Direct identification of a major autophosphorylation site on vascular endothelial growth factor receptor Flt-1 that mediates phosphatidylinositol 3'-kinase binding.

Progress has been made in our understanding of the mechanism by which the binding of vascular endothelial growth factor (VEGF) to cognate receptors induces a range of biological responses, but it is far from complete. Identification of receptor autophosphorylation sites will allow us to determine how activated VEGF receptors are coupled to specific downstream signalling proteins. In the present study, we have expressed human VEGF receptors in insect cells using the baculovirus expression system, identified a major autophosphorylation site on the VEGF receptor fms-like tyrosine kinase-1 (Flt-1) by HPLC-electrospray ionization (ESI)-MS, and characterized in vitro interactions between Flt-1 and phosphatidylinositol 3'-kinase (PI3-kinase). Infection of High 5 insect cells with Flt-1 recombinant virus resulted in the expression of a 170 kDa glycoprotein, which bound VEGF with a K(d) of 2 x 10(-10) M in intact insect cells. The overexpressed recombinant Flt-1 receptors exhibited tyrosine kinase activity and were constitutively phosphorylated. Analysis of Flt-1 tryptic peptides by HPLC-ESI-MS with selective phosphate ion monitoring identified a hexapeptide (YVNAFK; where single-letter amino-acid code has been used) containing a phosphotyrosine (pTyr) residue at position 1213. Using synthetic phosphopeptides, this pTyr residue was found to be directly involved in the binding of PI3-kinase in vitro even though it did not fall within a consensus pYM/VXM PI3-kinase binding motif. These results suggest that phosphorylated Flt-1 associates with PI3-kinase at pTyr(1213) to mediate the activation of this pathway in VEGF signalling.

Characterization of the VEGF binding site on the Flt-1 receptor.

The angiogenic growth factor VEGF binds to the receptor tyrosine kinases Flt-1 and KDR/Flk-1. Immunoglobulin (Ig)-like loop-2 of Flt-1 is involved in binding VEGF, but the contribution of other Flt-1 Ig-loops to VEGF binding remains unclear. We tested the ability of membrane-bound chimeras between the extracellular domain of Flt-1 and the cell adhesion molecule embigin to bind VEGF. VEGF bound as well to receptors containing Flt-1 loops 1-2 or 2-3 as it did to the entire Flt-1 extracellular domain. Chimeras containing only loop-2 of Flt-1 bound VEGF with 22-fold lower affinity. We conclude that high-affinity VEGF binding requires Ig-like loop-2 plus either loop-1 or loop-3. In addition, Flt-1 amino acid residues Arg-224 and Asp-231 were not essential for high-affinity binding of VEGF to membrane-bound Flt-1.

Androgen-dependent expression of fibroblast growth factor-1 in submaxillary gland of mouse.

We have purified a 16,000 dalton protein that stimulates growth of human umbilical cord vein-derived endothelial cells (HUV-EC) from mouse submaxillary glands by using heparin-Sepharose affinity and C4 reverse phase chromatography. The purified molecule was identified as an FGF-1 on the basis of its biological activities, its affinity for heparin and its N-terminal amino-acid sequence. The concentrations of FGF-1 in the submaxillary gland of male or testosterone-treated female mice were about 12 times those of untreated females or castrated males. The 2.3 and 4.1 kb FGF-1 mRNAs were expressed in the glands of male mice older than 4 weeks but not in the glands of female mice. These results suggest that FGF-1 may have important functions for growth, differentiation and development of mouse submaxillary glands, and it may act as an endocrine hormone.

A novel heparin-binding protein, HBp15, is identified as mammalian ribosomal protein L22.

A 15kDa-protein (HBp15) was purified from mouse submandibular gland and bovine brain by virtue of its heparin-binding property. The amino acid sequences of mouse and bovine HBp15 showed a high degree of homology to a sea urchin protein encoded by gene called "development specific protein 217." Using reverse transcription-polymerase chain reaction methods, cDNA clones for HBp15 were isolated from submandibular gland mRNA of mouse, human and pig, and sequenced. Database search of HBp15 showed that HBp15 also resembles yeast ribosomal protein YL31 in addition to the 217 protein. Using specific antibodies against HBp15, HBp15 was identified as mammalian ribosomal protein L22, for which no sequence information is available.

Intramitotic variation in proliferative potential: stochastic events in cellular aging.

A series of mitotic pairs of cells were isolated and the proliferative potential of each cell determined. Although the proliferative potentials of the two cells of a single pair were similar for most pairs examined, in a significant proportion large differences were observed. These results suggest a strong stochastic component in the somatic inheritance of proliferative potential. This observation provides the basis for refinement of a mathematical model for in vitro cellular aging.

In vitro proliferation and lifespan of bovine aorta endothelial cells: response to conditioned media.

The effect of conditioned medium on the growth rate of bovine aorta endothelial cells (CSC311) was examined. Media conditioned by several cell types (both immortal and normal) was found to increase the growth rate of CSC311 cells. The growth rate of CSC311 was increased even when the medium was conditioned by other CSC311 cells. The rate of growth of unstimulated CSC311 cells is 0.43 population doublings (p.d./day. Conditioned medium from several cell types gave a maximum stimulated growth rate of 1.3 p.d./day. In addition, we present evidence for an increase in the degree of stimulation by the growth-promoting activity found in conditioned medium with increasing in vitro age of CSC311 cells.

A general method for determining the replicative age of normal animal cell cultures.

The percentage of cells capable of forming colonies of at least a given size has been found to provide a reliable estimate of the remaining doubling potential of several human diploid fibroblast cell lines and for chick embryo fibroblasts. In five independently derived cell lines of human diploid fibroblasts we have found the same linear relationship between population doublings remaining and the percentage of cells able to form colonies of at least 16 cells. For chick embryo fibroblasts there is a linear relationship between the percentage of cells forming colonies of 64 or more cells and remaining proliferative potential. When human diploid fibroblasts were grown in medium containing hydrocortisone at 5 microgram/ml the increased in vitro lifespan was reflected in the colony size distribution long before the end of the in vitro lifespan.

Intraclonal variation in proliferative potential of human diploid fibroblasts: stochastic mechanism for cellular aging.

At several points during the growth of a clone of human embryonic lung fibroblasts in vitro, 100 to 200 cells were removed at random and the proliferative potential of each cell was determined. At each sample point, a wide variation in remaining population doubling ability was observed among the individual cells and the distributions of doubling potentials were distinctly bimodal. Furthermore, the two cells arising from a single mitosis differed in their ability to proliferate by as many as eight population doublings (256-fold in the number of cells produced). The results suggest that a stochastic process is responsible for determining the limited proliferative potential of human embryonic lung fibroblasts.