An efficient protein complex purification method for functional proteomics in higher eukaryotes

The ensemble of expressed proteins in a given cell is organized in multiprotein complexes. The identification of the individual components of these complexes is essential for their functional characterization. The introduction of the 'tandem affinity purification' (TAP) methodology substantially improved the purification and systematic genome-wide characterization of protein complexes in yeast. The use of this approach in higher eukaryotic cells has lagged behind its use in yeast because the tagged proteins are normally expressed in the presence of the untagged endogenous version, which may compete for incorporation into multiprotein complexes. Here we describe a strategy in which the TAP approach is combined with double-stranded RNA interference (RNAi) to avoid competition from corresponding endogenous proteins while isolating and characterizing protein complexes from higher eukaryotic cells. This strategy allows the determination of the functionality of the tagged protein and increases the specificity and the efficiency of the purification.     

Biotin tagging coupled with amino acid‐coded mass tagging for efficient and precise screening of interaction proteome in mammalian cells

In mammalian cells, when tandem affinity purification approach is employed, the existence of untagged endogenous target protein and repetitive washing steps together result in overall low yield of purified/stable complexes and the loss of weakly and transiently interacting partners of biological significance. To avoid the trade‐offs involving in methodological sensitivity, precision, and throughput, here we introduce an integrated method, biotin tagging coupled with amino acid‐coded mass tagging, for highly sensitive and accurate screening of mammalian protein–protein interactions. Without the need of establishing a stable cell line, using a short peptide tag which could be specifically biotinylated in vivo, the biotin‐tagged target/bait protein was then isolated along with its associates efficiently by streptavidin magnetic microbeads in a single step. In a pulled‐down complex amino acid‐coded mass tagging serves as “in‐spectra” quantitative markers to distinguish those bait‐specific interactors from non‐specific background proteins under stringent criteria. Applying this biotin tagging coupled with amino acid‐coded mass tagging approach, we first biotin‐tagged in vivo a multi‐functional protein family member, 14‐3‐3ε, which was expressed at close to endogenous level. Starting with approximately 20 millions of 293T cells which were significantly less than what needed for a tandem affinity purification run, 266 specific interactors of 14‐3‐3ε were identified in high confidence.     

A novel S3S‐TAP‐tag for the isolation of T‐cell interaction partners of adhesion and degranulation promoting adaptor protein

The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S‐tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C‐ or N‐terminally to the S3S‐tag. After retroviral transduction of a vector containing S3S‐tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP‐tagged protein close to endogenous levels. The combination of a cleavable S‐tag and a Strep‐tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture‐based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co‐purification of the known interaction partner Src kinase‐associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF‐H1 and the adaptor protein DOCK2 corroborates a link between ADAP‐mediated integrin regulation and the cytoskeleton.     

Quantitative profiling of in vivo-assembled RNA-protein complexes using a novel integrated proteomic approach

Identification of proteins in RNA-protein complexes is an important step toward understanding regulation of RNA-based processes. Because of the lack of appropriate methodologies, many studies have relied on the creation of in vitro assembled RNA-protein complexes using synthetic RNA and cell extracts. Such complexes may not represent authentic RNPs as they exist in living cells as synthetic RNA may not fold properly and nonspecific RNA-protein interactions can form during cell lysis and purification processes. To circumvent limitations in current approaches, we have developed a novel integrated strategy namely MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) to capture bona fide in vivo-assembled RNA-protein complexes. In this method, HB-tagged bacteriophage protein MS2 and stem-loop tagged target or control RNAs are co-expressed in cells. The tight association between MS2 and the RNA stem-loop tags allows efficient HB-tag based affinity purification of authentic RNA-protein complexes. Proteins associated with target RNAs are subsequently identified and quantified using SILAC-based quantitative mass spectrometry. Here the 1.2 kb internal ribosome entry site (IRES) from lymphoid enhancer factor-1 mRNA has been used as a proof-of-principle target RNA. An IRES target was chosen because of its importance in protein translation and our limited knowledge of proteins associated with IRES function. With a conventionally translated target RNA as control, 36 IRES binding proteins have been quantitatively identified including known IRES binding factors, novel interacting proteins, translation initiation factors (eIF4A-1, eIF-2A, and eIF3g), and ribosomal subunits with known noncanonical actions (RPS19, RPS7, and RPL26). Validation studies with the small molecule eIF4A-1 inhibitor Hippuristanol shows that translation of endogenous lymphoid enhancer factor-1 mRNA is especially sensitive to eIF4A-1 activity. Our work demonstrates that MS2 in vivo biotin tagged RNA affinity purification is an effective and versatile approach that is generally applicable for other RNA-protein complexes.     

Uncoupling of bait‐protein expression from the prey protein environment adds versatility for cell and tissue interaction proteomics and reveals a complex of CARP‐1 and the PKA Cβ1 subunit

An expression‐uncoupled tandem affinity purification assay is introduced which differs from the standard TAP assay by uncoupling the expression of the TAP‐bait protein from the target cells. Here, the TAP‐tagged bait protein is expressed in Escherichia coli and purified. The two concatenated purification steps of the classical TAP are performed after addition of the purified bait to brain tissue homogenates, cell and nuclear extracts. Without prior genetic manipulation of the target, upscaling, free choice of cell compartments and avoidance of expression triggered heat shock responses could be achieved in one go. By the strategy of separating bait expression from the prey protein environment numerous established, mostly tissue‐specific binding partners of the protein kinase A catalytic subunit Cβ1 were identified, including interactions in binary, ternary and quaternary complexes. In addition, the previously unknown small molecule inhibitor‐dependent interaction of Cβ1 with the cell cycle and apoptosis regulatory protein‐1 was verified. The uncoupled tandem affinity purification procedure presented here expands the application range of the in vivo TAP assay and may serve as a simple strategy for identifying cell‐ and tissue‐specific protein complexes.     

Homo-oligomeric complexes of the yeast alpha-factor pheromone receptor are functional units of endocytosis

alpha-Factor receptors from Saccharomyces cerevisiae are G-protein-coupled receptors containing seven transmembrane segments. Receptors solubilized with the detergent n-dodecyl beta-D-maltoside were found to sediment as a single 8S species in glycerol density gradients. When the membranes from cells coexpressing two differentially tagged receptors were solubilized with detergent and subjected to immunoprecipitation, we found that the antibodies specific for either epitope tag resulted in precipitation of both tagged species. Coprecipitation was not a consequence of incomplete detergent extraction because the abundant plasma membrane protein Pma1 did not coprecipitate with the receptors. Moreover, the receptor complexes were present prior to detergent extraction because coimmunoprecipitation was not observed when cells expressing the single tagged species were mixed prior to membrane preparation. Treatment of cultures with alpha-factor had little effect on the extent of oligomerization as judged by the sedimentation behavior of the receptor complexes and by the efficiency of coimmunoprecipitation. The ability of receptor complexes to undergo ligand-mediated endocytosis was evaluated by using membrane fractionation and fluorescence microscopy. Mutant receptors that fail to bind alpha-factor (Ste2-S184R) or lack the endocytosis signal (Ste2-T326) became competent for ligand-mediated endocytosis when they were expressed in cells containing wild-type receptors. Coimmunoprecipitation experiments indicated that the C-terminal cytoplasmic domain and intermolecular disulfide bonds were unnecessary for oligomer formation. We conclude that alpha-factor receptors form homo-oligomers and that these complexes are subject to ligand-mediated endocytosis. Furthermore, we show for the first time that unoccupied receptors participate in these endocytosis-competent complexes.     

Rapid purification of protein complexes from mammalian cells

The evaluation of the protein binding partner(s) of biologically important proteins is currently an area of intense research, especially since the development of the yeast two-hybrid assay. However, not all protein–protein interactions uncovered by this assay are biologically relevant and another confirmatory assay must be performed. Ideally, this assay should be rapid, versatile and performed under conditions which mimic the ‘normal’ physiological state as closely as possible. Towards this goal, we have constructed two eukaryotic expression vectors that facilitate the purification of a protein of interest, along with any associated proteins, from mammalian cells. These vectors incorporate the following features: (i) a tetracycline-responsive promoter so that the level of protein production can be regulated; (ii) an N-terminal glutathione S-transferase tag or a triple repeat of the HA1 epitope, to facilitate purification of the protein either by glutathione affinity chromatography or immunoprecipitation, respectively, followed by a multiple cloning site; (iii) the gene for the enhanced green fluorescent protein (for detection of the presence of the fusion protein and subcellular localization); (iv) a puromycin marker for the selection of stable transformants; (v) a truncated EBNA protein and oriP sequence for episomal replication of the vector. These latter two features permit expansion of small cultures of transfected cells under puromycin selection, thereby increasing the amount of tagged protein that can be purified. We show that these vectors can be used to direct the doxycycline-inducible expresssion of tagged proteins and to recover tagged CIP1–p21 protein complexes from HeLa cells. Furthermore, we show that these tagged p21-purified complexes contain both cyclin A and Cdk2, which are known to interact with p21, but not β-actin.     

Let's think again about using mammalian temperature‐sensitive mutants to investigate functional molecules—The perspectives from the studies on three mutants showing chromosome instability

This review evaluates the use of temperature‐sensitive (ts) mutants to investigate functional molecules in mammalian cells. A series of studies were performed in which mammalian cells expressing functional molecules were isolated from ts mutants using complementation by the introduction and expression of the responsible protein tagged with the green fluorescent protein. The results showed that chromosome instability and cell‐cycle arrest were caused by ts defects in the following three molecules: the largest subunit of RNA polymerase II, a protein involved in splicing, and ubiquitin‐activating enzyme. The cells expressing functional protein were then isolated by introducing the responsible gene tagged with the green fluorescent protein to complement the ts phenotype. These cells proved to be useful in analyzing the dynamics of RNA polymerase II in living cells. Analyses of the functional interaction between proteins involved in splicing were also useful in the investigation of ts mutants and their derivatives. In addition, these cells demonstrated the functional localization of ubiquitin‐activating enzyme in the nucleus. Mammalian ts mutants continue to show great potential to aid in understanding the functions of the essential molecules in cells. Therefore, it is highly important that studies on the identification and characterization of the genes responsible for the phenotype of a mutant are carried out.     

Isotope-differentiated binding energy shift tags (IDBEST™) for improved targeted biomarker discovery and validation

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST? (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.     

Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.     

Multinuclear magnetic resonance, electrospray ionization-mass spectroscopy, and parametric method 5 studies of a new derivative of gossypol with 2-thiophenecarbohydrazide as well as its complexes with LI+, Na+, K+, RB+, and Cs+ cations

A new derivative of racemic gossypol with 2-thiophenecarbohydrazide (GHHT) and its complexes with monovalent cations have been synthesized and studied by electrospray ionization-mass spectroscopy (ESI-MS), multinuclear nuclear magnetic resonance (NMR), as well as by the Parametric Method 5 (PM5) methods. It is demonstrated that GHHT forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by three types of intramolecular hydrogen bonds. The spectroscopic methods have provided clear evidence that GHHT and its complexes exist in the DMSO-d6 solution in the N-imine-N-imine tautomeric forms. The structures of the GHHT and its complexes with Li+, Na+, K+, Rb+, and Cs+ cations are visualized and discussed in detail.     

丛枝菌根真菌状巨孢囊霉孢子伴生细菌的绿色荧光蛋白标记及定殖分析

摘要：【目的】分析丛枝菌根（Arbuscualr Mycorrhizal, AM）真菌珠状巨孢囊霉(Gigaspora margarita) MAFF 520054孢子伴生细菌的定殖情况,明确其生态位点,以及为进一步分析其种群生态或功能提供信息。【方法】以载体pNF8（gfp-mut1）对6株珠状巨孢囊霉MAFF 520054 孢子伴生细菌进行绿色荧光蛋白（GFP）标记,并通过荧光显微镜和平板计数的方法研究标记菌株对真菌宿主的定殖位点和不同条件下的定殖数量动态。【结果】对粘状芽孢杆菌（Peanibacillus spp.）M060106-1和M061122-6、芽孢杆菌（Bacillus sp.）M061122-10和短小芽孢杆菌（Brevibacillus sp.）M061122-12成功进行了GFP标记,其均具有较好的质粒稳定性,且与出发株的基本性状一致,适合短期内进行环境定殖研究。所有菌株均能定殖珠状巨孢囊霉MAFF 520054孢子壁,而M061122-6和M061122-12还能够定殖其菌丝;不同pH值条件下,各菌株定殖珠状巨孢囊霉MAFF 520054孢子的数量动态均为先上升后下降,pH值对各菌株的定殖数量有不同的影响;各GFP菌株对低活力的珠状巨孢囊霉孢子定殖数量高于高活力的孢子,且对高活力孢子的定殖数量动态不同。【结论】分离的珠状巨孢囊霉孢子伴生细菌能够重新定殖其孢子,菌株的定殖能力受其特性及外界因子的影响,为进一步分析AM真菌伴生细菌的种群生态及功能提供了信息。     

T cell receptor ligation induces the formation of dynamically regulated signaling assemblies

Tcell antigen receptor (TCR) ligation initiates tyrosine kinase activation, signaling complex assembly, and immune synapse formation. Here, we studied the kinetics and mechanics of signaling complex formation in live Jurkat leukemic T cells using signaling proteins fluorescently tagged with variants of enhanced GFP (EGFP). Within seconds of contacting coverslips coated with stimulatory antibodies, T cells developed small, dynamically regulated clusters which were enriched in the TCR, phosphotyrosine, ZAP-70, LAT, Grb2, Gads, and SLP-76, excluded the lipid raft marker enhanced yellow fluorescent protein-GPI, and were competent to induce calcium elevations. LAT, Grb2, and Gads were transiently associated with the TCR. Although ZAP-70-containing clusters persisted for more than 20 min, photobleaching studies revealed that ZAP-70 continuously dissociated from and returned to these complexes. Strikingly, SLP-76 translocated to a perinuclear structure after clustering with the TCR. Our results emphasize the dynamically changing composition of signaling complexes and indicate that these complexes can form within seconds of TCR engagement, in the absence of either lipid raft aggregation or the formation of a central TCR-rich cluster.     

Surveying polypeptide and protein domain conformation and association with FlAsH and ReAsH

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT2 and ReAsH-EDT2. This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.     

Identification of novel Nicotinamide Phosphoribosyltransferase (NAMPT) inhibitors using computational approaches

Nicotinamide Phosphoribosyltransferase (NAMPT) is a rate-limiting enzyme in the biosynthesis of NAD. Cancer cells have elevated poly [ADP-Ribose] polymerase 1 (PARP) activity as well as the immense necessity of ATP: thereby consuming NAD at a higher rate than normal tissues. The perturbation of these intracellular processes is more sensitive and highly dependent on NAMPT to maintain the required NAD levels. Functional inhibition of NAMPT is, therefore, a promising drug target in therapeutic oncology. In this study, the importance of intermolecular contacts was realized based on contact occupancy and favorable energetic from molecular dynamic simulation to discern non-critical contacts of four different classes of potential NAMPT inhibitor bound complexes. Further, pharmacophore modeling, molecular docking, a quantum mechanical properties and MD simulation, as well as active site residual network communication were employed to identify potential leads. Present studies identified two leads, 2 and 3 which have better binding free energy compared to known inhibitors and showed stable hydrogen bonding and hydrophobic contacts with β barrel cavity lining residues in the active site of the dimer interface (A′B). Lead 2 containing fluorene as central core and lead 3 having phenyl-benzamide as a core showed stable moiety which was observed from electronic property analysis. Active site residual communication in identified leads bound complex also showed similarity to known inhibitor complexes. Compounds containing these moieties were not reported until now against NAMPT inhibition and can be considered as novel cores for future development of drugs to inhibit NAMPT function.     

A survey of cellulose microfibril patterns in dividing,expanding, and differentiating cells of Arabidopsis thaliana

Cellulose microfibrils are critical for plant cell specialization and function. Recent advances in live cell imaging of fluorescently tagged cellulose synthases to track cellulose synthesis have greatly advanced our understanding of cellulose biosynthesis. Nevertheless, cellulose deposition patterns remain poorly described in many cell types, including those in the process of division or differentiation. In this study, we used field emission scanning electron microscopy analysis of cryo-planed tissues to determine the arrangement of cellulose microfibrils in various faces of cells undergoing cytokinesis or specialized development, including cell types in which cellulose cannot be imaged by conventional approaches. In dividing cells, we detected microfibrillar meshworks in the cell plates, consistent with the concentration at the cell plate of cellulose synthase complexes, as detected by fluorescently tagged CesA6. We also observed a loss of parallel cellulose microfibril orientation in walls of the mother cell during cytokinesis, which corresponded with the loss of fluorescently tagged cellulose synthase complexes from these surfaces. In recently formed guard cells, microfibrils were randomly organized and only formed a highly ordered circumferential pattern after pore formation. In pit fields, cellulose microfibrils were arranged in circular patterns around plasmodesmata. Microfibrils were random in most cotyledon cells except the epidermis and were parallel to the growth axis in trichomes. Deposition of cellulose microfibrils was spatially delineated in metaxylem and protoxylem cells of the inflorescence stem, supporting recent studies on microtubule exclusion mechanisms.     

CellFIE: CRISPR- and Cell Fusion-based Two-hybrid Interaction Mapping of Endogenous Proteins

Numerous genetic methods facilitate the detection of binary protein–protein interactions (PPIs) by exogenous overexpression, which can lead to false results. Here, we describe CellFIE, a CRISPR- and cell fusion-based PPI detection method, which enables the mapping of interactions between endogenously tagged two-hybrid proteins. We demonstrate the specificity and reproducibility of CellFIE in a matrix mapping approach, validating the interactions of VCP with ASPL and UBXD1, and the self-interaction of TDP-43 under endogenous conditions. Furthermore, we show that CellFIE can be used to quantify changes of endogenous PPIs upon stress induction or drug treatment. For the first time, CellFIE facilitates systematic mapping of interactions between endogenously tagged proteins and represents a novel tool to characterize PPIs in live cells under dynamic conditions.     

Formation of stable homomeric and transient heteromeric bone morphogenetic protein (BMP) receptor complexes regulates Smad protein signaling

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. (a) All homomeric complexes (without or with BMP-2) were stable on the patch/FRAP time scale (minutes), whereas the heterocomplexes were transient, a difference that may affect signaling. (b) Patch/FRAP between HA- and myc-tagged BMPRII combined with competition by untagged BMPRIb showed that the heterocomplexes form at the expense of homodimers. (c) Stabilization of BMPRII·BMPRIb heterocomplexes (but not homomeric complexes) by IgG binding to same-tag receptors elevated phospho-Smad formation both without and with BMP-2. These findings suggest two mechanisms that may suppress the tendency of preformed BMP receptor hetero-oligomers to signal without ligand: (a) competition between homo- and heterocomplex formation, which reduces the steady-state level of the latter, and (b) the transient nature of the heterocomplexes, which limits the time during which BMPRI can be phosphorylated by BMPRII in the heterocomplex.