JCB: ARTICLE A concentration-dependent endocytic trap and sink mechanism converts Bmper from an activator to an inhibitor of Bmp signaling Rusty Kelley,1 Rongqin Ren,1 Xinchun Pi,1 Yaxu Wu,1 Isabel Moreno,1 Monte Willis,1 Martin Moser,1 Malcolm Ross,1 Monika Podkowa,2 Liliana Attisano,2 and Cam Patterson1 1Department of Medicine, Carolina Cardiovascular Biology Center, University of North Carolina, Chapel Hill, NC 27599 2Department of Biochemistry, Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada Y G O L O I B L L E C F O L A N R U O J E H T B gaster crossveinless 2, is a secreted factor that regu- mper, which is orthologous to Drosophila melano- lates Bmp activity in a tissue- and stage-dependent manner. Both pro- and anti-Bmp activities have been pos- tulated for Bmper, although the molecular mechanisms through which Bmper affects Bmp signaling are unclear. In this paper, we demonstrate that as molar concentrations of Bmper exceed Bmp4, Bmper dynamically switches from an activator to an inhibitor of Bmp4 signaling. Inhibition of Bmp4 through a novel endocytic trap-and-sink mecha- nism leads to the efﬁcient degradation of Bmper and Introduction Bmp pathways are tightly regulated at multiple levels of signal- ing to allow for diverse biological function. The Bmp signals that initiate target cell activation are strongly influenced by extra- cellular Bmp modulators (for review see Balemans and Van Hul, 2002). The extracellular cues that target these downstream Bmp signaling components to distinct endocytic pathways re- main unclear. Extracellular Bmp modulators, including Bmper (Bmp-binding endothelial cell precursor-derived regulator), a member of the Kielin-Chordin–related protein subfamily, are an important component of Bmp regulation as they help control the boundaries and sensitivity of Bmp signaling during many aspects of development (Michos et al., 2004; Rentzsch et al., 2006; Choi et al., 2007). Until recently, disparate reports of Bmper serving both as a pro- and anti-Bmp factor failed to sup- port a model that explained the dual nature of Bmper activity (Conley et al., 2000; Moser et al., 2003; Binnerts et al., 2004; Coles et al., 2004; Kamimura et al., 2004; Ralston and Blair, Bmp4 by the lysosome. Bmper-mediated internalization of Bmp4 reduces the duration and magnitude of Bmp4- dependent Smad signaling. We also determined that Noggin and Gremlin, but not Chordin, trigger endocyto- sis of Bmps. This endocytic transport pathway expands the extracellular roles of selective Bmp modulators to include intracellular regulation. This dosage-dependent molecular switch resolves discordances among studies that examine how Bmper regulates Bmp activity and has broad implica- tions for Bmp signal regulation by secreted mediators. 2005; Ikeya et al., 2006; Rentzsch et al., 2006; Serpe et al., 2008). Simple models of the biological role of Bmper do not re- solve the dissonance identified in these divergent systems. Recent data provide support for a model in which the ac- tivity of Cv-2 is biphasic with low levels of Cv-2 promoting and high levels inhibiting signaling during the formation of the crossveins in the Drosophila melanogaster wing. In this system, Cv-2 was found to selectively interact with Bmp ligands and receptors in a concentration-dependent manner (Serpe et al., 2008). The present study provides further evidence that Bmper behaves in a concentration-dependent manner. Bmper enhances Bmp4-mediated Smad activation at molar concentrations below that of Bmp4 in endothelial cells. Alternatively, when Bmper concentrations exceed those of Bmp4, they internalize inter- dependently into an endocytic shuttle to the lysosome for their dissolution. Interestingly, we found that Noggin and Gremlin, but not Chordin, can also trigger Bmp endocytosis, suggesting Correspondence to Cam Patterson: cpatters@med.unc.edu Abbreviations used in this paper: ES, embryonic stem; HCAEC, human coro- nary arterial endothelial cell; MEC, mouse endothelial cell; MEF, mouse embry- onic ﬁbroblast. © 2009 Kelley et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the ﬁrst six months after the publica- tion date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Cell Biol. Vol. 184 No. 4 597–609 www.jcb.org/cgi/doi/10.1083/jcb.200808064 JCB 597