The EMBO Journal (2010) 29, 1377–1388 | & 2010 European Molecular Biology Organization | Some Rights Reserved 0261-4189/10 www.embojournal.org VEGF receptor 2/-3 heterodimers detected in situ by proximity ligation on angiogenic sprouts THE EMBO JOURNAL EMBO open This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits distribution, and reproduction in any medium, provided the original author and source are credited. This license does not permit commercial exploitation without speciﬁc permission. Ingrid Nilsson1,7,9, Fuad Bahram1,9, Xiujuan Li1, Laura Gualandi1, Sina Koch 1 , 1 1 Malin Jarvius , Ola So¨ derberg , Andrey Anisimov2, Ivana Kholov a´3,8, Bronislaw Pytowski4, Megan Baldwin5, Seppo Yl a¨-Herttuala3, Kari Alitalo2, Johan Kreuger6 and Lena Claesson-Welsh1,* 1Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden, 2Molecular and Cancer Biology Research Program, University of Helsinki, Helsinki, Finland, 3Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland, 4Imclone Systems Corporation, New York, NY, USA, 5Vegenics Limited, Toorak, Victoria, Australia and 6Department of Medical Biochemistry and Microbiology, Biomedical Center, Uppsala University, Uppsala, Sweden The vascular endothelial growth factors VEGFA and VEGFC are crucial regulators of vascular development. They exert their effects by dimerization and activation of the cognate receptors VEGFR2 and VEGFR3. Here, we have used in situ proximity ligation to detect receptor complexes in intact endothelial cells. We show that both VEGFA and VEGFC potently induce formation of VEGFR2/- 3 heterodimers. Receptor heterodimers were found in both developing blood vessels and immature lymphatic structures in embryoid bodies. We present evidence that heterodimers frequently localize to tip cell ﬁlopodia. Interestingly, in the presence of VEGFC, heterodimers were enriched in the leading tip cells as compared with trailing stalk cells of growing sprouts. Neutralization of VEGFR3 to prevent heterodimer formation in response to VEGFA decreased the extent of angiogenic sprouting. We conclude that VEGFR2/-3 heterodimers on angiogenic sprouts induced by VEGFA or VEGFC may serve to positively regulate angiogenic sprouting. The EMBO Journal (2010) 29, 1377–1388. doi:10.1038/ emboj.2010.30; Published online 11 March 2010 Subject Categories: signal transduction Keywords: angiogenic sprouting; embryoid body; heterodimer; proximity ligation; VEGF receptor *Corresponding author. Department of Genetics and Pathology, Uppsala University, Rudbeck Laboratory, Dag Hammarskjoldsv. 20, Uppsala 751 85, Sweden. Tel.: þ 46 18 471 4363; Fax: þ 46 18 55 89 31; E-mail: lena.welsh@genpat.uu.se 7Present address: Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden 8Present address: Pathology, Laboratory Centre, Tampere University Hospital, Finland 9These authors contributed equally to this work Received: 10 October 2009; accepted: 15 January 2010; published online: 11 March 2010 & 2010 European Molecular Biology Organization Introduction The mammalian vascular endothelial growth factor (VEGF) family includes VEGFA, VEGFB, VEGFC, VEGFD and placenta growth factor (PlGF). These ligands bind in an overlapping pattern to three different receptor tyrosine kinases denoted VEGF receptor 1 (VEGFR1), VEGFR2 and VEGFR3 (Olsson et al, 2006). Although the VEGFRs are not exclusively ex- pressed on vascular cells, there is prominent expression and important function of VEGFR1 on hematopoietic and en- dothelial cells, of VEGFR2 on vascular endothelial cells and of VEGFR3 on vascular and lymphatic endothelial cells (LECs). The active VEGF/VEGFR signalling complexes also include co-receptors, such as heparan sulphate proteoglycans (Esko and Selleck, 2002) and neuropilins (Geretti et al, 2008). The VEGFRs transduce their effects according to the con- sensus scheme for receptor tyrosine kinases. Binding of ligand leads to dimerization of receptors. This close apposi- tion confers structural reorganization of the receptor intra- cellular domain and exposure of the kinase active site (Hubbard, 1999). Kinase activity catalyses the transfer of phosphate groups to tyrosine residues, which serve as sub- strates for the kinase. Such acceptor sites are found both on the partner in the dimer as well as on cytoplasmic signalling molecules. Tyrosine phosphorylation initiates signal trans- duction cascades, which ultimately become established as cellular responses such as survival, proliferation and motility. Certain VEGF ligands bind to more than one VEGFR, potentially allowing receptors to form heterodimers in addi- tion to homodimers. We have shown earlier that VEGFR2/-3 heterodimers are formed in cultured LECs in response to VEGFC (Dixelius et al, 2003). In accordance, processing of VEGFC during synthesis allows binding to both VEGFR2 and -3 (Joukov et al, 1997; Alitalo et al, 2005). Heterodimerization was accompanied by a loss of phosphorylation of C-terminal tyrosine residues in VEGFR3 (Dixelius et al, 2003). The underlying mechanism may involve different substrate spe- ciﬁcities of the VEGFR2 and VEGFR3 kinases. Alternatively, VEGFR3 may undergo a conformational change when en- gaged in a heterodimer with VEGFR2, and thereby, certain acceptor sites become hidden or otherwise inaccessible. Thus, it is important to determine the composition of receptor complexes formed under different conditions, as this will be of consequence for the biological response. Various sophisticated recombinant animal models have greatly aided our understanding of the biology of the VEGFRs. All three receptors are required for proper embryo- nic development. Gene inactivation of vegfr1 is lethal at E8.5–9 due to increased endothelial proliferation leading to obstruction of vessels (Fong et al, 1995, 1999). Inactivation of vegfr2 leads to arrest in endothelial differentiation, causing embryonic lethality at E8.5 (Shalaby et al, 1995). Inactivation The EMBO Journal VOL 29 | NO 8 | 2010 1377