FRET Reveals Novel Protein-Receptor Interaction of Bone Morphogenetic Proteins Receptors and Adaptor Protein 2 at the Cell Surface

Bone morphogenetic proteins (BMPs) are involved with a wide range of processes including apoptosis, differentiation, and proliferation. Several different pathways such as Smad, p38, and PI3/Akt are activated by BMPs. Signaling is transduced by BMP receptors (BMPRs) of type I and type II that are serine/threonine kinase receptors. BMPRs shuttle between membrane domains such as caveolae enriched with caveolin-1 β-isoform and caveolae of the caveolin-1 α/β-isoforms. It is hypothesized that there are other membrane domains to which the receptors localize. We used immunoprecipitation, Western blots, image cross-correlation spectroscopy, and fluorescence resonance energy transfer to investigate the interaction of BMPRs with proteins in clathrin-coated pits (CCPs). Our data indicate that these domains are associated with at least two of the BMPRs: BRIa and BRII. For the first time, to our knowledge, we showed what we believe are specific interactions between BRIa and BRII with a key component of CCPs, adaptor protein 2. Further, disruption of CCPs resulted in increased BRIa aggregation at the cell surface and activation of the BMP pathway even in the absence of BMP2. Therefore, CCPs seem to function as a negative regulatory membrane domain for BMP pathway activation.     

Caveolae regulate smad signaling as verified by novel imaging and system biology approaches

The contribution of caveolae in Bone Morphogenetic Protein 2 (BMP2) activated Smad signaling was quantified using a system biology approach. BMP2 plays crucial roles during processes such as hematopoiesis, embryogenesis, and skeletal development. BMP2 signaling is tightly regulated on the plasma membrane by its receptors. The localization of BMP receptors in caveolae and endocytosis through clathrin‐coated pits are thought to regulate the signaling; however the conclusions in the current literature are inconsistent. Therefore published literature was used to establish a mathematical model that was validated using confocal AFM (atomic force microscopy), confocal microscopy, and sucrose density centrifugation followed by Western blots, and reporter gene assays. The model and experiments confirmed that both caveolae and CCPs regulate the Smad‐dependent signaling pathway, however caveolae are centers at the plasma membrane where receptor–ligand interaction is crucial, Smad phosphorylation occurs, and a high degree of Smad signaling is regulated. This demonstrates a role for caveolae that needs to be considered and further studied. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Bone morphogenetic protein receptor type Ia localization causes increased BMP2 signaling in mice exhibiting increased peak bone mass phenotype

Bone morphogenetic protein 2 (BMP2) is a growth factor that initiates osteoblast differentiation. Recent studies show that BMP2 signaling regulates bone mineral density (BMD). BMP2 interacts with BMP receptor type Ia (BMPRIa) and type II receptor leading to the activation of the Smad signaling pathway. BMPRIa must shuttle between distinct plasma membrane domains, enriched of Caveolin‐1 alpha and Caveolin‐1 beta isoforms, and receptor activation occurs in these domains. Yet it remains unknown whether the molecular mechanism that regulates BMP2 signaling is driving mineralization and BMD. Therefore, the B6.C3H‐1‐12 congenic mouse model with increased BMD and osteoblast mineralization was utilized in this study. Using the family image correlation spectroscopy, we determined if BMP2 led to a significant re‐localization of BMPRIa to caveolae of the alpha/beta isoforms in bone marrow stromal cells (BMSCs) isolated from B6.C3H‐1‐12 mice compared to the C57BL/6J mice, which served as controls. The control, C57BL/6J mice, was selected due to only 4 Mb of chromosome 1 from the C3H/HeJ mouse was backcrossed to a C57BL/6J background. Using reporter gene assays, the B6.C3H‐1‐12 BMSCs responded to BMP2 with increased Smad activation. Furthermore, disrupting caveolae reduced the BMP2‐induced Smad signaling in BMSCs isolated from B6.C3H‐1‐12 and C57BL/6J. This study suggests for the first time a regulatory mechanism of BMPRIa signaling at the plasma membrane of BMSCs that (i) associated with genetic differences in the distal Chromosome 1 segment carried by the B6.C3H‐1‐12 congenic and (ii) contributes to increase BMD of the B6.C3H‐1‐12 compared to the C57BL/6J control mice. J. Cell. Physiol. 227: 2870–2879, 2012. © 2011 Wiley Periodicals, Inc.     

Inhibition of CK2 binding to BMPRIa induces C2C12 differentiation into osteoblasts and adipocytes

BMP2 is a growth factor that regulates the cell fate of mesenchymal stem cells into osteoblast and adipocytes. However, the detailed signaling pathways and mechanism are unknown. We previously reported a new interaction of Casein kinase II (CK2) with the BMP receptor type-Ia (BMPRIa) and demonstrated using mimetic peptides CK2.1, CK2.2 and CK2.3 that the release of CK2 from BMPRIa activates Smad signaling and osteogenesis. Previously, we showed that mutation of these CK2 sites on BMPRIa (MCK2.1 (476S-A), MCK2.2 (324S-A) and MCK2.3 (214S-A)) induced osteogenesis. However, one mutant MCK2.1 induced osteogenesis similar to overexpression of wild type BMPRIa, suggesting that the effect of this mutant on mineralization was due to overexpression. In this paper we investigated the signaling pathways involved in the CK2-BMPRIa mediated osteogenesis and identified a new signaling pathway activating adipogenesis dependent on the BMPRIa and CK2 association. Further the mechanism for adipogenesis and osteogenesis is specific to the CK2 interaction site on BMPRIa. In detail our data show that overexpression of MCK2.2 induced osteogenesis was dependent on Caveolin-1 (Cav1) and the activation of the Smad and mTor pathways, while overexpression of MCK2.3 induced osteogenesis was independent of Caveolin-1 without activation of Smad pathway. However, MCK2.3 induced osteogenesis via the MEK pathway. The adipogenesis induced by the overexpression of MCK2.2 in C2C12 cells was dependent on the p38 and ERK pathways as well as Caveolin-1. These data suggest that signaling through BMPRIa used two different signaling pathways to induce osteogenesis dependent on CK2. Additionally the data supports a signaling pathway initiated in caveolae and one outside of caveolae to induce mineralization. Moreover, they reveal the signaling pathway of BMPRIa mediated adipogenesis.     

Trapping of BMP receptors in distinct membrane domains inhibits their function in pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) are pleiotrophic growth factors that influence diverse processes such as skeletal development, hematopoiesis, and neurogenesis. They play crucial roles in diseases such as pulmonary arterial hypertension (PAH). In PAH, mutants of the BMP type II receptors (BMPR2) were detected, and their functions were impaired during BMP signaling. It is thought that expression levels of these receptors determine the fate of BMP signaling, with low levels of expression leading to decreased Smad activation in PAH. However, our studies demonstrate, for the first time, that the localization of receptors on the plasma membrane, in this case BMPR2, was misdirected. Three BMPR2 mutants, D485G, N519K, and R899X, which are known to be involved in PAH, were chosen as our model system. Our results show that all three BMPR2 mutants decreased BMP-dependent Smad phosphorylation and Smad signaling. Although the three mutants reached the cell membrane and their expression was lower than that of BMPR2, they formed smaller clusters and associated differently with membrane domains, such as caveolae and clathrin-coated pits. The disruption of these domains restored the Smad signaling of D485G and N519K to the level of wild-type BMPR2, showing that these mutants were trapped in the domains, rather than just expressed at a lower level on the surface. Therefore, new treatment options for PAH should also target receptor localization, rather than just expression level.     

Caveolin-1 regulates BMPRII localization and signaling in vascular smooth muscle cells

Recent studies demonstrate the interaction of BMPRII and caveolin-1 in various cell types. In this study we test the hypothesis that caveolin-1 interacts with and regulates BMPRII-dependent signaling in vascular smooth muscle cells. We demonstrate that BMPRII localizes to caveolae and directly interacts with caveolin-1 in mouse aortic smooth muscle cells. We demonstrate that this interaction is mediated by the caveolin-1 scaffolding domain and is regulated by caveolin-1 phosphorylation. Downregulation of caveolin-1 via siRNA resulted in a loss of BMP-dependent SMAD phosphorylation and gene regulation. Further studies revealed that loss of caveolin-1 results in decreased BMPRII membrane localization and decreased association of BMPRII with the type I BMP receptor BMPRIa. Dominant negative caveolin-1 decreased BMPRII membrane localization suggesting a role for caveolin-1 in BMPRII trafficking. Taken together, our findings establish caveolin-1 as an important regulator of downstream signaling and membrane targeting of BMPRII in vascular smooth muscle cells.     

Localization of the insulin receptor in caveolae of adipocyte plasma membrane.

The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with beta-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. beta-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.     

Endocytosis and trafficking of BMP receptors: Regulatory mechanisms for fine-tuning the signaling response in different cellular contexts

Signaling by bone morphogenetic protein (BMP) receptors is regulated at multiple levels in order to ensure proper interpretation of BMP stimuli in different cellular settings. As with other signaling receptors, regulation of the amount of exposed and signaling-competent BMP receptors at the plasma-membrane is predicted to be a key mechanism in governing their signaling output. Currently, the endocytosis of BMP receptors is thought to resemble that of the structurally related transforming growth factor-β (TGF-β) receptors, as BMP receptors are constitutively internalized (independently of ligand binding), with moderate kinetics, and mostly via clathrin-mediated endocytosis. Also similar to TGF-β receptors, BMP receptors are able to signal from the plasma membrane, while internalization to endosomes may have a signal modulating effect. When at the plasma membrane, BMP receptors localize to different membrane domains including cholesterol rich domains and caveolae, suggesting a complex interplay between membrane distribution and internalization. An additional layer of complexity stems from the putative regulatory influence on the signaling and trafficking of BMP receptors exerted by ligand traps and/or co-receptors. Furthermore, the trafficking and signaling of BMP receptors are subject to alterations in cellular context. For example, genetic diseases involving changes in the expression of auxiliary factors of endocytic pathways hamper retrograde BMP signals in neurons, and perturb the regulation of synapse formation. This review summarizes current understanding of the trafficking of BMP receptors and discusses the role of trafficking in regulation of BMP signals.     

Identification of receptors and signaling pathways for orphan bone morphogenetic protein/growth differentiation factor ligands based on genomic analyses

There are more than 30 human transforming growth factor beta/bone morphogenetic protein/growth differentiation factor (TGFbeta/BMP/GDF)-related ligands known to be important during embryonic development, organogenesis, bone formation, reproduction, and other physiological processes. Although select TGFbeta/BMP/GDF proteins were found to interact with type II and type I serine/threonine receptors to activate downstream Smad and other proteins, the receptors and signaling pathways for one-third of these TGFbeta/BMP/GDF paralogs are still unclear. Based on a genomic analysis of the entire repertoire of TGFbeta/BMP/GDF ligands and serine/threonine kinase receptors, we tested the ability of three orphan BMP/GDF ligands to activate a limited number of phylogenetically related receptors. We characterized the dimeric nature of recombinant GDF6 (also known as BMP13), GDF7 (also known as BMP12), and BMP10. We demonstrated their bioactivities based on the activation of Smad1/5/8-, but not Smad2/3-, responsive promoter constructs in the MC3T3 cell line. Furthermore, we showed their ability to induce the phosphorylation of Smad1, but not Smad2, in these cells. In COS7 cells transfected with the seven known type I receptors, overexpression of ALK3 or ALK6 conferred ligand signaling by GDF6, GDF7, and BMP10. In contrast, transfection of MC3T3 cells with ALK3 small hairpin RNA suppressed Smad signaling induced by all three ligands. Based on the coevolution of ligands and receptors, we also tested the role of BMPRII and ActRIIA as the type II receptor candidates for the three orphan ligands. We found that transfection of small hairpin RNA for BMPRII and ActRIIA in MC3T3 cells suppressed the signaling of GDF6, GDF7, and BMP10. Thus, the present approach provides a genomic paradigm for matching paralogous polypeptide ligands with a limited number of evolutionarily related receptors capable of activating specific downstream Smad proteins.     

Smad1 stabilization and delocalization in response to the blockade of BMP activity

Signaling at the plasma membrane receptors is generally terminated by some form of feedback regulation, such as endocytosis and/or degradation of the receptors. BMP-Smad1 signaling can also be attenuated by BMP-induced expression of the inhibitory Smads, which are negative regulators of Smad1 transactivation activity and/or BMP antagonists. Here, we report on a novel Smad1 regulation mechanism that occurs in response to the blockade of BMP activity. Lowering the serum levels or antagonizing BMPs with noggin led to upregulation of Smad1 at the protein level in several cell lines, but not to upregulation of Smad5, Smad8 or Smad2/3. The Smad1 upregulation occurs at the level of protein stabilization. Upregulated Smad1 was relocalized to the perinuclear region. These alterations seem to affect the dynamics and amplitude of BMP2-induced Smad1 reactivation. Our findings indicate that depleting or antagonizing BMPs leads to Smad1 stabilization and relocalization, thus revealing an unexpected regulatory mechanism for BMP-Smad1 signaling.     

Tob proteins enhance inhibitory Smad-receptor interactions to repress BMP signaling

Tob inhibits bone morphogenetic protein (BMP) signaling by interacting with receptor-regulated Smads in osteoblasts. Here we provide evidence that Tob also interacts with the inhibitory Smads 6 and 7. A yeast two-hybrid screen identified Smad6 as a protein interacting with Tob. Tob co-localizes with Smad6 at the plasma membrane and enhances the interaction between Smad6 and activated BMP type I receptors. Furthermore, we have isolated Xenopus Tob2, and show that it cooperates with Smad6 in inducing secondary axes when expressed in early Xenopus embryos. Finally, Tob and Tob2 cooperate with Smad6 to inhibit endogenous BMP signaling in Xenopus embryonic explants and in cultured mammalian cells. Our results provide both in vitro and in vivo evidence that Tob inhibits endogenous BMP signaling by facilitating inhibitory Smad functions.     

Dullard promotes degradation and dephosphorylation of BMP receptors and is required for neural induction

Bone morphogenetic proteins (BMPs) regulate multiple biological processes, including cellular proliferation, adhesion, differentiation, and early development. In Xenopus development, inhibition of the BMP pathway is essential for neural induction. Here, we report that dullard, a gene involved in neural development, functions as a negative regulator of BMP signaling. We show that Dullard promotes the ubiquitin-mediated proteosomal degradation of BMP receptors (BMPRs). Dullard preferentially complexes with the BMP type II receptor (BMPRII) and partially colocalizes with the caveolin-1-positive compartment, suggesting that Dullard promotes BMPR degradation via the lipid raft-caveolar pathway. Dullard also associates with BMP type I receptors and represses the BMP-dependent phosphorylation of the BMP type I receptor. The phosphatase activity of Dullard is essential for the degradation of BMP receptors and neural induction in Xenopus. Together, these observations suggest that Dullard is an essential inhibitor of BMP receptor activation during Xenopus neuralization.     

Casein Kinase 2 β-Subunit Is a Regulator of Bone Morphogenetic Protein 2 Signaling

Bone morphogenetic proteins (BMPs) play a crucial role during embryonic development and regulate processes as diverse as neurogenesis, skeletal formation, and hematopoesis. They signal through a hetero-oligomer complex of BMP receptors. Binding of the ligand to the receptors activates several pathways, including Smad and p38. BMP signaling is controlled in the extracellular space, the plasma membrane, and the intracellular space; however, the mechanism of receptor signaling at the plasma membrane and proteins that regulate this process still need to be identified. The experiments presented here identify the protein kinase casein kinase II (CK2) as a BMP receptor type Ia (BRIa) interacting protein. Fluorescence resonance energy transfer revealed that this interaction occurs at the plasma membrane. BMP2 stimulation of C2C12 cells leads to the release of CK2 from BRIa. Blocking this interaction with specific peptides that inhibit the binding sites for CK2 on BRIa demonstrated a redistribution of BRIa on the plasma membrane. Signaling was initiated once CK2 was released from BRIa, leading to the mineralization of C2C12 cells. These data suggest that CK2 is a negative regulator of BMP signaling and osteoblast differentiation.     

Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bone morphogenetic protein co-receptor

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-beta, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor.     

Selective inhibitory effects of Smad6 on bone morphogenetic protein type I receptors

The inhibitory Smads, Smad6 and Smad7, play pivotal roles in negative regulation of transforming growth factor-beta (TGF-beta) family signaling as feedback molecules as well as mediators of cross-talk with other signaling pathways. Whereas Smad7 acts as a ubiquitous inhibitor of Smad signaling, Smad6 has been shown to effectively inhibit bone morphogenetic protein (BMP) signaling but only weakly TGF-beta/activin signaling. In the present study, we have found that Smad6 inhibits signaling from the activin receptor-like kinase (ALK)-3/6 subgroup in preference to that from the ALK-1/2 subgroup of BMP type I receptors. The difference is attributable to the interaction of Smad6 with these BMP type I receptors. The amino acid residues responsible for Smad6 sensitivity of ALK-3 were identified as Arg-238, Phe-264, Thr-265, and Ala-269, which map to the N-terminal lobe of the ALK-3 kinase domain. Although Smad6 regulates BMP signaling through multiple mechanisms, our findings suggest that interaction with type I receptors is a critical step in the function of Smad6.