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Structure/function analysis of VEGF receptors

English title Structure/function analysis of VEGF receptors
Applicant Ballmer-Hofer Kurt
Number 130463
Funding scheme Project funding (Div. I-III)
Research institution Paul Scherrer Institut
Institution of higher education Paul Scherrer Institute - PSI
Main discipline Cellular Biology, Cytology
Start/End 01.05.2010 - 30.04.2011
Approved amount 138'333.00
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All Disciplines (2)

Discipline
Cellular Biology, Cytology
Structural Research

Keywords (8)

receptor; tyrosine kinase; angiogenesis; VEGF; protein structure; small angle scattering; x-ray crystallography; spheroid culture

Lay Summary (English)

Lead
Lay summary
Background: Vascular Endothelial Growth Factors (VEGFs) constitute a family of proteins, VEGF-A through -F and Placenta Growth Factor, PlGF, that regulate blood and lymphatic vessel development and homeostasis. Vessel development is tightly regulated maintaining adequate blood supply and lymph drainage in normal, healthy organisms. Aberrant VEGF signaling plays a role in various diseases such as in atherosclerosis, diabetic retinopathy, macular degeneration, lymphoproliferative and rheumatoid disease, neural degeneration and in tumor growth and metastasis. VEGFs bind to three variants of type V receptor tyrosine kinase growth factor receptors (RTKs), VEGF receptor-1, -2 and -3 (VEGFR-1, -2, -3). Each VEGF isoform binds to specific receptor subtypes that activate particular signaling pathways creating distinct biological output. Ligand binding to the extracellular ligand-binding domain (ECD) leads to receptor dimerization followed by structural changes in the intracellular kinase domain that unleash its enzyme activity and promote signaling to downstream targets. Specific aims: RTKs such as VEGFRs play a fundamental role in normal development and in many diseases. A prominent example is the presence of mutated receptors in tumor cells that is a hallmark of many types of highly aggressive malignancies such as breast, colon and stomach cancer. Tight regulation of VEGF signaling is mandatory for proper development of higher organisms and no mutations in VEGF or VEGFR genes have been identified so far in humans or mice. We investigate the mechanisms responsible for VEGFR activation combining biochemical and cell biological experiments with an analysis of protein structure. Access to clinical data for cancer-associated VEGFR-2 ‘single nucleotide polymorphism’ (SNP) variants from a collaborating lab will allow us to assess the role of recently discovered receptor ECD variants.Experimental design and first results: We designed a series of VEGFR-2 mutants with altered extracellular or transmembrane domains to unravel the mechanism of receptor activation. We mainly investigate the ECD, in particular immunoglobulin homology domains 4 and 7, that are required for receptor dimerization as shown earlier by our group. In a collaborative, more clinically oriented project, we found naturally occurring receptor variants (SNPs) with amino acid changes in the ECD that had increased activity. These receptor variants, together with the ECD mutants described above, will be further studied in endothelial cells to assess ligand-stimulated receptor activity and signaling to downstream signaling molecules. Ligand binding to the ECD is also studied at the structural level comprising low resolution methods such as electron microscopy and small angle solution scattering as well as high resolution protein crystallography. A first high resolution structure of immunoglobulin homology domains 2-3 bound to VEGF-C was obtained recently in a collaborative effort. Structure determination of additional ECD constructs bound to either VEGF-A or -E is ongoing. Finally, to investigate the role of dimerization in kinase activation we created a series of transmembrane domain (TMD) receptor mutants capable to dimerize the receptor in the absence of ligand. Our data show that receptor dimerization is necessary, but not sufficient for receptor activation, and suggest that specific alignment of receptor monomers is an absolute requirement for receptor kinase activation.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A.
Markovic-Mueller Sandra, Stuttfeld Edward, Asthana Mayanka, Weinert Tobias, Bliven Spencer, Goldie Kenneth N, Kisko Kaisa, Capitani Guido, Ballmer-Hofer Kurt (2017), Structure of the Full-length VEGFR-1 Extracellular Domain in Complex with VEGF-A., in Structure (London, England : 1993), 25(2), 341-352.
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.
Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist.
Grillo Elisabetta, Ravelli Cosetta, Corsini Michela, Ballmer-Hofer Kurt, Zammataro Luca, Oreste Pasqua, Zoppetti Giorgio, Tobia Chiara, Ronca Roberto, Presta Marco, Mitola Stefania (2016), Monomeric gremlin is a novel vascular endothelial growth factor receptor-2 antagonist., in Oncotarget, 7(23), 35353-68.
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.
VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread.
Li Xiujuan, Padhan Narendra, Sjöström Elisabet O, Roche Francis P, Testini Chiara, Honkura Naoki, Sáinz-Jaspeado Miguel, Gordon Emma, Bentley Katie, Philippides Andrew, Tolmachev Vladimir, Dejana Elisabetta, Stan Radu V, Vestweber Dietmar, Ballmer-Hofer Kurt, Betsholtz Christer, Pietras Kristian, Jansson Leif, Claesson-Welsh Lena (2016), VEGFR2 pY949 signalling regulates adherens junction integrity and metastatic spread., in Nature communications, 7, 11017-11017.

Associated projects

Number Title Start Funding scheme
147530 Functional analysis of novel VEGF receptor 2 ligands for application in tumour imaging and anti-angiogenic therapy 01.04.2013 Marie Heim-Voegtlin grants
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)
133810 Upgrading the biophysical facility at PSI with analytical ultracentrifuge equipped with fluorescence detection system. 01.12.2010 R'EQUIP
134208 Functional analysis of novel VEGF receptor 2 ligands for application in tumour imaging and anti-angiogenic therapy 01.04.2011 Marie Heim-Voegtlin grants
116507 Structure/function analysis of VEGF receptors 01.05.2007 Project funding (Div. I-III)
121315 Upgrading the light microscopy facility at PSI with total internal reflection fluorescence microscopy (TIRFM) 01.07.2008 R'EQUIP

Abstract

ZUSAMMENFASSUNGBackground: Vascular Endothelial Growth Factors (VEGFs) constitute a family of proteins, VEGF-A through -F and Placenta Growth Factor, PlGF, that regulate blood and lymphatic vessel development and homeostasis. Vessel development is tightly regulated maintaining adequate blood supply and lymph drainage in normal, healthy organisms. Aberrant VEGF signaling plays a role in various diseases such as in atherosclerosis, diabetic retinopathy, macular degeneration, lymphoproliferative and rheumatoid disease, neural degeneration and in tumor growth and metastasis. VEGFs bind to three variants of type V receptor tyrosine kinase growth factor receptors (RTKs), VEGF receptor-1, -2 and -3 (VEGFR-1, -2, -3). Each VEGF isoform binds to specific receptor subtypes that activate particular signaling pathways creating distinct biological output. Ligand binding to the extracellular ligand-binding domain (ECD) leads to receptor dimerization followed by structural changes in the intracellular kinase domain that unleash its enzyme activity and promote signaling to downstream targets. Specific aims: RTKs such as VEGFRs play a fundamental role in normal development and in many diseases. A prominent example is the presence of mutated receptors in tumor cells that is a hallmark of many types of highly aggressive malignancies such as breast, colon and stomach cancer. Tight regulation of VEGF signaling is mandatory for proper development of higher organisms and no mutations in VEGF or VEGFR genes have been identified so far in humans or mice. We investigate the mechanisms responsible for VEGFR activation combining biochemical and cell biological experiments with an analysis of protein structure. Access to clinical data for cancer-associated VEGFR-2 ‘single nucleotide polymorphism’ (SNP) variants from a collaborating lab will allow us to assess the role of recently discovered receptor ECD variants.Experimental design and first results: We designed a series of VEGFR-2 mutants with altered extracellular or transmembrane domains to unravel the mechanism of receptor activation. We mainly investigate the ECD, in particular immunoglobulin homology domains 4 and 7, that are required for receptor dimerization as shown earlier by our group. In a collaborative, more clinically oriented project, we found naturally occurring receptor variants (SNPs) with amino acid changes in the ECD that had increased activity. These receptor variants, together with the ECD mutants described above, will be further studied in endothelial cells to assess ligand-stimulated receptor activity and signaling to downstream signaling molecules. Ligand binding to the ECD is also studied at the structural level comprising low resolution methods such as electron microscopy and small angle solution scattering as well as high resolution protein crystallography. A first high resolution structure of immunoglobulin homology domains 2-3 bound to VEGF-C was obtained recently in a collaborative effort. Structure determination of additional ECD constructs bound to either VEGF-A or -E is ongoing. Finally, to investigate the role of dimerization in kinase activation we created a series of transmembrane domain (TMD) receptor mutants capable to dimerize the receptor in the absence of ligand. Our data show that receptor dimerization is necessary, but not sufficient for receptor activation, and suggest that specific alignment of receptor monomers is an absolute requirement for receptor kinase activation.
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