Gregg Crumley

Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation

Heparin is required for fibroblast growth factor (FGF) stimulation of biological responses. Using isothermal titration calorimetry, we show that acidic FGF (aFGF) forms a 1:1 complex with the soluble extracellular domain of FGF receptor (FGFR). Heparin exerts its effect by binding to many molecules of aFGF. The resulting aFGF-heparin complex can bind to several receptor molecules, leading to FGFR dimerization. In two cell lines lacking endogenous heparan sulfate, exogenous heparin is required for FGFR dimerization, tyrosine kinase activation, c-fos mRNA transcription, and cell proliferation. Moreover, a synthetic heparin analog that binds monovalently to aFGF blocks FGFR dimerization, activation, and signaling via FGFR. We propose that heparin causes oligomerization of aFGF such that its binding to FGFR results in dimerization and activation. This represents a novel mechanism for transmembrane signaling and may account for the action of many heparin-bound growth factors.

Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic fibroblast growth factors.

The fibroblast growth factor (FGF) family consists of at least seven closely related polypeptide mitogens which exert their activities by binding and activation of specific cell surface receptors. Unanswered questions have been whether there are multiple FGF receptors and what factors determine binding specificity and biological response. We report the complete cDNA cloning of two human genes previously designated flg and bek. These genes encode two similar but distinct cell surface receptors comprised of an extracellular domain with three immunoglobulin‐like regions, a single transmembrane domain, and a cytoplasmic portion containing a tyrosine kinase domain with a typical kinase insert. The expression of these two cDNAs in transfected NIH 3T3 cells led to the biosynthesis of proteins of 150 kd and 135 kd for flg and bek, respectively. Direct binding experiments with radiolabeled acidic FGF (aFGF) or basic FGF (bFGF), inhibition of binding with native growth factors, and Scatchard analysis of the binding data indicated that bek and flg bind either aFGF or bFGF with dissociation constants of (2‐15) x 10(‐11) M. The high affinity binding of two distinct growth factors to each of two different receptors represents a unique double redundancy without precedence among polypeptide growth factor‐receptor interactions.

Ligand-induced transphosphorylation between different FGF receptors.

Recent evidence shows that different fibroblast growth factors (FGF) bind with similar high affinities to two FGF receptors (FGFR) called flg and bek. In order to explore the mechanism of FGFR tyrosine autophosphorylation, we have generated cell lines which co‐express a kinase‐negative mutant of FGFR and an active form of FGFR. The following transfected NIH 3T3 cells were generated: (i) cells which express a shorter truncated form of bek (two Ig domains) together with a kinase‐negative mutant of full length bek (bek K517A), (ii) cells which express wild‐type bek together with kinase‐negative flg (flg K514A) and (iii) cells co‐expressing wild‐type flg together with bek K517A. Immunoprecipitations with either bek‐or flg‐specific antisera followed by immunoblotting indicated that the double transfectants express the desired receptor species. The addition of acidic FGF (aFGF) to the various cell lines followed by immunoprecipitation with anti‐FGFR antibodies and immunoblotting with anti‐phosphotyrosine specific antibodies indicated that aFGF induces tyrosine phosphorylation of the kinase‐negative FGFR mutants. These results show that tyrosine autophosphorylation of the kinase‐negative FGFR is mediated by a transphosphorylation mechanism and that both homologous (bek‐‐‐‐bek) and heterologous (bek‐‐‐‐flg and flg‐‐‐‐bek) transphosphorylation occurs in living cells. Recent evidence shows that tyrosine autophosphorylation of receptors with tyrosine kinase activities is essential for mediating interactions with signaling molecules. Therefore, heterologous transphosphorylation could amplify the response of cells to various forms of FGFs and their cognate receptors.

BEK, a receptor for multiple members of the fibroblast growth factor (FGF) family, maps to human chromosome 10q25.3→q26

The gene for the fibroblast growth factor receptor BEK was assigned to human chromosome 10 by applying polymerase chain reaction techniques to DNAs from a panel of human x rodent somatic cell hybrids. The gene was further localized to 10q25.3----q26 by in situ hybridization.

Effect of heparin on the binding affinity of acidic FGF for the cloned human FGF receptors, flg and bek.

Heparin potentiates the mitogenic activity of acidic fibroblast growth factor (aFGF) by 20-100 fold but mechanisms detailing this potentiation have not yet been fully elucidated. We report that heparin increases the binding affinity of aFGF for the two cloned and overexpressed human FGF receptors, flg and bek, by 2-3 fold. This increase in binding affinity, together with previous data demonstrating a 3-5 fold increase in the stability of aFGF, are likely to account for a significant portion of heparins potentiation of aFGF activity observed in biological assay systems.

Modulation of expression of the human gamma interferon gene in E.coli by site-directed mutagenesis

Plasmids expressing 2 forms of human immune interferon (IFN-gamma) in E. coli have been constructed: 1) pIFNTacI which expresses IFN-gamma with an N-terminal amino acid sequence of met-cys-tyr-cys-gln-, and 2) pIFNTacII which is a derivative of pIFNTacI from which the 9 base pairs (bp) coding for the cys-tyr-cys have been deleted. Quantitation of Western blots showed that approximately 10-fold more IFN-gamma was produced in cells harboring pIFNTacII (7.5% of total cellular protein) as compared to pIFNTacI. The IFN-gamma expressed in E. coli pIFNTacII is biologically active and routinely recoverable at 10(9) units per liter. When examined microscopically, IPTG induced E. coli harboring either plasmid construction contains prominent cytoplasmic inclusion bodies.

Analysis and Transfection of Human Acidic Fibroblast Growth Factor (aFGF) cDNA Clones and Structural Analysis of the Human aFGF Gene reveals Features Shared within a Family of Homologous Proteins

“Acidic fibroblast growth factor” (aFGF) refers to a group of micro-heterogeneous anionic polypeptides which are mitogenic for a variety of cells derived from the embryonic mesoderm and neuroectoderm, most notably endothelial cells [reviewed in (1)]. The micro-heterogeneity is due to specific proteolytic cleavages which result in polypeptides of 134, 140, and 154 residues from the 155 residue primary translation product (2,3). aFGF was originally isolated from neural tissue [reviewed in (4)] however its isolation from non-neural tissues such as kidney (5), heart (6), smooth muscle cells (7), bone (8) and foreskin fibroblasts (7) demonstrates a wider tissue distribution than originally thought. aFGF shows 55% amino acid sequence identity with basic fibroblast growth factor (bFGF), a group of micro-heterogeneous cationic polypeptides derived from similar cleavages of a 155 residue primary translation produce (9). The broad range of biological effects of aFGF and bFGF are very similar (see below). Cellular receptors having tyrosine kinase activity have been identified which interact with both aFGF and bFGF (10–13). Three new oncogenes have recently been identified — hst (14,15), int-2 (16,17) and FGF-5 (18) which encode proteins homologous to aFGF and bFGF.