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Structural and functional analysis of the extracellular domain of VEGF receptors

English title Structural and functional analysis of the extracellular domain of VEGF receptors
Applicant Ballmer-Hofer Kurt
Number 152908
Funding scheme Project funding (Div. I-III)
Research institution Paul Scherrer Institut
Institution of higher education Paul Scherrer Institute - PSI
Main discipline Biochemistry
Start/End 01.09.2014 - 31.08.2016
Approved amount 405'026.00
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All Disciplines (5)

Discipline
Biochemistry
Cellular Biology, Cytology
Structural Research
Biophysics
Molecular Biology

Keywords (10)

receptor tyrosine kinase; protein crystallography; allosteric inhibition; angiogenesis; signaling; DARPin; antibody; protein structure; electron microscopy; antibody

Lay Summary (German)

Lead
Strukturelle und funktionelle Analyse des Aktivierungsmechanismus von VEGF RezeptortyrosinkinasenStructural and functional analysis of VEGF receptor tyrosine kinase activation
Lay summary

Blut- und Lymphgefässe gewährleisten die Versorgung eines Organismus mit Sauerstoff und Nährstoffen. Viele Krankheiten, wie Degenerationserkrankungen des Auges, der Gefässe  oder Gelenke, sowie das Wachstum von Tumoren sind von erhöhter Produktion gefäss-bildender Wachstumsfaktoren wie Vascular Endothelial Growth Factor (VEGF) begleitet. VEGF bindet und aktiviert Rezeptoren auf den die Gefässe auskleidenden Endothelzellen. Blockieren des VEGF resp. der VEGF Rezeptoren kann krankhaftes Gefässwachstum verhindern.

Wir untersuchen die Struktur der VEGF Rezeptoren und den molekularen Mechanismus ihrer Aktivierung mit Hilfe verschiedener Techniken wie Elektronenmikroskopie, Kleinwinkelstreuung und Röntgenkristallographie. Wir konnten zeigen, dass die Bindung des Liganden, also des VEGF, zur Rezeptordimerisierung und in der Folge zur Aenderung der Rezeptorstruktur führt. Diese Strukturänderung führt innert Sekunden zur Aktivierung der intrazellulären Rezeptordomäne, einer sogenannten Tyrosinkinase. Die aktivierte Tyrosinkinase aktiviert dann durch direkte Phosphorylierung spezifischer Signalmoleküle eine Vielzahl zellulärer Signalwege in der Zelle und stimuliert so z.B. die Zellmigration und Zellteilung. Die präzise Kenntnis der Molekülstruktur des VEGF Rezeptors hat es uns ermöglicht eine neue Klasse von Inhibitoren zu entwickeln. Diese Inhibitoren binden an die extrazelluläre Seite der Rezeptoren und verändern deren Struktur nur geringfügig. Trotzdem sind sie in der Lage, die Aktivierung der sich im Zellinnern befindenden Rezeptorkinase fast vollständig zu blockieren, sie wirken also als ‚allosterische‘ Inhibitoren. Neben der verfeinerten Analyse der Rezeptorstruktur untersuchen wir auch die Wirksamkeit dieser neuen Inhibitoren sowohl in Zellkulturen wie auch in Tiermodellen.

Direct link to Lay Summary Last update: 08.08.2014

Responsible applicant and co-applicants

Employees

Publications

Publication
Highly efficient baculovirus-mediated multigene delivery in primary cells.
Mansouri Maysam, Bellon-Echeverria Itxaso, Rizk Aurélien, Ehsaei Zahra, Cianciolo Cosentino Chiara, Silva Catarina S, Xie Ye, Boyce Frederick M, Davis M Wayne, Neuhauss Stephan C F, Taylor Verdon, Ballmer-Hofer Kurt, Berger Imre, Berger Philipp (2016), Highly efficient baculovirus-mediated multigene delivery in primary cells., in Nature communications, 7, 11529-11529.
VEGFR-2 conformational switch in response to ligand binding.
Sarabipour Sarvenaz, Ballmer-Hofer Kurt, Hristova Kalina (2016), VEGFR-2 conformational switch in response to ligand binding., in eLife, 5, 13876-13876.
Subcellular object quantification with Squassh3C and SquasshAnalyst.
Rizk Aurélien, Mansouri Maysam, Ballmer-Hofer Kurt, Berger Philipp (2015), Subcellular object quantification with Squassh3C and SquasshAnalyst., in BioTechniques, 59(5), 309-12.
Vascular endothelial growth factor-A165b prevents diabetic neuropathic pain and sensory neuronal degeneration.
Hulse Richard P, Beazley-Long Nicholas, Ved Nikita, Bestall Samuel M, Riaz Hamza, Singhal Priya, Ballmer Hofer Kurt, Harper Steve J, Bates David O, Donaldson Lucy F (2015), Vascular endothelial growth factor-A165b prevents diabetic neuropathic pain and sensory neuronal degeneration., in Clinical science (London, England : 1979), 129(8), 741-56.
Cell lines expressing recombinant transmembrane domain-activated receptor kinases as tools for drug discovery.
Weber Holger, Müller Daniel, Müller Melanie, Ortiz Alexandra, Birkle Marianne, Umber Sarah, Ketterer Constance, Siedentopf Oliver, Feger Daniel, Totzke Frank, Kubbutat Michael, Schaechtele Christoph, Ballmer-Hofer Kurt, Ehlert Jan Erik, Graeser Ralph (2014), Cell lines expressing recombinant transmembrane domain-activated receptor kinases as tools for drug discovery., in Journal of biomolecular screening, 19(10), 1350-61.
Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation.
Manni Sandro, Kisko Kaisa, Schleier Thomas, Missimer Jack, Ballmer-Hofer Kurt (2014), Functional and structural characterization of the kinase insert and the carboxy terminal domain in VEGF receptor 2 activation., in FASEB journal : official publication of the Federation of American Societies for Experimental Biolog, 28(11), 4914-23.
Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation.
Manni Sandro, Mineev Konstantin S, Usmanova Dinara, Lyukmanova Ekaterina N, Shulepko Mikhail A, Kirpichnikov Mikhail P, Winter Jonas, Matkovic Milos, Deupi Xavier, Arseniev Alexander S, Ballmer-Hofer Kurt (2014), Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation., in Structure (London, England : 1993), 22(8), 1077-89.
Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy.
Oltean Sebastian, Qiu Yan, Ferguson Joanne K, Stevens Megan, Neal Chris, Russell Amy, Kaura Amit, Arkill Kenton P, Harris Kirstie, Symonds Clare, Lacey Katja, Wijeyaratne Lihini, Gammons Melissa, Wylie Emma, Hulse Richard P, Alsop Chloe, Cope George, Damodaran Gopinath, Betteridge Kai B, Ramnath Raina, Satchell Simon C, Foster Rebecca R, Ballmer-Hofer Kurt, Donaldson Lucy F, Barratt Jonathan (2014), Vascular Endothelial Growth Factor-A165b Is Protective and Restores Endothelial Glycocalyx in Diabetic Nephropathy., in Journal of the American Society of Nephrology : JASN, 26(8), 1889-904.

Collaboration

Group / person Country
Types of collaboration
Marco Presta, University of Brescia Italy (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Division of Pre-Clinical Oncology School of Clini Nottingham NG7 2UH Great Britain and Northern Ireland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
K. Alitalo, Biomedicum Helsinki Finland Finland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Lena Claesson Welsh Rudland institute Uppsala Sweden (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication

Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Wie unterhalten sich die Zellen in unserem Körper miteinander? German-speaking Switzerland 2014

Associated projects

Number Title Start Funding scheme
135276 Structural and functional analysis of the activation mechanism of receptor tyrosine kinases of the VEGF and Angiopoietin receptor family 01.09.2011 Project funding (Div. I-III)

Abstract

Background: The human genome encodes 58 receptor tyrosine kinases (RTKs) that give rise to signal output regulating a plethora of cellular activities such as survival, growth, migration, and differentiation. RTKs play fundamental roles in normal development and tissue homeostasis, and aberrant regulation of RTKs is the cause of many diseases. We study type V RTKs, Vascular Endothelial Growth Factor Receptors, VEGF receptor-1, -2 and -3, which are activated by VEGF family ligands. VEGFs comprise a family of more than 30 proteins encoded by five genes. VEGFs regulate blood and lymphatic vessel development maintaining adequate blood supply and lymph drainage in higher organisms. Each VEGF isoform binds to distinct receptor subtypes creating specific biological output upon activation of multiple intracellular signaling molecules. Ligand binding to the extracellular domain (ECD) promotes structural changes that stimulate receptor dimerization, followed by activation of the intracellular RTK domain. Tight regulation of VEGF signaling is essential for vascular homeostasis in higher organisms. Not surprisingly, various diseases such as atherosclerosis, diabetic retinopathy, macular degeneration, lymphoproliferative and rheumatoid disease, neural degeneration and tumor growth and metastasis are associated with aberrant signaling by VEGF receptors. VEGF receptors are thus clinically relevant, and several inhibitory molecules aimed at blocking VEGF signaling are in use today as promising drugs.Recent achievements: We determined the structure of the extracellular ligand-binding domain of VEGFR-2 by X-ray crystallography and generated low resolution structural models of the full length ligand-bound ECD of VEGFR-1, -2 , and -3, applying small angle solution scattering (SAXS) and single particle negative stain electron microscopy (EM). We showed that distinct subdomains of the receptor ECD, which is composed of seven immunoglobulin-homology domains (Ig-domains), fulfill very specific tasks in receptor activation; domains 2 and 3 comprise the ligand binding site while domains 4-7 form homotypic contacts resulting from ligand-mediated structural rearrangement of the ECD. These homotypic contacts are indispensable for VEGF receptor activation and signaling. We propose that they are required for the correct orientation of the intracellular kinase domains in active dimers. Based on our structural information, we developed Designed Ankyrin Repeat proteins (DARPins) and scFv antibody fragments specifically binding to domains 4-7. Several of these ECD binders block VEGFR-2 activation, but not dimerization. These molecules represent thus a novel class of allosteric inhibitors. We apply them as tracers for tumor vessel imaging in vivo in mice using Single Photon Emission Computed Tomography (SPECT). Future plans: We plan to generate high resolution structures of the full length VEGFR-1 and -2 ECD in complex with ligand and currently reach a resolution of about 4 Å. This structural information will allow to design low Mr inhibitors disrupting the receptor contacts in domains 4-7 and thus to inhibit receptor activity. Based on our study of the VEGFR-2 TMD structure we will also investigate membrane-associated full length ECD constructs of VEGFR-1 and -2. This will reveal the mechanism responsible for tight steric coupling of the ECD to the intracellular kinase domain and generate the first structure of a membrane-bound RTK ECD. These data will create new insights into transmembrane signaling by RTKs and bare promise for developing novel receptor-inhibitory drugs.
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