Figure 8. Bmp4 endocytosis is mediated by Bmper, Gremlin, and Noggin, but not Chordin. A panel of secreted Bmp factors (6.0 nM) were evaluated for their effects on Bmp4 (0.6 nM) and on Bmper-mediated Bmp4 internalization and signaling at several time points. A Western analysis showing the effects of active recombinant Chordin, Noggin, and Gremlin on the activity of Bmp4 alone or with cleaved full-length Bmper was measured 10 min (a) and 1.5 h (b) after treatment. Protein internalization and Smad activation were measured for each of these Bmp factors: Chordin (top), Noggin (middle), and Gremlin (bottom). (c) Pulse-chase experiment in MECs treated with Bmp4, Bmper, Noggin, and combinations of Bmp4 and Bmper (top), Bmp4 and Noggin (middle), and Bmper and Chordin (bottom). After treating MECs with recombinant protein(s) using the same experimental conditions and pro- tein concentrations used in panel a, MECs were either chased with cold media or cold media containing chloroquine that lack recombinant protein(s). (d) A model of Bmper-mediated Bmp4 internalization. are available describing Bmp degradation (Entchev et al., 2000; Degnin et al., 2004). Lysosomal- and proteasomal-dependent degradation of Bmp4 within the presecretory pathway was shown to occur after the initial cleavage step of the inactive Bmp4 precursor and before the final cleavage step that stabilizes the mature secreted form (Degnin et al., 2004). Postsecretory regulation of Dpp through an endocytic mechanism that traffics DPP to the lysosome for degradation during Drosophila devel- opment has also been described. The present study provides the first example of targeted intracellular Bmp degradation by a se- creted regulator of Bmp signaling and indicates that this endo- cytic mechanism is broadly used by Bmp regulators such as Bmper, Noggin, and Gremlin to fine-tune Bmp regulation. Indications that endocytic internalization mechanisms modulate Bmp signaling were first suggested by studies in Dro- sophila showing that a Dynamin-dependent retrieval process participates in creating an inverse gradient of the Bmp modula- tor Sog (a Chordin homologue), which fine-tunes concentration gradients for Dpp in the extracellular space (Srinivasan et al., 2002). Receptors for Bmp family members have also been shown to internalize through distinct endocytic pathways. In the case of TGF- receptors, caveolin-dependent internalization mechanisms target the receptors for proteasomal degradation, whereas clathrin-dependent internalization alternatively is required for appropriate Smad activation (Di Guglielmo et al., 2003). Constitutive internalization and degradation of type I and II Bmp receptors via clathrin-mediated endocytosis, and to a lesser extent internalization of BmprII by a caveolae- dependent route, have also been established (Hartung et al., 2006). Our experimental observations extend the role of endo- cytic mechanisms within the Bmp signaling pathway by demon- strating mutually triggered clathrin-dependent endocytosis 606 JCB • VOLUME 184 • NUMBER 4 • 2009