02234nas a2200157   4500008004100000245010800041210007000149260001500219520165500234100003201889700001801921700002101939700002001960700002301980856007302003       2015                        eng d00aSubstrate efflux propensity is the key determinant of iPLA-β-mediated glycerophospholipid hydrolysised0 aSubstrate efflux propensity is the key determinant of iPLAβmedia  c2015/02/233 aA-type phospholipases (PLAs) are key players in glycerophospholipid (GPL)homeostasis and in mammalian cells, Ca2+-independent PLA-beta (iPLAβ) in particular has been implicated in this essential process.However, the regulation of this enzyme,which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory nonhomeostatic) PLAs. To study if this is the case with iPLAβ as well a mass-spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn1 and sn2 acyl binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLAβ- mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.1 aBatchu, Krishna, Chaithanya1 aHokynar, Kati1 aJeltsch, Michael1 aMattonet, Kenny1 aSomerharju, Pentti  uhttp://www.jbc.org/content/early/2015/02/23/jbc.M115.642835.abstract03178nas a2200517   4500008004100000020001400041245009100055210006900146260001200215300001800227490000800245520156900253653002401822653001801846653002501864653002701889653004001916653001101956653001201967653002501979653002202004653002802026653002702054653001302081653002002094653002802114653003202142653002802174653003402202653001902236653004102255653005002296653002202346100002602368700002002394700001702414700002102431700002102452700002102473700002902494700002202523700002302545700002402568700001802592856005002610       2013                        eng d  a1091-649000aStructural and mechanistic insights into VEGF receptor 3 ligand binding and activation0 aStructural and mechanistic insights into VEGF receptor 3 ligand   c08/2013  a12960 - 129650 v1103 aVascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are key drivers of blood and lymph vessel formation in development, but also in several pathological processes. VEGF-C signaling through VEGFR-3 promotes lymphangiogenesis, which is a clinically relevant target for treating lymphatic insufficiency and for blocking tumor angiogenesis and metastasis. The extracellular domain of VEGFRs consists of seven Ig homology domains; domains 1-3 (D1-3) are responsible for ligand binding, and the membrane-proximal domains 4-7 (D4-7) are involved in structural rearrangements essential for receptor dimerization and activation. Here we analyzed the crystal structures of VEGF-C in complex with VEGFR-3 domains D1-2 and of the VEGFR-3 D4-5 homodimer. The structures revealed a conserved ligand-binding interface in D2 and a unique mechanism for VEGFR dimerization and activation, with homotypic interactions in D5. Mutation of the conserved residues mediating the D5 interaction (Thr446 and Lys516) and the D7 interaction (Arg737) compromised VEGF-C induced VEGFR-3 activation. A thermodynamic analysis of VEGFR-3 deletion mutants showed that D3, D4-5, and D6-7 all contribute to ligand binding. A structural model of the VEGF-C/VEGFR-3 D1-7 complex derived from small-angle X-ray scattering data is consistent with the homotypic interactions in D5 and D7. Taken together, our data show that ligand-dependent homotypic interactions in D5 and D7 are essential for VEGFR activation, opening promising possibilities for the design of VEGFR-specific drugs.10aAmino Acid Sequence10aBinding Sites10aBinding, Competitive10aCrystallography, X-Ray10aElectrophoresis, Polyacrylamide Gel10aHumans10aLigands10aMicroscopy, Electron10aModels, Molecular10aMolecular Sequence Data10aMultiprotein Complexes10aMutation10aProtein Binding10aProtein Multimerization10aProtein Structure, Tertiary10aScattering, Small Angle10aSequence Homology, Amino Acid10aThermodynamics10aVascular Endothelial Growth Factor C10aVascular Endothelial Growth Factor Receptor-310aX-Ray Diffraction1 aLeppänen, Veli-Matti1 aTvorogov, Denis1 aKisko, Kaisa1 aProta, Andrea, E1 aJeltsch, Michael1 aAnisimov, Andrey1 aMarkovic-Mueller, Sandra1 aStuttfeld, Edward1 aGoldie, Kenneth, N1 aBallmer-Hofer, Kurt1 aAlitalo, Kari  uhttp://www.pnas.org/content/110/32/12960.long02103nas a2200241   4500008004100000245010100041210006900142260001400211300001400225490000800239520135400247100002601601700002101627700002101648700002001669700001501689700002101704700001901725700002601744700002401770700001801794856004901812       2011                        eng d00aStructural determinants of vascular endothelial growth factor-D receptor binding and specificity0 aStructural determinants of vascular endothelial growth factorD r  c2011/Feb/  a1507 - 150 v1173 aVascular endothelial growth factors (VEGFs) and their tyrosine kinase receptors (VEGFR-1-3) are central mediators of angiogenesis and lymphangiogenesis. VEGFR-3 ligands VEGF-C and VEGF-D are produced as precursor proteins with long N- and C-terminal propeptides and show enhanced VEGFR-2 and VEGFR-3 binding on proteolytic removal of the propeptides. Two different proteolytic cleavage sites have been reported in the VEGF-D N-terminus. We report here the crystal structure of the human VEGF-D Cys117Ala mutant at 2.9 Å resolution. Comparison of the VEGF-D and VEGF-C structures shows similar extended N-terminal helices, conserved overall folds, and VEGFR-2 interacting residues. Consistent with this, the affinity and the thermodynamic parameters for VEGFR-2 binding are very similar. In comparison with VEGF-C structures, however, the VEGF-D N-terminal helix was extended by 2 more turns because of a better resolution. Both receptor binding and functional assays of N-terminally truncated VEGF-D polypeptides indicated that the residues between the reported proteolytic cleavage sites are important for VEGF-D binding and activation of VEGFR-3, but not of VEGFR-2. Thus, we define here a VEGFR-2-specific form of VEGF-D that is angiogenic but not lymphangiogenic. These results provide important new insights into VEGF-D structure and function.1 aLeppänen, Veli-Matti1 aJeltsch, Michael1 aAnisimov, Andrey1 aTvorogov, Denis1 aAho, Kukka1 aKalkkinen, Nisse1 aToivanen, Pyry1 aYlä-Herttuala, Seppo1 aBallmer-Hofer, Kurt1 aAlitalo, Kari  uhttp://view.ncbi.nlm.nih.gov/pubmed/2114808502522nas a2200241   4500008004100000245008800041210006900129260001200198300001400210490000800224520178600232100002602018700002102044700002102065700002102086700002102107700002002128700002102148700002002169700002402189700001802213856004902231       2010                        eng d00aStructural determinants of growth factor binding and specificity by VEGF receptor 20 aStructural determinants of growth factor binding and specificity  c02/2010  a2425 - 300 v1073 aVascular endothelial growth factors (VEGFs) regulate blood and lymph vessel formation through activation of three receptor tyrosine kinases, VEGFR-1, -2, and -3. The extracellular domain of VEGF receptors consists of seven immunoglobulin homology domains, which, upon ligand binding, promote receptor dimerization. Dimerization initiates transmembrane signaling, which activates the intracellular tyrosine kinase domain of the receptor. VEGF-C stimulates lymphangiogenesis and contributes to pathological angiogenesis via VEGFR-3. However, proteolytically processed VEGF-C also stimulates VEGFR-2, the predominant transducer of signals required for physiological and pathological angiogenesis. Here we present the crystal structure of VEGF-C bound to the VEGFR-2 high-affinity-binding site, which consists of immunoglobulin homology domains D2 and D3. This structure reveals a symmetrical 22 complex, in which left-handed twisted receptor domains wrap around the 2-fold axis of VEGF-C. In the VEGFs, receptor specificity is determined by an N-terminal alpha helix and three peptide loops. Our structure shows that two of these loops in VEGF-C bind to VEGFR-2 subdomains D2 and D3, while one interacts primarily with D3. Additionally, the N-terminal helix of VEGF-C interacts with D2, and the groove separating the two VEGF-C monomers binds to the D2/D3 linker. VEGF-C, unlike VEGF-A, does not bind VEGFR-1. We therefore created VEGFR-1/VEGFR-2 chimeric proteins to further study receptor specificity. This biochemical analysis, together with our structural data, defined VEGFR-2 residues critical for the binding of VEGF-A and VEGF-C. Our results provide significant insights into the structural features that determine the high affinity and specificity of VEGF/VEGFR interactions.1 aLeppänen, Veli-Matti1 aProta, Andrea, E1 aJeltsch, Michael1 aAnisimov, Andrey1 aKalkkinen, Nisse1 aStrandin, Tomas1 aLankinen, Hilkka1 aGoldman, Adrian1 aBallmer-Hofer, Kurt1 aAlitalo, Kari  uhttp://view.ncbi.nlm.nih.gov/pubmed/2014511602377nas a2200265   4500008004100000245014000041210006900181260001100250300001100261490000600272520152800278100001901806700002101825700002401846700002001870700002301890700002501913700002001938700002101958700001801979700001801997700002502015700002202040856004902062       2010                        eng d00aSuppressive effects of vascular endothelial growth factor-B on tumor growth in a mouse model of pancreatic neuroendocrine tumorigenesis0 aSuppressive effects of vascular endothelial growth factorB on tu  c2010//  ae141090 v53 aBACKGROUND: The family of vascular endothelial growth factors (VEGF) contains key regulators of blood and lymph vessel development, including VEGF-A, -B, -C, -D, and placental growth factor. The role of VEGF-B during physiological or pathological angiogenesis has not yet been conclusively delineated. Herein, we investigate the function of VEGF-B by the generation of mouse models of cancer with transgenic expression of VEGF-B or homozygous deletion of Vegfb. METHODOLOGY/PRINCIPAL FINDINGS: Ectopic expression of VEGF-B in the insulin-producing β-cells of the pancreas did not alter the abundance or architecture of the islets of Langerhans. The vasculature from transgenic mice exhibited a dilated morphology, but was of similar density as that of wildtype mice. Unexpectedly, we found that transgenic expression of VEGF-B in the RIP1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis retarded tumor growth. Conversely, RIP1-Tag2 mice deficient for Vegfb presented with larger tumors. No differences in vascular density, perfusion or immune cell infiltration upon altered Vegfb gene dosage were noted. However, VEGF-B acted to increase blood vessel diameter both in normal pancreatic islets and in RIP1-Tag2 tumors. CONCLUSIONS/SIGNIFICANCE: Taken together, our results illustrate the differences in biological function between members of the VEGF family, and highlight the necessity of in-depth functional studies of VEGF-B to fully understand the effects of VEGFR-1 inhibitors currently used in the clinic.1 aAlbrecht, Imke1 aKopfstein, Lucie1 aStrittmatter, Karin1 aSchomber, Tibor1 aFalkevall, Annelie1 aHagberg, Carolina, E1 aLorentz, Pascal1 aJeltsch, Michael1 aAlitalo, Kari1 aEriksson, Ulf1 aChristofori, Gerhard1 aPietras, Kristian  uhttp://view.ncbi.nlm.nih.gov/pubmed/2112484101998nas a2200265   4500008004100000245012000041210006900161260001400230300001400244490000700258520123100265100001601496700001501512700001501527700001601542700001501558700001701573700001201590700001601602700001801618700001501636700001701651700001501668856004901683       2001                        eng d00aSignalling via vascular endothelial growth factor receptor-3 is sufficient for lymphangiogenesis in transgenic mice0 aSignalling via vascular endothelial growth factor receptor3 is s  c2001/Mar/  a1223 - 310 v203 aVascular endothelial growth factor receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-2, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal keratinocytes under the keratin 14 promoter. Both transgenes induced the growth of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these growth factors act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.1 aVeikkola, T1 aJussila, L1 aMakinen, T1 aKarpanen, T1 aJeltsch, M1 aPetrova, T V1 aKubo, H1 aThurston, G1 aMcDonald, D M1 aAchen, M G1 aStacker, S A1 aAlitalo, K  uhttp://view.ncbi.nlm.nih.gov/pubmed/1125088900578nas a2200181   4500008004100000245008300041210006900124260003800193100001600231700001700247700001600264700001700280700001700297700001400314700001600328700001600344856003600360       2001                        eng d00aSignalling via VEGFR-3 is sufficient for lymphangiogenesis in transgenic mice.0 aSignalling via VEGFR3 is sufficient for lymphangiogenesis in tra  aMiami Beach, Floridac2001/10/29/1 aJussila, L.1 aVeikkola, T.1 aJeltsch, M.1 aThurston, G.1 aMcDonald, D.1 aAchen, M.1 aStacker, S.1 aAlitalo, K.  uhttps://jeltsch.org/Jussila2001