John H. Walker

Intrinsic Tyrosine Kinase Activity is Required for Vascular Endothelial Growth Factor Receptor 2 Ubiquitination, Sorting and Degradation in Endothelial Cells

The human endothelial vascular endothelial growth factor receptor 2 (VEGFR2/kinase domain region, KDR/fetal liver kinase‐1, Flk‐1) tyrosine kinase receptor is essential for VEGF‐mediated physiological responses including endothelial cell proliferation, migration and survival. How VEGFR2 kinase activation and trafficking are co‐coordinated in response to VEGF‐A is not known. Here, we elucidate a mechanism for endothelial VEGFR2 response to VEGF‐A dependent on constitutive endocytosis co‐ordinated with ligand‐activated ubiquitination and proteolysis. The selective VEGFR kinase inhibitor, SU5416, blocked the endosomal sorting required for VEGFR2 trafficking and degradation. Inhibition of VEGFR2 tyrosine kinase activity did not block plasma membrane internalization but led to endosomal accumulation. Lysosomal protease activity was required for ligand‐stimulated VEGFR2 degradation. Activated VEGFR2 codistributed with the endosomal hepatocyte growth factor‐regulated tyrosine kinase substrate (Hrs)/signal‐transducing adaptor molecule (STAM) complex in a ligand and time‐dependent manner, implying a role for this factor in sorting of ubiquitinated VEGFR2. Increased tyrosine phosphorylation of the Hrs subunit in response to VEGF‐A links VEGFR2 activation and Hrs/STAM function. In contrast, VEGFR2 in quiescent cells was present on both the endothelial plasma membrane and early endosomes, suggesting constitutive recycling between these two compartments. This pathway was clathrin‐linked and dependent on the AP2 adaptor complex as the A23 tyrphostin inhibited VEGFR2 trafficking. We propose a mechanism whereby the transition of endothelial VEGFR2 from a constitutive recycling itinerary to a degradative pathway explains ligand‐activated receptor degradation in endothelial cells. This study outlines a mechanism to control the VEGF‐A‐mediated response within the vascular system.

The lectin-like oxidized low-density-lipoprotein receptor: a pro-inflammatory factor in vascular disease

Scavenger receptors are membrane glycoproteins that bind diverse ligands including lipid particles, phospholipids, apoptotic cells and pathogens. LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is increasingly linked to atherosclerotic plaque formation. Transgenic mouse models for LOX-1 overexpression or gene knockout suggests that LOX-1 contributes to atherosclerotic plaque formation and progression. LOX-1 activation by oxidized LDL (low-density lipoprotein) binding stimulates intracellular signalling, gene expression and production of superoxide radicals. A key question is the role of leucocyte LOX-1 in pro-atherogenic lipid particle trafficking, accumulation and signalling leading to differentiation into foam cells, necrosis and plaque development. LOX-1 expression is elevated within vascular lesions and a serum soluble LOX-1 fragment appears diagnostic of patients with acute coronary syndromes. LOX-1 is increasingly viewed as a vascular disease biomarker and a potential therapeutic target in heart attack and stroke prevention.

The regulation of neurotransmitter secretion by protein kinase C

The effect of protein kinase C(PKC) on the release of neurotransmitters from a number preparations, including sympathetic nerve endings, brain slices, synaptosomes, and neuronally derived cell lines, is considered. A comparison is drawn between effects of activation of PKC on neurotransmitter release from small synaptic vesicles and large dense-cored vesicles. The enhancement of neurotransmitter release is discussed in relation to the effect of PKC on:1.Rearrangement of the F-actin-based cytoskeleton, including the possible role of MARCKS in this process, to allow access of large dense-cored vesicles to release sites on the plasma membrane.2.Phosphorylation of key components in the SNAP/SNARE complex associated with the docking and fusion of vesicles at site of secretion.3.Ion channel activity, particularly Ca2+ channels.

Ligand-Stimulated VEGFR2 Signaling is Regulated by Co-Ordinated Trafficking and Proteolysis

Vascular endothelial growth factor A (VEGF‐A)‐induced signaling through VEGF receptor 2 (VEGFR2) regulates both physiological and pathological angiogenesis in mammals. However, the temporal and spatial mechanism underlying VEGFR2‐mediated intracellular signaling is not clear. Here, we define a pathway for VEGFR2 trafficking and proteolysis that regulates VEGF‐A‐stimulated signaling and endothelial cell migration. Ligand‐stimulated VEGFR2 activation and ubiquitination preceded proteolysis and cytoplasmic domain removal associated with endosomes. A soluble VEGFR2 cytoplasmic domain fragment displayed tyrosine phosphorylation and activation of downstream intracellular signaling. Perturbation of endocytosis by the depletion of either clathrin heavy chain or an ESCRT‐0 subunit caused differential effects on ligand‐stimulated VEGFR2 proteolysis and signaling. This novel VEGFR2 proteolysis was blocked by the inhibitors of 26S proteasome activity. Inhibition of proteasome activity prolonged VEGF‐A‐induced intracellular signaling to c‐Akt and endothelial nitric oxide synthase (eNOS). VEGF‐A‐stimulated endothelial cell migration was dependent on VEGFR2 and VEGFR tyrosine kinase activity. Inhibition of proteasome activity in this assay stimulated VEGF‐A‐mediated endothelial cell migration. VEGFR2 endocytosis, ubiquitination and proteolysis could also be stimulated by a protein kinase C‐dependent pathway. Thus, removal of the VEGFR2 carboxyl terminus linked to phosphorylation, ubiquitination and trafficking is necessary for VEGF‐stimulated endothelial signaling and cell migration.

Annexins—New family of Ca2+-regulated-phospholipid binding protein

Calcium and phospholipid binding proteins have been identified and localized by immunocytochemistry in a wide range of cells and tissues. Two of these proteins (calpactins) also bind F-actin and are substrates for tyrosine kinases. The similar membrane-binding properties of these molecules arise from conserved amino acid sequences and a model is proposed for the tertiary structure of a common calcium and phospholipid binding domain.

The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non‐receptor proteins including the receptor‐associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one‐ and two‐dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane‐bound acetylcholine receptor and the cytoskeleton.

The use of the human neuroblastoma SH-SY5Y to study the effect of second messengers on noradrenaline release

1. Recent data suggesting that the human neuroblastoma SH-SY5Y is a suitable cell line in which to study the effect of second messengers on NA release are discussed in the context of current views on exocytosis. 2. Release of NA is evoked by depolarization, as well as activation of muscarinic (M3) and bradykinin (B2) receptors in SH-SY5Y cells which have not been differentiated by the addition of growth factors. 3. Evoked release is enhanced by activation of protein kinase C. 4. Activation of protein kinase C decreases the changes in intracellular calcium evoked by carbachol, bradykinin and 100 mM K+. 5. SH-SY5Y express N-type and L-type voltage sensitive Ca2+ channels. L-Type Ca(2+)-channels are coupled to NA release under conditions of weak depolarization. However with strong depolarization (100 mM K+) both L-type and N-type channels are involved. 6. Muscarinic- and neuropeptide Y receptors are coupled to the inhibition of Ca2+ channel activity.

Identification of calcium-dependent phospholipid-binding proteins in higher plant cells

Calcium‐dependent phospholipid‐binding proteins of apparent M r 33 000 and 35 000 were isolated from suspension cultures of tomato cells. Two‐dimensional gel electrophoresis showed the proteins to have isoelectric points of approx. 5.7 and 5.6, respectively. In the presence of calcium, both proteins bound to liposomes formed from a mixture of phosphatidylserine and phosphatidylcholine, but not to liposomes of phosphatidylcholine alone. Both proteins showed immunological similarities to previously characterized calcium‐dependent phospholipid‐binding proteins (annexins) from Torpedo marmorata and mammalian species. The protein of M r 33 000 cross‐reacted with three separate antisera raised to the annexin Torpedo calelectrin, whereas that of M r 35 000 cross‐reacted with antisera to the bovine annexins p68 and p32/34. We suggest that the two proteins may represent the first identification in higher plants of the annexin family of calcium‐dependent phospholipid‐binding proteins.

LOX-1 scavenger receptor mediates calcium-dependent recognition of phosphatidylserine and apoptotic cells

The LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) scavenger receptor regulates vascular responses to oxidized-low-density-lipoprotein particles implicated in atherosclerotic plaque formation. LOX-1 is closely related to C-type lectins, but the mechanism of ligand recognition is not known. Here we show that human LOX-1 recognizes a key cellular phospholipid, PS (phosphatidylserine), in a Ca2+-dependent manner, both in vitro and in cultured cells. A recombinant, folded and glycosylated LOX-1 molecule binds PS, but not other phospholipids. LOX-1 recognition of PS was maximal in the presence of millimolar Ca2+ levels. Mg2+ was unable to substitute for Ca2+ in LOX-1 binding to PS, indicating a Ca2+-specific requirement for bivalent cations. LOX-1-mediated recognition of PS-containing apoptotic bodies was dependent on Ca2+ and was decreased to background levels by bivalent-cation chelation, LOX-1-blocking antibodies or PS-containing liposomes. The LOX-1 membrane protein is thus a Ca2+-dependent phospholipid receptor, revealing novel recognition of phospholipids by mammalian lectins.

The Ca2+-dependent lipid binding domain of P120GAP mediates protein-protein interactions with Ca2+-dependent membrane-binding proteins. Evidence for a direct interaction between annexin VI and P120GAP.

The CaLB domain is a 43-amino acid sequence motif found in a number of functionally diverse signaling proteins including three Ras-specific GTPase activating proteins (GAPs). In the Ras GTPase activating protein, P120GAP, this domain has the ability to confer membrane association in response to intracellular Ca2+ elevation. Here we have isolated three proteins, p55, p70, and p120, which interact with the P120GAP CaLB domain in vitro. We identify p70 as the Ca2+-dependent phospholipid-binding protein annexin VI. Using co-immunoprecipitation studies, we have shown that the interaction between P120GAP and annexin VI is also detectable in rat fibroblasts, suggesting that this interaction may have a physiological role in vivo. Thus, the CaLB domain in P120GAP appears to have the ability to direct specific protein-protein interactions with Ca2+-dependent membrane-associated proteins. In addition, annexin VI is known to have tumor suppressor activity. Therefore, it is possible that the interaction of annexin VI with P120GAP may be important in the subsequent modulation of p21ras activity.