Sreenivasan Ponnambalam

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.

CHARACTERIZATION AND REGULATION OF CONSTITUTIVE TRANSPORT INTERMEDIATES INVOLVED IN TRAFFICKING FROM THE TRANS -GOLGI NETWORK

Transport vesicles or containers (TCs) mediate constitutive protein transport between the trans‐Golgi network (TGN) and the plasma membrane. A key question is the nature and regulation of these transport containers or intermediates. We have used a trans ‐Golgi network resident, TGN38, to investigate TC formation. TGN38 is a recycling membrane glycoprotein that moves to the cell surface via constitutive membrane traffic and returns via the endosomal pathway. Anin vitro assay to measure TC formation was devised using rat liver Golgi membranes, cytosolic factors and ATP. Transport intermediates containing TGN38 were produced and found to be smooth vesicles and tubules of up to 200nm in length. These membrane‐enclosed structures contain different constitutively secreted membrane glycoproteins, including molecules involved in immune functions such as MHC Class I and the polymeric Ig receptor, showing that these intermediates correspond to TCs that have been previously identified in vivo. Importantly, TC formation can be stimulated or inhibited by protein kinase and phosphatase inhibitors, showing regulation by intracellular signalling pathways.

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.

Clathrin light chains : arrays of protein motifs that regulate coated-vesicle dynamics

Polymerization of clathrin triskelions into clathrin coats and subsequent disassembly by the heat shock protein hsc70 control receptor-mediated pathways of intracellular transport. The clathrin light chains are major regulatory elements in these processes. These polypeptides consist of linear arrays of functional domains with distinctive sequence motifs. Comparison of unicellular and multicellular eukaryotes reveals differences in the numbers of clathrin light chains and in the functional domains they contain.

Constitutive protein secretion from the trans -Golgi network to the plasma membrane (Review)

Constitutive secretion is used to deliver newly synthesized proteins to the cell surface and to the extracellular milieu. The trans -Golgi network is a key station along this route that mediates sorting of proteins into distinct transport pathways, aided in part by clathrin and adaptor proteins. Subsequent movement of proteins to the plasma membrane can occur either directly or via the endocytic pathway. Moreover, multiple, parallel pathways from the trans -Golgi network to the plasma membrane appear to exist, not only in complex, polarized cells such as epithelial cells and neurons, but also in relatively simple cells such as fibroblasts. In addition to typical secretory vesicles, these pathways involve both small, pleiomorphic transport containers and relatively large tubular-saccular carriers that travel along cytoskeletal tracks. While production and movement of these membranous structures are typically described as constitutive, recent studies have revealed that these key steps in secretion are tightly regulated by Ras-superfamily GTPases, members of the protein kinase D family and tethering complexes such as the exocyst.

Scavenger Receptor Structure and Function in Health and Disease

Scavenger receptors (SRs) are a ‘superfamily’ of membrane-bound receptors that were initially thought to bind and internalize modified low-density lipoprotein (LDL), though it is currently known to bind to a variety of ligands including endogenous proteins and pathogens. New family of SRs and their properties have been identified in recent years, and have now been classified into 10 eukaryote families, defined as Classes A-J. These receptors are classified according to their sequences, although in each class they are further classified based in the variations of the sequence. Their ability to bind a range of ligands is reflected on the biological functions such as clearance of modified lipoproteins and pathogens. SR members regulate pathophysiological states including atherosclerosis, pathogen infections, immune surveillance, and cancer. Here, we review our current understanding of SR structure and function implicated in health and disease.

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.

Scavenger Receptors and Their Potential as Therapeutic Targets in the Treatment of Cardiovascular Disease

Scavenger receptors act as membrane-bound and soluble proteins that bind to macromolecular complexes and pathogens. This diverse supergroup of proteins mediates binding to modified lipoprotein particles which regulate the initiation and progression of atherosclerotic plaques. In vascular tissues, scavenger receptors are implicated in regulating intracellular signaling, lipid accumulation, foam cell development, and cellular apoptosis or necrosis linked to the pathophysiology of atherosclerosis. One approach is using gene therapy to modulate scavenger receptor function in atherosclerosis. Ectopic expression of membrane-bound scavenger receptors using viral vectors can modify lipid profiles and reduce the incidence of atherosclerosis. Alternatively, expression of soluble scavenger receptors can also block plaque initiation and progression. Inhibition of scavenger receptor expression using a combined gene therapy and RNA interference strategy also holds promise for long-term therapy. Here we review our current understanding of the gene delivery by viral vectors to cells and tissues in gene therapy strategies and its application to the modulation of scavenger receptor function in atherosclerosis.

Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.