A mutant form of vascular endothelial growth factor (VEGF) that lacks VEGF receptor-2 activation retains the ability to induce vascular permeability.

Vascular endothelial growth factor (VEGF) is a major mediator of vasculogenesis and angiogenesis both during development and in pathological conditions. VEGF has a variety of effects on vascular endothelium, including the ability to stimulate endothelial cell mitogenesis, and the potent induction of vascular permeability. These activities are at least in part mediated by binding to two high affinity receptors, VEGFR-1 and VEGFR-2. In this study we have made mutations of mouse VEGF in order to define the regions that are required for VEGFR-2-mediated functions. Development of a bioassay, which responds only to signals generated by cross-linking of VEGFR-2, has allowed evaluation of these mutants for their ability to activate VEGFR-2. One mutant (VEGF0), which had amino acids 83-89 of VEGF substituted with the analogous region of the related placenta growth factor, demonstrated significantly reduced VEGFR-2 binding compared with wild type VEGF, indicating that this region was required for VEGF-VEGFR-2 interaction. Intriguingly, when this mutant was evaluated in a Miles assay for its ability to induce vascular permeability, no difference was found when compared with wild type VEGF. In addition we have shown that the VEGF homology domain of the structurally related growth factor VEGF-D is capable of binding to and activating VEGFR-2 but has no vascular permeability activity, indicating that VEGFR-2 binding does not correlate with permeability activity for all VEGF family members. These data suggest different mechanisms for VEGF-mediated mitogenesis and vascular permeability and raise the possibility of an alternative receptor mediating vascular permeability.

A model for the activation of the epidermal growth factor receptor kinase involvement of an asymmetric dimer?

The binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, which activates the intracellular kinase domain. Homology models of the inactive and active forms of the EGF-receptor kinase domains have been derived, and these models suggest that the active form can be stabilized by the interaction of helix C and the surrounding area in one receptor monomer with one of two possible complementary surfaces on a second receptor monomer. Both hydrophobic interaction sites are strongly conserved within the EGF-receptor family but not in other tyrosine kinases. Two of the three predicted kinase dimers are symmetric; the other is asymmetric and is predicted to contain only one active kinase. One of the symmetric models and the asymmetric model would account for the effects of two mutations in helix C (Y740F and V741G) on kinase activity. They also provide an explanation for previously reported dominant negative mutants of the EGF receptor and have interesting implications for the signaling through homo- and heterodimers of the family members: EGF receptor, erbB2, erbB3, and erbB4.

The human A33 antigen is a transmembrane glycoprotein and a novel member of the immunoglobulin superfamily.

The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and > 95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.

Clinical experience with famciclovir against hepatitis B virus.

Famciclovir (FCV, the oral form of penciclovir, PCV) is a potent antiviral agent of hepatitis B virus (HBV) and is currently in phase III clinical trials. In this review, we examine the outcome of FCV treatment in preventing recurrent HBV in patients post transplantation. Resistance to FCV has now been documented in this setting, in which reduced sensitivity to FCV was associated with mutations upstream from the conserved 'YMDD' motif in the HBV polymerase gene. These mutations are in a region which has been designated as the B domain in RNA-dependent polymerases. To understand these mutations we have developed a model of the catalytic regions of the HBV polymerase and located mutations selected during antiviral treatment on this model.

Phosphorylation events associated with different states of activation of a hepatic cardiolipin/protease-activated protein kinase. Structural identity to the protein kinase N-type protein kinases.

Cardiolipin- or protease-activated protein kinase, isolated from rat liver cytosol and originally named liver PAK-1, was found to be the natural form of protein kinase N (PKN) by comparing the sequences of 43 tryptic peptides of the purified liver enzyme and determining the corresponding liver cDNA sequence. These analyses also identified (i) Arg-546 as the major site of proteolytic activation, (ii) the protease resistance of the C-terminal extension beyond the catalytic domain, and (iii) in vivo stoichiometric phosphorylation of Thr-778 in the mature enzyme. Homology modeling of the catalytic domain indicated that phosphothreonine 778 functions as an anchoring site similar to Thr-197 in cAMP-dependent protein kinase, which stabilizes an active site compatible with preferred substrate sequences of PAK-1/PKN. Sigmoidal autophosphorylation kinetics and increased S6-(229-239) peptide kinase activity following preincubation with ATP suggested phosphorylation-dependent activation of PAK-1/PKN. The onset of activation corresponded with phosphorylation of the regulatory domain site Ser-377 (located within a spectrin homology region), followed by Thr-504 (within a limited protein kinase C homology region), and, to a lesser extent, Thr-64 (in the RhoA-binding region). Several additional sites in the hinge region adjacent to a PEST protein degradation signal were selectively autophosphorylated following cardiolipin activation. Overall, these observations suggest that the regulation of this class of protein kinase involves complex interactions among phosphorylation-, lipid-, and other ligand-dependent activation events.

Structure-function relationships for the EGF/TGF-alpha family of mitogens.

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-alpha) are ligands for the EGF-receptor and act as mitogens for a variety of tissues. TGF-alpha, in particular, has been implicated as an autocrine growth factor for several cancer cell lines. Over the last 10 years many groups have examined the structure-function relationships in EGF/TGF-alpha in attempts to develop antagonists or agonists. In this review the results of these studies are summarised and related to the three-dimensional structure of EGF/TGF-alpha. The difficulties associated with the purification and characterisation of analogues of EGF/TGF-alpha and with the biological assays are discussed. It is clear that these difficulties have, in some cases, led to apparently contradicting results. The available binding data indicate that the receptor interaction surface for EGF/TGF-alpha might encompass one complete side of the molecule with a few strong binding determinants, in particular Arg41 and Leu47. The arginine at position 41 is the most critical residue and its full hydrogen-bonding capacity is needed for strong binding of EGF/TGF-alpha to the EGF-receptor. As this side of the molecule consists of residues from both the N- and C-terminal domain, it seems unlikely that agonists or antagonists can be developed on the basis of short peptides taken from the primary sequence. This concept is supported by the available binding and activity data.