VEGF receptor heterodimers I Nilsson et al of vegfr3 is lethal slightly later, at E10.5, due to the lack of remodelling of the vascular network (Dumont et al, 1998). In subsequent development, VEGFR3 is critical for lymphatic vessel function. Thus, during a restricted phase in early development, both VEGFR2 and VEGFR3 serve critical functions in the formation of the vascular tree. A number of cell types co-express VEGFR2 and VEGFR3, potentially allowing formation of heterodimers. Expression of VEGFR3 is induced during angiogenic sprouting in the adult, both in physiological and pathological conditions (Tammela et al, 2008). In differentiating stem cell cultures, VEGFR3 is expressed by subpopulations of endothelial cells in growing vessels, some of which may transdifferentiate to become early lymphatic structures (Kreuger et al, 2006; Adams and Alitalo, 2007). In addition, VEGFR2 may be variably expressed by LECs (Petrova et al, 2002), and on collecting lymphatic vessels that otherwise are characterized by high expression levels of VEGFR3 (Saaristo et al, 2002). Here, we investigate the function of VEGFR2/-3 hetero- dimerization in intact endothelial cells and angiogenic sprouts, using a newly developed in situ proximity ligation assay (in situ PLA) (Soderberg et al, 2006; Jarvius et al, 2007). This method is based on the use of oligonucleotide- conjugated antibodies, which when brought in close proxi- mity by binding to epitopes on, for example, dimerized VEGFRs, allow a rolling-circle ampliﬁcation detected by a ﬂuorescently labelled probe. We show that endogenous unmanipulated VEGFRs form heterodimers in response to VEGFA or VEGFC. We present evidence that heterodimers are present on tip cell ﬁlopodia, and that neutralization of VEGFR3 to prevent heterodimer formation induced by VEGFA leads to a marked decrease in sprouting activity. It is concluded that VEGFR2/-3 heterodimers have a signiﬁcant function in the positive regulation of angiogenic sprouting. Results Detection of VEGFC-induced VEGFR2/-3 heterodimers in endothelial cells by immunoprecipitation To study the induction of VEGFR2/-3 heterodimers, we examined receptor expression in different endothelial cell cultures. On reducing gels, VEGFR3 migrates as three mole- cular weight species, 195, 175 and 125 kDa (Pajusola et al, 1994). The mature, cell surface expressed VEGFR2 appears as a 250 kDa band on immunoblots. Using porcine aortic endothelial (PAE) cells transfected to express VEGFR3 as control, we compared expression of receptors in primary human saphenous and umbilical vein-derived endothelial cells (HSaVECs and HUVECs). Both cell types co-expressed VEGFR2 and VEGFR3 (Figure 1A); we chose HSaVECs for further experimentation. Treatment of HSaVECs with processed VEGFC, followed by immunoprecipitation of VEGFR2 and immunoblotting for VEGFR3, showed complex formation between the receptors. Figure 1B displays the prominent 125 kDa band and the weaker 195–175 kDa VEGFR3 bands in the VEGFR2 immu- noprecipitate. Heterodimers were not detected in response to VEGFA in this assay (Figure 1B, upper panel). The effect of VEGFA on VEGFR2 tyrosine phosphorylation was more pro- minent than that of VEGFC (Figure 1B, middle panel), in agreement with the data from earlier biochemical analyses, 1378 The EMBO Journal VOL 29 | NO 8 | 2010 Figure 1 Induction of VEGFR2/-3 heterodimers in co-expressing endothelial cells. (A) Total cell lysates derived from HUVECs, HSaVECS or PAE cells; either untransfected or expressing VEGFR3 were immunoblotted to detect VEGFR3 (left panel). After reduction of disulphide bridges, VEGFR3 migrates as three species of apparent mw 195, 175 and 125 kDa. PAE, HUVEC and HSaVEC lysates were also immunoblotted to detect VEGFR2, which migrates as two species around the 250 kDa marker (right panel). (B) HSaVEC lysates from cells treated or not with VEGFA or VEGFC for 8 min were used for immunoprecipitation (ip) of VEGFR2 followed by immunoblotting for VEGFR3 (upper panel) to detect receptor het- erodimerization. This was followed by immunoblotting to detect phosphorylated VEGFR2 (middle panel) using the 4G10 mAb, and immunoblotting to show equal loading of VEGFR2 (lower panel). (C) Immunoprecipitation of VEGFR3 from HSaVECs treated as in (B) followed by immunoblotting to detect co-immunoprecipitation of VEGFR2 (upper panel), phosphorylation of VEGFR3 (middle panel) and VEGFR3 loading (lower panel). showing higher afﬁnity of VEGFA than VEGFC, for binding to VEGFR2 (Joukov et al, 1997). Similarly, treatment with VEGFC but not with VEGFA, allowed detection of the 250 kDa VEGFR2 band in immuno- blots of VEGFR3 immunoprecipitates from HSaVECs (Figure 1C, upper panel). These data show that VEGFR2 and VEGFR3 form heterodimeric signalling complexes in primary cells in response to VEGFC. In situ PLA reveals VEGFR2/-3 heterodimerization in response to VEGFA or VEGFC To demonstrate the formation of heterodimers in intact cells, we used in situ PLA (see schematic outline in Figure 2A). We used an experimental design where cells were incubated with VEGFA, VEGFC or vehicle for 8 min, ﬁxed and then probed with primary antibodies raised in different species and direc- ted towards the intracellular domains of VEGFR2 or VEGFR3. & 2010 European Molecular Biology Organization