxidized to oxalacetate and pyruvate in proliferating cells and, therefore, the efficient utilization of glutamine as a respiratory fuel during cell proliferation.

Extracellular regulation of fibroblast multiplication. Quantitative differences in nutrient and serum factor requirements for multiplication of normal and SV40 virus-transformed human lung cells.

The principles of Henri-Michaelis-Menten kinetic analysis were applied to directly relate the concentration of serum growth factors and individual nutrients in the culture medium to the multiplication rate of a population of normal (N-HLF) and SV40 virus-transformed (SV-HLF) human lung fibroblasts. When all nutrient concentrations were optimal and in steady state, the concentration of serum factors that was required to support a half-maximal rate of proliferation of both N-HLF and SV-HLF was similar. When the serum factor concentration was optimal and constant, SV-HLF cells exhibited a reduced requirement (p less than 0.001) for 12 of 27 individual nutrients that were examined. Serum factors control the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of N-HLF (McKeehan, W. L., and McKeehan, K. A. (1980) Proc. Natl, Acad, Sci, U. S. A. 77, 3417-3421). SV-HLF exhibited a constitutively reduced requirement for Ca2+, K+, and Mg2+ which partially removed the requirement for the 3 ions for multiplication of SV-HLF from the control of serum factors. The results suggest that SV40 virus transformation confers a growth advantage on human lung fibroblasts by alteration of their quantitative requirements for specific nutrients.

Calcium, magnesium, and serum factors in multiplication of normal and transformed human lung fibroblasts.

Serum factors determine the extracellular requirement for both Ca2+ and Mg2+ for multiplication of normal human lung fibroblasts in vitro. Serum factors also affect the extracellular Ca2+ requirement for transformed fibroblasts but to a different extent than for normal cells. Transformed cells exhibit a reduced requirement for both Ca2+ and Mg2+ for multiplication. The apparent reduction in Ca2+ requirement of transformed cells is dependent on the level of serum factors in the medium. The reduced Mg2+ requirement for transformed cells is more striking than the loss of Ca2+ and independent of the level of serum factors in the medium. A sequential effector relationship among serum factors, Ca2+ and Mg2+, in a proliferative control system for normal cells is proposed. Alteration or bypass of an intracellular Mg2+-requiring process is proposed as a major lesion in the transformed cells. This alteration causes an observed loss of requirements for both Ca2+ and serum factors for the multiplication of transformed cells.

Serum factors modify the cellular requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for multiplication of normal human fibroblasts.

The rate of multiplication of a population of cultured human lung fibroblasts is determined by the level of common nutrients and serum factors in the medium. By application of the principles of Henri--Michaelis--Menten kinetic analysis to cell growth in vitro, the relationship between the level of a macromolecular fraction of serum that contains growth factors and the concentration of individual nutrient that is required to support a half-maximal rate of cell multiplication was explored. The requirement for the majority of individual nutrients is independent of the level of serum factors in the medium. This includes all 20 amino acids, glucose, purines, pyrimidines, polyamines, choline, and inositol. In contrast, serum factors determine the cellular requirement for Ca2+, K+, Mg2+, Pi, and 2-oxocarboxylic acids. These nutrients are most likely involved in cellular processes related to the mechanism by which growth factors in serum control the cell multiplication rate.