Figure 3. Bmp4 and Bmper are recipro- cally regulated by internalization. MECs were treated with recombinant Bmper (6 nM) and/or Bmp4 (0.6 nM) and their lysates were sub- jected to the following analysis. (a) A Western blot showing enhanced Bmper and Bmp4 in- ternalization in the presence of one another (lane 4). Addition of a Bmp4 neutralizing anti- body prevented their internalization (lane 5). (b) MECs were infected with adenoviruses ex- pressing GFP, wild-type Bmp receptor II (WT), or a tailless form of Bmp receptor II (Tailless) for 24 h before cotreatment with 0.6 nM Bmp4 and 6 nM Bmper for 1.5 h. (c) Kinetic analysis of the effects of Bmper (6 nM) on Bmp4 (0.6 nM) activity and Bmp4 internalization after treat- ing MECs with Bmp4 and/or Bmper for 48 h. (d) A direct comparison of internalized Bmper and Bmp4 after a pulse with recombinant Bmp proteins and a chase with cold media or media containing chloroquine. (e) Cell lysates from MECs pulsed with Bmp4 alone (0.6 nM) and with Bmper (6 nM) for 2, 24, and 48 h were measured by Western analysis for Bmp4 in conditioned media before and after retreat- ing the cells at 22 and 46 h (24p and 48p, respectively). (f) A Western analysis of Bmper, Bmp4, and p-Smad using lysates from MECs treated as follows: HEK-293 conditioned media from untreated cells (UT CM); untrans- fected HEK-293 cells spiked with 6 nM of re- combinant full-length cleaved Bmper (rBmper) and/or 0.6 nM of recombinant Bmp4 (Bmp4); and conditioned media from HEK-293 cells ex- pressing the amino fragment (N terminus), car- boxy fragment (C terminus), and a proteolytic cleavage mutant of Bmper (PC mutant). MECs with Bmper (6 nM) and Bmp4 (0.6 nM) with or without a Bmp4 monoclonal antibody that prevents Bmp4 binding to its re- ceptor and neutralizes Bmp4-mediated Smad phosphorylation (Fig. 3 a). If Bmp ligands and Bmp receptors participate during Bmper internalization, then their respective presence or inhibition would have an effect on endocytosis. Bmp4 alone significantly activated Smad and was weakly detected in whole cell lysates. Interestingly, Bmper and Bmp4 internalization dramatically in- creased in the presence of one another, whereas Bmp4-mediated Smad activation was diminished in the presence of Bmper. The Bmp4 neutralizing antibody abolished Bmper-mediated Bmp4 internalization and Smad activation, suggesting that Bmper initi- ates Bmp4 internalization through a receptor-dependent process. To rule out the possibility that the Bmp4 neutralizing antibody disrupts the ability of Bmper to bind Bmp4, we immunoprecipi- tated Bmp4 in the presence of Bmper using an antibody against Myc-Bmper, both in the absence and presence of the Bmp4 neu- tralizing antibody (Fig. S1 b). The neutralizing antibody did not affect the ability of Bmper to bind Bmp4. These data suggest that the neutralizing antibody and Bmper bind to Bmp4 through ex- clusive binding sites and that the interaction of a Bmp4–Bmper complex with Bmp receptors at the cell surface is prevented by the neutralizing antibody. Accordingly, we were able to signifi- cantly reduce the amount of internalized Bmper and Bmp4 and rescue Bmp4-mediated Smad activity by ectopically expressing a tailless form of Bmp receptor II in endothelial cells (Fig. 3 b). The mutant BmprII in this construct lacks a cytoplasmic extension downstream of the kinase domain, including sequences required for endocytosis, and emulates the naturally occurring splice vari- ant of this receptor (Lee-Hoeflich et al., 2004). It is important to note that we were able to express these proteins via adenoviral transduction with an efficiency of 40–50% in endothelial cells, so that the effects observed by Western blotting are an underesti- mate of the consequences of receptor endocytosis inhibition. To clarify the relationships of Bmp4 and Bmper on Smad phosphorylation, we evaluated the temporal effects of Bmper on Bmp4 activity using pulse and pulse-chase experiments (Fig. 3, c–e). When treated with Bmp4 (0.6 nM) alone, Smad activa- tion was detected for at least 24 h in MECs (Fig. 3 c, left). Attenuation of Smad phosphorylation coincided with the slow accumulation of Bmp4 in cell lysates. Bmper (6 nM) alone ac- cumulated in MEC lysates over a 2-h interval and Smad phos- phorylation was not detected (Fig. 3 c, middle). When MECs were treated with both Bmp4 (0.6 nM) and Bmper (6 nM), Bmp4 internalization was markedly enhanced and accelerated with kinetics that closely paralleled those of Bmper, which in turn internalized more efficiently in the presence of Bmp4 (Fig. 3 c, right). In the presence of Bmper, Bmp4-dependent Smad phosphorylation returned to baseline much more rapidly, within 2 h (Fig. 3 c and Fig. S1, c and d). Collectively, these data indicate that Bmper regulates Bmp4 at least in part by accelerat- ing the removal of extracellular Bmp4. We reasoned that, similar to Bmper, internalized Bmp4 might also be sorted for lysosomal degradation as a mechanism 600 JCB • VOLUME 184 • NUMBER 4 • 2009