VEGF receptor heterodimers I Nilsson et al Of note, VEGFR2/-3 heterodimerization also increased signiﬁcantly (25-fold) when cells were treated with VEGFA for 8 min (see Figure 2B and C). Therefore, despite the fact that VEGFA fails to induce receptor heterodimerization as detected by co-immunoprecipitation, VEGFR2/-3 heterodi- mers were induced by VEGFA in the in situ PLA. To show the speciﬁcity of the reactions, we preincubated cells with antibodies previously shown to speciﬁcally neu- tralize either human VEGFR2; IMC-1121b (Zhu et al, 2003) or human VEGFR3; hF4-3C5 (Persaud et al, 2004), by binding to the ligand-binding part of the extracellular domains. As shown in Figure 2C, blocking with either of the neutralizing antibodies quenched the appearance of heterodimers to the level of basal heterodimerization recorded in unstimulated cells. Different VEGFA isoforms (VEGFA121, 145, 165 and 189) were analysed for their ability to induce VEGFR2/-3 hetero- dimers. Isoforms with a higher propensity for binding to heparan sulphate and extracellular matrix, that is VEGFA145 and VEGFA189 (Kawamura et al, 2008), induced heterodimers more efﬁciently than the relatively more solu- ble isoforms VEGFA121 and VEGFA165 (Figure 2D). In repeated experiments, however, VEGFC induced heterodimer formation more efﬁciently than any of the VEGFA isoforms. Further, VEGFD also induced VEGFR2/-3 heterodimeriza- tion, in agreement with that processed human VEGFD, but not mouse VEGFD, binds to VEGFR2 (Baldwin et al, 2001). The effect was less efﬁcient than that of VEGFC but more prominent than that of VEGFA. To control for the speciﬁcity, we analysed the effect of PDGFB, which does not bind to either of the VEGF receptors. As shown in Figure 2E, VEGFR2/-3 heterodimers were not induced by PDGFB. Binding of ligand induces internalization and degradation of receptor tyrosine kinases. Figure 2F shows HSaVECs treated with VEGFC for different time periods, followed by in situ PLA. Formation of heterodimers peaked at 10 min and the ligand-induced heterodimers remained detectable for up to 2 h. With prolonged incubations, the in situ PLA signals were lost, in agreement with the clearance of receptors. Combined, these data provide evidence that heterodimers are formed in a ligand-dependent and speciﬁc manner in intact cells. Detection of homodimerization of VEGFRs by in situ PLA We next examined the pattern of VEGFR2 and VEGFR3 homodimerization induced by VEGFA and VEGFC. For this purpose, monoclonal antibodies against either VEGFR2 or VEGFR3 were divided in pools, which were ligated with either a ‘plus’ oligonucleotide or a ‘minus’ oligonucleotide. The PLA reaction indicating for example VEGFR2 homo- dimers, would occur only as a result of close proximity of a plus-ligated antibody with a minus-ligated antibody, whereas pairs consisting of plus–plus or minus–minus ligated anti- bodies would not give rise to PLA signals (see Figure 3A for a schematic outline). Consequently, we could score only 50% of the actual homodimerization events, namely when anti- bodies combined in plus–minus and minus–plus constella- tions. Moreover, we could not directly compare the relative extent of receptor homo- and heterodimerization, as different combinations of antibodies had to be used to detect the different receptor complexes. As shown in Figure 3B, VEGFA and VEGFC consistently induced heterodimers and VEGFC was about 3–4-fold more Figure 3 VEGFR2/-3 homo- and heterodimers induced by VEGFA or VEGFC. (A) Schematic outline of primary antibody ligation with oligonucleotide plus and minus strands to detect VEGFR homodimers. Only pairing of antibodies with plus and minus strands allow initiation of the rolling-circle ampliﬁcation. (B) VEGFR2/-3 heterodimerization. HSaVECs were treated for 8 min with either VEGFA or VEGFC. Heterodimerization was 3–4-fold more efﬁciently induced by VEGFC. n ¼ 6. (C) VEGFR2 homodimers. Equal mixtures of VEGFR2 monoclonal antibodies ligated with plus and minus strands of oligonucleotides (as outlined in A) were used to detect VEGFR2 homodimers on HSaVECs treated for 8 min with VEGFA or VEGFC as above. n ¼ 6. (D) VEGFR3 homodimers. Equal mixtures of VEGFR3 monoclonal antibodies ligated with plus and minus strands of oligonucleotides were used to detect VEGFR3 homodimers on HSaVECs treated for 8 min with VEGFA or VEGFC. n ¼ 6. (E) Schematic outline of VEGFR homo- and heterodimerization induced by VEGFA or VEGFC. Note that the relative distribution of homodimers versus heterodimers cannot be accurately determined due to the inherent difference in afﬁnity of different antibodies. Asterisks in panels B–D indicate the degree of signiﬁcance (***Po0.001). 1380 The EMBO Journal VOL 29 | NO 8 | 2010 & 2010 European Molecular Biology Organization