The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. Bone morphogenetic proteins (BMPs) 2 are members of the transforming growth factor-␤ (TGF-␤) cytokine superfamily (1-3). They play critical roles in numerous biological processes, including development, differentiation, and tissue regeneration in embryonic and mature tissues (4 -8), and have been implicated in disorders such as primary pulmonary hypertension (9, 10), brachydactyly-type dysostosis (11-13), juvenile polyposis syndrome (14 -16), and cancer (17). BMPs signal via two receptor Ser/Thr kinases, type I (BMPRIa and -Ib) and type II (BMPRII; as well as the type II activin receptors ActRII and ActRIIb) (5, 18 -20). Unlike the related TGF-␤ receptors where type I (T␤RI) fails to bind ligand and type II (T␤RII) is the high affinity receptor, BMPRII binds BMP-2 only weakly, whereas BMPRI binds the ligand with high affinity (20 -24).Using immunofluorescence co-patching and co-immunoprecipitation studies, we have demonstrated that even prior to ligand binding the BMP receptors exist at the cell surface as a mixed population largely comprising monomers but also containing homodimers and heteromeric complexes (the latter are termed preformed complexes (PFCs)) (18,20,25,26). Following ligand binding to PFCs (25), BMPRII phosphorylates BMPRI, which proceeds to phosphorylate Smad1/5/8; they then bind to Smad4 and translocate to the nucleus where they regulate the expression of specific target genes (2, 27). BMPs can also signal via non-Smad pathways, which appear to be initiated mainly by ligand-induced BMP receptor heterocomplexes, reflecting a different oligomerization mode (5,19,25,26,28,29).The crystal structures of BMP-2 in complex with the ectodomain (ECD) of BMPRIa (23) and of BMP-7 with the ECD of the ActRII (30, 31) show a dimeric ligand in complex with two receptors (homodimers). Recent studies (24,32) reported the structures of the ternary heteromeric complexes of BMP-2 with the ECDs of BMPRIa and ActRII or ActRIIb. Interestingly, these heterocomplexes differ from the ternary complex of TGF-␤3 with the ECDs of T␤RI and T␤RII (33) because only the TGF-␤ receptor heterocomplex displays a direct contact between T␤RI and T␤RII that contributes to the interactions (20).Although structural studies have shown that th...

Integration of multiple signals into the canonical BMP/Smad pathway poses a big challenge during the course of embryogenesis and tissue homeostasis. Here, we show that cyclic guanosine 3',5'-monophosphate (cGMP)-dependent kinase I (cGKI) modulates BMP receptors and Smads, providing a novel mechanism enhancing BMP signalling. cGKI, a key mediator of vasodilation and hypertension diseases, interacts with and phosphorylates the BMP type II receptor (BMPRII). In response to BMP-2, cGKI then dissociates from the receptors, associates with activated Smads, and undergoes nuclear translocation. In the nucleus, cGKI binds with Smad1 and the general transcription factor TFII-I to promoters of BMP target genes such as Id1 to enhance transcriptional activation. Accordingly, cGKI has a dual function in BMP signalling: (1) it modulates BMP receptor/Smad activity at the plasma membrane and (2) after redistribution to the nucleus, it further regulates transcription as a nuclear co-factor for Smads. Consequently, cellular defects caused by mutations in BMPRII, found in pulmonary arterial hypertension patients, were compensated through cGKI, supporting the positive action of cGKI on BMP-induced Smad signalling downstream of the receptors.

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BackgroundBone morphogenetic proteins (BMPs) are involved in a plethora of cellular processes in embryonic development and adult tissue homeostasis. Signaling specificity is achieved by dynamic processes involving BMP receptor oligomerization and endocytosis. This allows for spatiotemporal control of Smad dependent and non-Smad pathways. In this study, we investigate the spatiotemporal regulation within the BMP-induced Smad transcriptional pathway.Methodology/Principal FindingsHere we discriminate between Smad signaling events that are dynamin-dependent (i.e., require an intact endocytic pathway) and dynamin-independent. Inhibition of dynamin-dependent endocytosis in fluorescence microscopy and fractionation studies revealed a delay in Smad1/5/8 phosphorylation and nuclear translocation after BMP-2 stimulation of C2C12 cells. Using whole genome microarray and qPCR analysis, we identified two classes of BMP-2 induced genes that are differentially affected by inhibition of endocytosis. Thus, BMP-2 induced gene expression of Id1, Id3, Dlx2 and Hey1 is endocytosis-dependent, whereas BMP-2 induced expression of Id2, Dlx3, Zbtb2 and Krt16 is endocytosis-independent. Furthermore, we demonstrate that short term inhibition of endocytosis interferes with osteoblast differentiation as measured by alkaline phosphatase (ALP) production and qPCR analysis of osteoblast marker gene expression.Conclusions/SignificanceOur study demonstrates that dynamin-dependent endocytosis is crucial for the concise spatial activation of the BMP-2 induced signaling cascade. Inhibition of endocytic processes during BMP-2 stimulation leads to altered Smad1/5/8 signaling kinetics and results in differential target gene expression. We show that interfering with the BMP-2 induced transcriptional network by endocytosis inhibition results in an attenuation of osteoblast differentiation. This implies that selective sensitivity of gene expression to endocytosis provides an additional mechanism for the cell to respond to BMP in a context specific manner. Moreover, we suggest a novel Smad dependent signal cascade induced by BMP-2, which does not require endocytosis.

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