Figure 4. Bmper and Bmp4 internalize through a clathrin-dependent mechanism. (a) MECs were treated with 50 μM chlorproma- zine to disrupt internalization into endosomes and 10 mM methyl- -cyclodextrin to disrupt lipid raft–caveolar internalization. (b) Transmis- sion electron microscopy sequentially captured after cotreatment of MEC with 0.6 nM Bmp4 (detected with 5-nm gold immunoparticles) and 6 nM Bmper (detected with 25-nm gold immunoparticles). Colocalization (arrows) is demonstrated at discreet locations on the cell membrane after a 5-min pulse with Bmp4 and Bmper (i and ii), coalescing along clathrin- coated invaginations (arrow) after a 30-min pulse (iii), and endocytic sorting after 1.5 h of treatment (iv and v). to suppress its signaling. After loading exogenous proteins into cells, Bmper and Bmp4 were rapidly degraded during a chase period with cold media (Fig. 3 d, top). In contrast, chasing in the presence of chloroquine prevented lysosome-dependent degrada- tion of both Bmp4 and Bmper with similar kinetics (Fig. 3 d, bot- tom), whereas the proteasome inhibitor MG-132 had no affect on Bmper or Bmp4 stability (not depicted). These data suggest that Bmper dampens the magnitude and duration of Bmp4-dependent Smad activation by restricting Bmp4 availability through endo- cytosis and raise the possibility (explored subsequently) that Bmper and Bmp4 colocalize during endosomal trafficking. To determine whether the levels of extracellular Bmp are reduced by Bmper-mediated endocytosis, we measured the dis- appearance of Bmp4 from conditioned media and its accumula- tion within endothelial cells. Extracellular Bmp4 was efficiently removed from conditioned media within 24 h in the presence of Bmper (Fig. 3 e). When MECs were retreated with Bmp4 and Bmper for 2 h before 24- and 48-h harvest time points, Bmp4 in- ternalization remained comparable to that of acute treatment. These data indicate that the extracellular pools of Bmper and Bmp4 are being depleted concomitant with endocytosis and this process does not cause loss of the cell surface machinery (that may include recycling Bmp receptors) responsible for internaliz- ing Bmp4 and Bmper. Both human and zebrafish Bmper are proteolytically pro- cessed into two fragments that remain associated by disulfide bonds (Binnerts et al., 2004; Rentzsch et al., 2006). To test whether proteolytic processing of Bmper affects Bmp4 internal- ization and signaling activity in endothelial cell culture, we com- pared wild-type Bmper to three mutant forms of Bmper protein: one containing only the C-terminal fragment, another containing the N-terminal fragment, and a full-length proteolytic cleavage mutant. These myc-tagged constructs were expressed in HEK-293 cells, and the conditioned media from these cells were used to treat MECs. Wild-type Bmper behaved identically to recombi- nant Bmper in conditioned media from untransfected HEK-293 cells (unpublished data). Likewise the C-terminal fragment had no impact on Bmp4 signaling, whereas the N-terminal fragment and the proteolytic cleavage mutant efficiently inhibited Bmp4 signaling as measured by Smad phosphorylation (Fig. 3 f). These data suggest that the N-terminal half of Bmper is required to trap Bmp4 extracellularly to prevent Smad activation through ma- chinery that have been suggested by previous studies (Zhang et al., 2007) and that the C-terminal half of Bmper is further re- quired to sink or internalize Bmp4 into the cell to more efficiently degrade Bmp4 and prevent signaling. Although the physiological relevance of Bmper cleavage is poorly understood, the present data suggest that Bmper proteolysis may differentially regulate Bmp activity in part by regulating its endocytosis. Bmper and Bmp4 internalize through a clathrin-dependent mechanism To explore the mechanism of Bmper internalization, we pre- treated MECs with pharmacological agents that inhibit clathrin- dependent internalization into endosomes (chlorpromazine) or that disrupt lipid raft–caveolar internalization (methyl- - cyclodextrin; Fig. 4 a; Di Guglielmo et al., 2003; Hartung et al., 2006). Endosomal transport was almost completely attenuated by chlorpromazine (17 μM), whereas methyl- -cyclodextrin (10 mM; and also the caveolin-dependent inhibitors nystatin and filipin [unpublished data]) had no effect. Collectively, this study supports a clathrin-dependent, caveolin-independent mechanism for Bmper internalization. Consistent with this con- clusion, there were no differences in the ability of caveolin-1 lung endothelial cells (Lin et al., 2007) to internalize Bmp4 in the presence of Bmper compared with their wild-type ENDOCYTIC REGULATION OF BMP SIGNALING BY BMPER • Kelley et al. 601