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• W2941014077 abstract "Vascular endothelial growth factor receptor 2 (VEGFR2) controls angiogenesis and is critically important for normal human development and cancer progression. A recent finding that VEGFR2 can dimerize in the absence of ligand raises the question whether VEGF binds to either VEGFR2 monomers or dimers or to both. Although VEGF–VEGFR2 effective binding constants have been measured, these prior measurements have not discriminated between the association state of the receptor. Because ligand binding is coupled to receptor dimerization, this coupling lends complexity to a seemingly straightforward problem. Here, we unravel this complexity by applying a rigorous thermodynamics approach and performing binding measurements over a broad range of receptor and ligand concentrations. By applying a global fitting procedure to a large data set, we reveal a 45-fold difference between VEGF binding affinities for monomeric and dimeric forms of VEGFR2. Vascular endothelial growth factor receptor 2 (VEGFR2) controls angiogenesis and is critically important for normal human development and cancer progression. A recent finding that VEGFR2 can dimerize in the absence of ligand raises the question whether VEGF binds to either VEGFR2 monomers or dimers or to both. Although VEGF–VEGFR2 effective binding constants have been measured, these prior measurements have not discriminated between the association state of the receptor. Because ligand binding is coupled to receptor dimerization, this coupling lends complexity to a seemingly straightforward problem. Here, we unravel this complexity by applying a rigorous thermodynamics approach and performing binding measurements over a broad range of receptor and ligand concentrations. By applying a global fitting procedure to a large data set, we reveal a 45-fold difference between VEGF binding affinities for monomeric and dimeric forms of VEGFR2. Vascular endothelial growth factor receptor 2 (VEGFR2) 2The abbreviations used are: VEGFR2vascular endothelial growth factor receptor 2VEGFvascular endothelial growth factorRTKreceptor tyrosine kinaseECextracellularTMtransmembraneFSIfully quantified spectral imagingscVEGFsingle-chain derivative of vascular endothelial growth factorMmonomersDdimersLMligand-bound monomersLDligand-bound dimersrecreceptorsAF594Alexa Fluor 594YFPyellow fluorescent proteinHEKhuman embryonic kidney. is a 151-kDa member of the receptor tyrosine kinase (RTK) family (1Olsson A.K. Dimberg A. Kreuger J. Claesson-Welsh L. VEGF receptor signalling—in control of vascular function.Nat. Rev. Mol. Cell Biol. 2006; 7 (16633338): 359-37110.1038/nrm1911Crossref PubMed Scopus (2455) Google Scholar, 2Shibuya M. Claesson-Welsh L. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.Exp. Cell Res. 2006; 312 (16336962): 549-56010.1016/j.yexcr.2005.11.012Crossref PubMed Scopus (851) Google Scholar3Koch S. Tugues S. Li X. Gualandi L. Claesson-Welsh L. Signal transduction by vascular endothelial growth factor receptors.Biochem. J. 2011; 437 (21711246): 169-18310.1042/BJ20110301Crossref PubMed Scopus (637) Google Scholar). This receptor is expressed on the surface of endothelial cells and controls angiogenesis, the formation of new blood vessels from existing vasculature, as well as vasculogenesis, the de novo formation of new blood vessels in tissues (4Ferrara N. Gerber H.P. LeCouter J. The biology of VEGF and its receptors.Nat. Med. 2003; 9 (12778165): 669-67610.1038/nm0603-669Crossref PubMed Scopus (7855) Google Scholar, 5Jeltsch M. Leppänen V.M. Saharinen P. Alitalo K. Receptor tyrosine kinase-mediated angiogenesis.Cold Spring Harb. Perspect. Biol. 2013; 5 (24003209)a009183 10.1101/cshperspect.a009183Crossref PubMed Scopus (113) Google Scholar6Qutub A.A. Mac Gabhann F. Karagiannis E.D. Vempati P. Popel A.S. Multiscale models of angiogenesis.IEEE Eng. Med. Biol. Mag. 2009; 28 (19349248): 14-3110.1109/MEMB.2009.931791Crossref PubMed Scopus (129) Google Scholar). Thus, VEGFR2 plays a critical role in human development and in cancer progression and is a valuable drug target. Indeed, therapies that inhibit VEGFR2 and angiogenesis would be applicable to many solid tumors, which need oxygen to grow, whereas proangiogenic therapies would be beneficial in the treatment of ischemia, such as in coronary artery disease, stroke, and chronic wounds (7Nessa A. Latif S.A. Siddiqui N.I. Hussain M.A. Bhuiyan M.R. Hossain M.A. Akther A. Rahman M. Angiogenesis-a novel therapeutic approach for ischemic heart disease.Mymensingh Med. J. 2009; 18 (19623159): 264-272PubMed Google Scholar, 8Mac Gabhann F. Qutub A.A. Annex B.H. Popel A.S. Systems biology of pro-angiogenic therapies targeting the VEGF system.Wiley Interdiscip. Rev. Syst. Biol. Med. 2010; 2 (20890966): 694-70710.1002/wsbm.92Crossref PubMed Scopus (74) Google Scholar9Takahashi H. Shibuya M. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions.Clin. Sci. 2005; 109 (16104843): 227-24110.1042/CS20040370Crossref PubMed Scopus (719) Google Scholar). vascular endothelial growth factor receptor 2 vascular endothelial growth factor receptor tyrosine kinase extracellular transmembrane fully quantified spectral imaging single-chain derivative of vascular endothelial growth factor monomers dimers ligand-bound monomers ligand-bound dimers receptors Alexa Fluor 594 yellow fluorescent protein human embryonic kidney. VEGFR2, like most RTKs, consists of an extracellular (EC) domain, a single-pass α-helical transmembrane (TM) domain, and an intracellular kinase domain (10Roskoski Jr., R. VEGF receptor protein-tyrosine kinases: structure and regulation.Biochem. Biophys. Res. Commun. 2008; 375 (18680722): 287-29110.1016/j.bbrc.2008.07.121Crossref PubMed Scopus (212) Google Scholar). VEGFR2’s EC domain is one of the largest of the RTK family and is composed of seven Ig-like domains, known as subunits D1–D7. Subunits D2 and D3 serve as the binding sites for activating ligands. As in the case of most RTKs, VEGFR2 dimerization and ligand binding are required for its activation (11Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103 (11057895): 211-22510.1016/S0092-8674(00)00114-8Abstract Full Text Full Text PDF PubMed Scopus (3530) Google Scholar, 12Lemmon M.A. Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2010; 141 (20602996): 1117-113410.1016/j.cell.2010.06.011Abstract Full Text Full Text PDF PubMed Scopus (3135) Google Scholar13He L. Hristova K. Physical-chemical principles underlying RTK activation, and their implications for human disease.Biochim. Biophys. Acta. 2012; 1818 (21840295): 995-100510.1016/j.bbamem.2011.07.044Crossref PubMed Scopus (45) Google Scholar). Dimeric, ligand-bound receptors efficiently cross-phosphorylate each other on specific intracellular tyrosines, which serve as docking sites for intracellular adaptor proteins (1Olsson A.K. Dimberg A. Kreuger J. Claesson-Welsh L. VEGF receptor signalling—in control of vascular function.Nat. Rev. Mol. Cell Biol. 2006; 7 (16633338): 359-37110.1038/nrm1911Crossref PubMed Scopus (2455) Google Scholar, 2Shibuya M. Claesson-Welsh L. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.Exp. Cell Res. 2006; 312 (16336962): 549-56010.1016/j.yexcr.2005.11.012Crossref PubMed Scopus (851) Google Scholar, 11Schlessinger J. Cell signaling by receptor tyrosine kinases.Cell. 2000; 103 (11057895): 211-22510.1016/S0092-8674(00)00114-8Abstract Full Text Full Text PDF PubMed Scopus (3530) Google Scholar, 14Matsumoto T. Claesson-Welsh L. VEGF receptor signal transduction.Sci. STKE. 2001; 2001 (11741095): re2110.1126/stke.2001.112.re21Crossref PubMed Scopus (429) Google Scholar). Adaptor protein binding initiates cytoplasmic signaling cascades controlling endothelial cell survival, proliferation, and motility (1Olsson A.K. Dimberg A. Kreuger J. Claesson-Welsh L. VEGF receptor signalling—in control of vascular function.Nat. Rev. Mol. Cell Biol. 2006; 7 (16633338): 359-37110.1038/nrm1911Crossref PubMed Scopus (2455) Google Scholar). The ligands that bind VEGFR2 (VEGF-A and processed forms of VEGF-C and VEGF-D) are released by cells under hypoxic conditions and direct angiogenesis (2Shibuya M. Claesson-Welsh L. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis.Exp. Cell Res. 2006; 312 (16336962): 549-56010.1016/j.yexcr.2005.11.012Crossref PubMed Scopus (851) Google Scholar, 4Ferrara N. Gerber H.P. LeCouter J. The biology of VEGF and its receptors.Nat. Med. 2003; 9 (12778165): 669-67610.1038/nm0603-669Crossref PubMed Scopus (7855) Google Scholar, 15Gerhardt H. Golding M. Fruttiger M. Ruhrberg C. Lundkvist A. Abramsson A. Jeltsch M. Mitchell C. Alitalo K. Shima D. Betsholtz C. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia.J. Cell Biol. 2003; 161 (12810700): 1163-117710.1083/jcb.200302047Crossref PubMed Scopus (2109) Google Scholar). Of these ligands, VEGF-A exhibits the highest binding affinity for VEGFR2 and is considered the most potent angiogenic agent. VEGF-A is a disulfide-linked, antiparallel homodimer (16Muller Y.A. Christinger H.W. Keyt B.A. de Vos A.M. The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 Å resolution: multiple copy flexibility and receptor binding.Structure. 1997; 5 (9351807): 1325-133810.1016/S0969-2126(97)00284-0Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar) that exists as four different isoforms (121, 165, 189, and 206 amino acids long). Although the binding strengths of all VEGF-A isoforms to VEGFR2 are thought to be the same, these isoforms differ in their interactions with the extracellular matrix and the coreceptors on the surface of cells (14Matsumoto T. Claesson-Welsh L. VEGF receptor signal transduction.Sci. STKE. 2001; 2001 (11741095): re2110.1126/stke.2001.112.re21Crossref PubMed Scopus (429) Google Scholar, 17Cross M.J. Claesson-Welsh L. FGF and VEGF function in angiogenesis: signalling pathways, biological responses and therapeutic inhibition.Trends Pharmacol. Sci. 2001; 22 (11282421): 201-20710.1016/S0165-6147(00)01676-XAbstract Full Text Full Text PDF PubMed Scopus (806) Google Scholar18Vempati P. Popel A.S. Mac Gabhann F. Extracellular regulation of VEGF: isoforms, proteolysis, and vascular patterning.Cytokine Growth Factor Rev. 2014; 25 (24332926): 1-1910.1016/j.cytogfr.2013.11.002Crossref PubMed Scopus (207) Google Scholar, 19Klagsbrun M. Takashima S. Mamluk R. The role of neuropilin in vascular and tumor biology.Adv. Exp. Med. Biol. 2002; 515 (12613541): 33-4810.1007/978-1-4615-0119-0_3Crossref PubMed Scopus (175) Google Scholar20Zachary I.C. How neuropilin-1 regulates receptor tyrosine kinase signalling: the knowns and known unknowns.Biochem. Soc. Trans. 2011; 39 (22103491): 1583-159110.1042/BST20110697Crossref PubMed Scopus (59) Google Scholar). VEGF-A121 is the smallest isoform of VEGF-A; however, it contains the full VEGFR2-binding site but lacks the sites mediating the interaction with the extracellular matrix and other coreceptors. It has been found that VEGFR2 can exist in either a monomeric or a dimeric form on the plasma membrane of cells, even in the absence of ligand (21Sarabipour S. Ballmer-Hofer K. Hristova K. VEGFR-2 conformational switch in response to ligand binding.Elife. 2016; 5 (27052508)e13876 10.7554/eLife.13876Crossref PubMed Scopus (81) Google Scholar, 22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar23King C. Wirth D. Workman S. Hristova K. Cooperative interactions between VEGFR2 extracellular Ig-like subdomains ensure VEGFR2 dimerization.Biochim. Biophys. Acta Gen. Subj. 2017; 1861 (28847506): 2559-256710.1016/j.bbagen.2017.08.021Crossref PubMed Scopus (7) Google Scholar). The existence of VEGFR2 monomers and dimers on the plasma membrane suggests that the formation of VEGF-bound, active dimers can occur through several different pathways (see Fig. 1). In one pathway, the monomeric receptors dimerize on the plasma membrane, upon which a ligand may then bind to the preformed dimer and activate it. Alternatively, a ligand may bind to a monomer of VEGFR2. This liganded monomer may then dimerize with an unliganded VEGFR2 monomer to form an active liganded VEGFR2 dimer. At very high VEGF concentrations, a third pathway exists in which two ligand-bound VEGFR2 monomers can dimerize to form an active dimer upon release of one of the bound VEGF ligands. The prevalence of these pathways in an experiment designed to measure VEGF–VEGFR2 binding affinities will depend on the VEGFR2 ligand-free monomer–dimer association constant and thus on the total concentration of VEGFR2 on the plasma membrane of cells, on the possibly different binding affinities of VEGF for monomeric and dimeric forms of VEGFR2, and on the concentration of free VEGF in the medium around the cells. There are previous reports of measurements of effective VEGF binding constants, but they do not discriminate between the association state of the receptor on the surface of the cell because they do not account for the surface density dependence of VEGFR2 dimer formation on the plasma membrane (24Terman B.I. Dougher-Vermazen M. Carrion M.E. Dimitrov D. Armellino D.C. Gospodarowicz D. Böhlen P. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.Biochem. Biophys. Res. Commun. 1992; 187 (1417831): 1579-158610.1016/0006-291X(92)90483-2Crossref PubMed Scopus (1402) Google Scholar25Waltenberger J. Claesson-Welsh L. Siegbahn A. Shibuya M. Heldin C.H. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor.J. Biol. Chem. 1994; 269 (7929439): 26988-26995Abstract Full Text PDF PubMed Google Scholar, 26Kilpatrick L.E. Friedman-Ohana R. Alcobia D.C. Riching K. Peach C.J. Wheal A.J. Briddon S.J. Robers M.B. Zimmerman K. Machleidt T. Wood K.V. Woolard J. Hill S.J. Real-time analysis of the binding of fluorescent VEGF165a to VEGFR2 in living cells: effect of receptor tyrosine kinase inhibitors and fate of internalized agonist-receptor complexes.Biochem. Pharmacol. 2017; 136 (28392095): 62-7510.1016/j.bcp.2017.04.006Crossref PubMed Scopus (39) Google Scholar27Peach C.J. Kilpatrick L.E. Friedman-Ohana R. Zimmerman K. Robers M.B. Wood K.V. Woolard J. Hill S.J. Real-time ligand binding of fluorescent VEGF-A isoforms that discriminate between VEGFR2 and NRP1 in living cells.Cell Chem. Biol. 2018; 25 (30057299): 1208-1218.e510.1016/j.chembiol.2018.06.012Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Thus, most methods used to assess ligand binding to cell surface receptors cannot provide an answer as to which of the pathways is preferentially utilized in the VEGF–VEGFR2 system. Here, we overcome these prior methodological limitations by employing the fully quantified spectral imaging (FSI) methodology (22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar) to directly measure the surface density of expressed VEGFR2, the surface density of bound VEGF on the same cell, and the free VEGF concentration in the buffer surrounding the cells. Imaging many cells at different free VEGF concentrations enables the use of a rigorous thermodynamics approach and global fitting to simultaneously determine the separate affinities of VEGF for monomeric and dimeric forms of VEGFR2. To perform these measurements, we use a commercially available Alexa Fluor 594–labeled version of a recombinant single-chain derivative of vascular endothelial growth factor (scVEGF) (28Anderson C.R. Rychak J.J. Backer M. Backer J. Ley K. Klibanov A.L. scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis.Invest. Radiol. 2010; 45 (20733505): 579-58510.1097/RLI.0b013e3181efd581Crossref PubMed Scopus (107) Google Scholar). Although VEGF-A is a disulfide-linked dimer of two separate chains, scVEGF is an engineered fusion protein that combines two fragments, each composed of amino acids 3–112 of VEGF-A121, that are cloned consecutively in a head-to-tail fashion. By creating a single recombinant protein, Backer and Backer (29Backer M.V. Backer J.M. Targeting endothelial cells overexpressing VEGFR-2: selective toxicity of Shiga-like toxin-VEGF fusion proteins.Bioconjug. Chem. 2001; 12 (11716701): 1066-107310.1021/bc015534jCrossref PubMed Scopus (46) Google Scholar) were able to introduce an N-terminal 15-amino-acid tag containing a unique cysteine residue that could be used for site-specific attachment of a single fluorophore despite the presence of 16 native cysteines. scVEGF is a fully functional form of VEGF-A121 as it binds and activates VEGFR2 just like endogenous VEGFs. It is widely used in translational research and has already proven its utility as an imaging and a therapeutic agent (28Anderson C.R. Rychak J.J. Backer M. Backer J. Ley K. Klibanov A.L. scVEGF microbubble ultrasound contrast agents: a novel probe for ultrasound molecular imaging of tumor angiogenesis.Invest. Radiol. 2010; 45 (20733505): 579-58510.1097/RLI.0b013e3181efd581Crossref PubMed Scopus (107) Google Scholar, 30Blankenberg F.G. Levashova Z. Goris M.G. Hamby C.V. Backer M.V. Backer J.M. Targeted systemic radiotherapy with scVEGF/177Lu leads to sustained disruption of the tumor vasculature and intratumoral apoptosis.J. Nucl. Med. 2011; 52 (21890879): 1630-163710.2967/jnumed.111.091629Crossref PubMed Scopus (14) Google Scholar, 31Wang H. Gao H. Guo N. Niu G. Ma Y. Kiesewetter D.O. Chen X. Site-specific labeling of scVEGF with fluorine-18 for positron emission tomography imaging.Theranostics. 2012; 2 (22768028): 607-61710.7150/thno.4611Crossref PubMed Scopus (29) Google Scholar). We utilize a thermodynamic cycle that accounts for all of the different forms of VEGFR2 that can exist in the absence and presence of VEGF: monomers (M), dimers (D), ligand-bound monomers (LM), and ligand-bound dimers (LD) as shown in Fig. 1A. This model assumes that one molecule of VEGF can bind either a VEGFR2 monomer or a VEGFR2 dimer. This thermodynamic cycle includes all the possible pathways leading from unliganded VEGFR2 monomers to active, VEGF-bound VEGFR2 dimers. Along the top of the cycle, VEGFR2 can undergo unliganded dimerization with affinity KR in units of (receptors/μm2)−1. KR=[D][M]2(Eq. 1) VEGF can then bind to the preformed dimer of VEGFR2 with affinity KLD having inverse molar units (m−1). KLD=[LD][L]free[D](Eq. 2) Alternatively, a VEGF ligand in solution can bind to a monomer of VEGFR2 with affinity KLM (m−1) (moving from the top left position in the thermodynamic cycle down in a counterclockwise direction in Fig. 1A). KLM=[LM][L]free[M](Eq. 3) Moving right in the cycle, a VEGF-bound VEGFR2 monomer, LM, can dimerize with an unliganded VEGFR2 monomer, M, again forming the ligand-bound, active dimeric form of VEGFR2 with an affinity of KLMD, having units of (receptors/μm2)−1. KLMD=[LD][LM][M](Eq. 4) Traversing further counterclockwise around the cycle, under saturating VEGF conditions two liganded VEGFR2 monomers, (LM), can combine to form a liganded dimer of VEGFR2 (LD) only upon release of a VEGF molecule into solution. This process is described by the association constant KLM–LD with units of m·(receptors/μm2)−1. KLM−LD=[LD][L]free[LM]2(Eq. 5) Experimentally, we measure the total surface density of YFP-labeled VEGFR2, [T]. [T]=[M]+2[D]+[LM]+2[LD](Eq. 6) We also measure the total bound surface density of scVEGF-AF594 on the same cell. [L]bound=[LM]+[LD](Eq. 7) Finally, we measure the free VEGF concentration in the medium surrounding the cells, [L]free. Using the association constants defined above, we write the total concentration of VEGF2, [T], in terms of the free VEGF concentration and the concentration of monomeric VEGFR2. [T]=2KR(1+[L]freeKLD)[M]2+(1+[L]freeKLM)[M](Eq. 8) Next, the bound surface density of VEGF (Equation 7) is written in terms of the monomeric VEGFR2 surface density, the free VEGF concentration, and the association constants. [L]bound=[L]free[M](KLM+[M]KLDKR)(Eq. 9) When [M] is determined from Equation 8 and substituted into Equation 9, Equation 9 provides a link between the measured parameters, KR, [L]free, and [T], and the unknown binding constants, KLM and KLD. Note that KR has been measured previously (22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar), and the measured value is given in Table 1. Thus, there are two unknowns in these experiments, KLM and KLD, and measurements of [T] and [L]bound for many cells at different [L]free concentrations yield an overdetermined system of equations that is used to determine the best-fit values of the monomeric and dimeric binding affinities, KLM and KLD.Table 1Thermodynamic parameters describing VEGF binding to VEGFR2Association constantECTM VEGFR2Full-length VEGFR2KLM (m−1)9.6 × 107 ± 1.8 × 1079.6 × 107 ± 1.8 × 107KLD (m−1)4.3 × 109 ± 0.6 × 1094.3 × 109 ± 0.6 × 109KRaMeasured previously (21, 22). ((rec/μm2)−1)3.7 × 10−4 ± 0.6 × 10−43.0 × 10−2 ± 2.1 × 10−2KLMD = KR·KLD/KLM ((rec/μm2)−1)1.7 × 10−2 ± 3.9 × 10−31.3 ± 0.3KLM–LD = KR·KLD/KLM2 (m·(rec/μm2)−1)1.7 × 10−10 ± 1.3 × 10−91.4 × 10−8 ± 1.2 × 10−8a Measured previously (21Sarabipour S. Ballmer-Hofer K. Hristova K. VEGFR-2 conformational switch in response to ligand binding.Elife. 2016; 5 (27052508)e13876 10.7554/eLife.13876Crossref PubMed Scopus (81) Google Scholar, 22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar). Open table in a new tab We seek to measure the two-dimensional VEGFR2 surface density in the plasma membranes of HEK293T cells as well as the concentration (surface density) of the bound scVEGF. To achieve this goal, we subject the cells to reversible osmotic swelling as described previously (22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar, 32Singh D.R. Ahmed F. Sarabipour S. Hristova K. Intracellular domain contacts contribute to Ecadherin constitutive dimerization in the plasma membrane.J. Mol. Biol. 2017; 429 (28549925): 2231-224510.1016/j.jmb.2017.05.020Crossref PubMed Scopus (17) Google Scholar, 33King C. Wirth D. Workman S. Hristova K. Interactions between NRP1 and VEGFR2 molecules in the plasma membrane.Biochim. Biophys. Acta Biomembr. 2018; 1860 (29630862): 2118-212510.1016/j.bbamem.2018.03.023Crossref PubMed Scopus (19) Google Scholar). This treatment is required for quantitative determination of the concentration of receptors and bound ligands on the surface of the cells as the complex topology of the resting plasma membrane prevents the conversion of effective 3D concentrations into 2D membrane protein surface densities (22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar). The osmotic swelling is completely reversible (34Singh D.R. Cao Q. King C. Salotto M. Ahmed F. Zhou X.Y. Pasquale E.B. Hristova K. Unliganded EphA3 dimerization promoted by the SAM domain.Biochem. J. 2015; 471 (26232493): 101-10910.1042/BJ20150433Crossref PubMed Scopus (39) Google Scholar, 35Singh D.R. Ahmed F. King C. Gupta N. Salotto M. Pasquale E.B. Hristova K. EphA2 receptor unliganded dimers suppress EphA2 pro-tumorigenic signaling.J. Biol. Chem. 2015; 290 (26363067): 27271-2727910.1074/jbc.M115.676866Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar36Sinha B. Köster D. Ruez R. Gonnord P. Bastiani M. Abankwa D. Stan R.V. Butler-Browne G. Vedie B. Johannes L. Morone N. Parton R.G. Raposo G. Sens P. Lamaze C. et al.Cells respond to mechanical stress by rapid disassembly of caveolae.Cell. 2011; 144 (21295700): 402-41310.1016/j.cell.2010.12.031Abstract Full Text Full Text PDF PubMed Scopus (602) Google Scholar) and does not change the molecular interactions of VEGFR2 ECTM constructs in the plasma membrane (22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar). We use a VEGFR2 construct, VEGFR2 ECTM-YFP, in which the kinase domain is substituted with YFP attached via a (GGS)5 flexible linker to the TM domain (see Discussion below). Fluorescent AF594-scVEGF is added to the hypotonic swelling medium, and cells expressing VEGFR2 are imaged under two-photon excitation with the OptiMis spectral detection system (37Raicu V. Stoneman M.R. Fung R. Melnichuk M. Jansma D.B. Pisterzi L.F. Rath S. Fox M. Wells J.W. Saldin D.K. Determination of supramolecular structure and spatial distribution of protein complexes in living cells.Nat. Photonics. 2009; 3: 107-11310.1038/nphoton.2008.291Crossref Scopus (88) Google Scholar, 38Biener G. Stoneman M.R. Acbas G. Holz J.D. Orlova M. Komarova L. Kuchin S. Raicu V. Development and experimental testing of an optical micro-spectroscopic technique incorporating true line-scan excitation.Int. J. Mol. Sci. 2013; 15 (24378851): 261-27610.3390/ijms15010261Crossref PubMed Scopus (46) Google Scholar). As shown in Fig. 2A, YFP is intracellularly located, whereas AF594-scVEGF is bound to the extracellular domain of VEGFR2. Fig. 2B shows a schematic of the experimental setup. Measurements of the plasma membrane VEGFR2 surface densities, bound VEGF surface densities, and free VEGF concentrations in the media are performed with the FSI methodology. As described previously (22King C. Stoneman M. Raicu V. Hristova K. Fully quantified spectral imaging reveals in vivo membrane protein interactions.Integr. Biol. 2016; 8 (26787445): 216-22910.1039/c5ib00202hCrossref Scopus (61) Google Scholar), FSI enables the measurements of the surface density of fluorophore-labeled membrane proteins in 2–3-μm-size patches by performing two scans, a “donor scan” to excite YFP at 960 nm and an “acceptor scan” to excite AF594 at 800 nm. The fluorescence absorbance and emission spectra of YFP and AF594 are shown in Fig. 2C. FSI also allows for the measurement of the three-dimensional concentration of freely diffusing, fluorescent moieties, in this case free AF594-scVEGF, in the solution surrounding the cells. Fig. 2, D and E, show three transiently transfected HEK293T cells that have been swollen with hypotonic medium in the presence of AF594-scVEGF. The three cells are each expressing different levels of VEGFR2 ECTM-YFP, as indicated by the differing VEGFR2 ECTM-YFP intensities. Fig. 2D, top, shows the cells under excitation at 960 nm, such that YFP attached to VEGFR2 ECTM is primarily excited. Large stretches of homogenous membrane fluorescence are visible, with the protein primarily localized in the plasma membrane. Fig. 2D, bottom, shows the same cells under excitation at 800 nm, exciting primarily AF594-scVEGF. AF594-scVEGF is highly concentrated on the surface of the cells, but no Alexa Fluor 594 fluorescence originates from the intracellular space. Thus, the ligand is not endocytosed as expected, primarily because cell swelling inhibits endocytosis (39Rauch C. Farge E. Endocytosis switch controlled by transmembrane osmotic pressure and phospholipid number asymmetry.Biophys. J. 2000; 78 (10827982): 3036-304710.1016/S0006-3495(00)76842-1Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Furthermore, the VEGFR2 ECTM construct lacks a kinase domain and does not undergo internalization. On cells with no measurable VEGFR2 ECTM-YFP fluorescence, we see no measurable AF594-scVEGF fluorescence (Fig. 3), indicating that nonspecific binding of VEGF provides a minimal contribution to our measurements. We measured the surface densities of VEGFR2 ECTM-YFP and bound AF594-scVEGF and the free AF594-scVEGF concentration in 12 independent experiments (at different free ligand concentrations) with a total of 387 cells. The free VEGF concentrations were varied across 3 orders of magnitude, from 0.21 to 42.4 nm, and were measured directly with the FSI method (Fig. 4). These experiments provided a total of 661 data points at the 12 free VEGF concentrations. Fig. 2E shows the measured total apparent FRET efficiency between the intracellular YFP of VEGFR2 ECTM and the extracellularly bound AF594-scVEGF as a function of total VEGFR2 surface density. The FRET efficiency is zero for all concentrations of VEGFR2. This is expected because YFP (the FRET donor) and the AF594 (the FRET acceptor) are further than 10 nm apart when VEGF is bound to the distal region of VEGFR2’s EC domain (see Fig. 2A). Indeed, the plasma membrane is ∼5 nm thick on its own, and the intracellular YFP is a large β-barrel connected to VEGFR2 TM domains via a 15-amino-acid (GGS)5 flexible linker, whereas VEGF is bound to the extracellular D2 and D3 subunits of VEGFR2. Thus, the negligible FRET efficiencies measured for all VEGFR2 surface densities and all free VEGF concentrations serve as an important control for these experiments. Fig. 5 shows the bound AF" @default.
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• W2941014077 title "Direct measurements of VEGF–VEGFR2 binding affinities reveal the coupling between ligand binding and receptor dimerization" @default.
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