Human vascular endothelial growth factor B: characterization of recombinant isoforms and generation of neutralizing monoclonal antibodies.

1. The vascular endothelial growth factor (VEGF) family is a focus of interest with respect to novel therapies for cardiovascular disease. Members of this family bind differentially to three receptor tyrosine kinases, namely VEGF-R1, -R2 and -R3, and to the semaphorin receptors neuropilin 1 and 2. The role of VEGF-R1 and the factors that interact exclusively with this receptor (VEGF-B and placenta growth factor) has remained controversial. 2. To further elucidate the role of VEGF-B in blood vessel formation and function, we have expressed, purified and refolded both naturally occurring VEGF-B isoforms and a truncated amino acid 10-108 form. All refolded proteins have been demonstrated to bind to VEGF-R1 with appropriate kinetics in biosensor-based analysis. 3. Robust cell assays for VEGF-R1 ligands, such as VEGF-B, have been problematic. We have developed an assay based on a chimeric receptor consisting of extracellular domains 1-4 of VEGF-R1 and the transmembrane and intracellular domains of gp130. The cell line expresses luciferase to high levels 24 h after exposure to VEGF-A and both refolded VEGF-B167 and the short 10-108 isoform have been demonstrated to be active in this assay. 4. The novel cell-based assay, in combination with a variety of immunochemical approaches, has been used to identify and characterize monoclonal antibodies that neutralize VEGF-B activity.

Turn up the HEAT.

The recently determined crystal structure of the PR65/A subunit of protein phosphatase 2A reveals the architecture of proteins containing HEAT repeats. The structural properties of this solenoid protein explain many functional characteristics and account for the involvement of solenoids as scaffold, anchoring and adaptor proteins.