VEGF receptor heterodimers I Nilsson et al Figure 6 VEGFA-induced angiogenic sprouting is mediated by VEGFR2 homodimers and VEGFR2/-3 heterodimers. (A) Effect of VEGFR3 neutralization. VEGFA- or VEGFC-induced EB cultures in 3D collagen were treated with or without antibodies neutralizing the function of VEGFR3. Scale bar ¼ 300 mm. (B) Quantiﬁcation of sprouting area. Cultures treated with VEGFA or VEGFC in the absence or presence of neutralizing VEGFR3 antibodies as shown in panel (A) were quantiﬁed. n ¼ 5. Asterisks indicate degree of signiﬁcance (**Po0.01). (C) Activation of VEGFR2. Immunoblotting for phosphorylated VEGFR2 (pY1175) from HSaVEC cultures treated with VEGFA in the presence and absence of neutralizing VEGFR3 antibodies, for different time periods (left panel). Middle panel shows blotting for VEGFR2 and lower panel shows equal loading of b-actin. Densitometric scanning (right panel) of bands showed no decrease in VEGFR2 activation by the VEGFR3 antibodies. Using in situ PLA, we made two novel and important discoveries concerning the composition of receptor complexes induced by VEGFA or VEGFC. First, VEGFC was able to induce formation of VEGFR3 homodimers and VEGFR2/-3 heterodi- mers, but not VEGFR2 homodimers. VEGFC has been shown to have the highest afﬁnity for VEGFR3, but processed mouse VEGFC also binds to VEGFR2, although with lower afﬁnity (Joukov et al, 1997) and theoretically, VEGFR2 homodimer- ization by VEGFC could therefore be anticipated. Indeed, VEGFC forms co-crystals with VEGFR2 immunoglobulin-like domains 2 and 3 (Leppa¨nen et al, 2010). However, co-expres- sion of VEGFR3 may block VEGFR2 homodimerization by VEGFC. Moreover, it is conceivable that the assembly and folding of the ligand-receptor complex is inﬂuenced by co- receptors such as neuropilins and proteoglycans. Thus, neu- ropilin2 has been shown to associate with VEGFR3 in a 1384 The EMBO Journal VOL 29 | NO 8 | 2010 VEGFC-dependent manner (Karkkainen et al, 2001; Favier et al, 2006; Karpanen et al, 2006). The second important ﬁnding reported here is that VEGFA, which binds only to VEGFR2 and not to VEGFR3, induced VEGFR2/-3 heterodi- mers. In contrast, VEGFA did not induce co-immunoprecipita- tion of VEGFR2 and VEGFR3 from monocultures of HSaVECs. A tendency for VEGFA-induced VEGFR2/-3 heterodimerization was, however, detected by co-immunoprecipitation in the more complex 2D EB model (Figure 4B). VEGFA-induced heterodimeric complexes may be less stable than those in- duced by VEGFC as VEGFC binds directly to both VEGFR2 and VEGFR3, whereas VEGFA binds only to VEGFR2. Such loosely bonded heterodimers would more efﬁciently be detected by the PLA methodology, which does not require that complexes withstand the stress of detergent lysis and stringent wash protocols. & 2010 European Molecular Biology Organization