Dimeric ligands of the transforming growth factor-beta (TGF-beta) superfamily signal across cell membranes in a distinctive manner by assembling heterotetrameric complexes of structurally related serine/threonine-kinase receptor pairs. Unlike complexes of the bone morphogenetic protein (BMP) branch that apparently form due to avidity from membrane localization, TGF-beta complexes assemble cooperatively through recruitment of the low-affinity (type I) receptor by the ligand-bound high-affinity (type II) pair. Here we report the crystal structure of TGF-beta3 in complex with the extracellular domains of both pairs of receptors, revealing that the type I docks and becomes tethered via unique extensions at a composite ligand-type II interface. Disrupting the receptor-receptor interactions conferred by these extensions abolishes assembly of the signaling complex and signal transduction (Smad activation). Although structurally similar, BMP and TGF-beta receptors bind in dramatically different modes, mediating graded and switch-like assembly mechanisms that may have coevolved with branch-specific groups of cytoplasmic effectors.

Transforming growth factor-beta (TGF-beta) is the prototype of a large family of structurally related cytokines that play key roles in maintaining cellular homeostasis by signaling through two classes of functionally distinct Ser/Thr kinase receptors, designated as type I and type II. TGF-beta initiates receptor assembly by binding with high affinity to the type II receptor. Here, we present the 2.15 A crystal structure of the extracellular ligand-binding domain of the human TGF-beta type II receptor (ecTbetaR2) in complex with human TGF-beta3. ecTbetaR2 interacts with homodimeric TGF-beta3 by binding identical finger segments at opposite ends of the growth factor. Relative to the canonical 'closed' conformation previously observed in ligand structures across the superfamily, ecTbetaR2-bound TGF-beta3 shows an altered arrangement of its monomeric subunits, designated the 'open' conformation. The mode of TGF-beta3 binding shown by ecTbetaR2 is compatible with both ligand conformations. This, in addition to the predicted mode for TGF-beta binding to the type I receptor ectodomain (ecTbetaR1), suggests an assembly mechanism in which ecTbetaR1 and ecTbetaR2 bind at adjacent positions on the ligand surface and directly contact each other via protein--protein interactions.

Helminth parasites defy immune exclusion through sophisticated evasion mechanisms, including activation of host immunosuppressive regulatory T (Treg) cells. The mouse parasite Heligmosomoides polygyrus can expand the host Treg population by secreting products that activate TGF-β signalling, but the identity of the active molecule is unknown. Here we identify an H. polygyrus TGF-β mimic (Hp-TGM) that replicates the biological and functional properties of TGF-β, including binding to mammalian TGF-β receptors and inducing mouse and human Foxp3+ Treg cells. Hp-TGM has no homology with mammalian TGF-β or other members of the TGF-β family, but is a member of the complement control protein superfamily. Thus, our data indicate that through convergent evolution, the parasite has acquired a protein with cytokine-like function that is able to exploit an endogenous pathway of immunoregulation in the host.

Crystallographic analysis of macromolecules depends on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals can provide a simple path for high-resolution structure determination by serial femtosecond crystallography or the cryoEM method MicroED.

TGFβ family ligands, which include the TGFβs, BMPs, and activins, signal by forming a ternary complex with type I and type II receptors. For TGFβs and BMPs, structures of ternary complexes have revealed differences in receptor assembly. However, structural information for how activins assemble a ternary receptor complex is lacking. We report the structure of an activin class member, GDF11, in complex with the type II receptor ActRIIB and the type I receptor Alk5. The structure reveals that receptor positioning is similar to the BMP class, with no interreceptor contacts; however, the type I receptor interactions are shifted toward the ligand fingertips and away from the dimer interface. Mutational analysis shows that ligand type I specificity is derived from differences in the fingertips of the ligands that interact with an extended loop specific to Alk4 and Alk5. The study also reveals differences for how TGFβ and GDF11 bind to the same type I receptor, Alk5. For GDF11, additional contacts at the fingertip region substitute for the interreceptor interactions that are seen for TGFβ, indicating that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFβ signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFβ family, different mechanisms regulate type I receptor binding and specificity, providing a molecular explanation for how the activin class accommodates low-affinity type I interactions without the requirement of cooperative receptor interactions.

To study the role of the GTPase dynamin in GLUT4 intracellular recycling, we have overexpressed dynamin؊1 wild type and a GTPase-negative mutant (K44A) in primary rat adipose cells. Transfection was accomplished by electroporation using an hemagglutinin (HA)-tagged GLUT4 as a reporter protein. In cells expressing HA-GLUT4 alone, insulin results in an Ϸ7-fold increase in cell surface anti-HA antibody binding. Studies with wortmannin indicate that the kinetics of HA-GLUT4-trafficking parallel those of the native GLUT4 and in addition, that newly synthesized HA-GLUT4 goes to the plasma membrane before being sorted into the insulin-responsive compartments. Short term (4 h) coexpression of dynamin-K44A and HA-GLUT4 increases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. Under conditions of maximal expression of dynamin-K44A (24 h), most or all of the intracellular HA-GLUT4 appears to be present on the cell surface in the basal state, and insulin has no further effect. Measurements of the kinetics of HA-GLUT4 endocytosis show that dynamin-K44A blocks internalization of the glucose transporters. In contrast, expression of dynamin wild type decreases the amount of cell surface HA-GLUT4 in both the basal and insulin-stimulated states. These data demonstrate that the endocytosis of GLUT4 is largely mediated by processes which require dynamin.In adipose cells, GLUT4 glucose transporters are constantly recycling between an intracellular compartment and the plasma membrane (1-4). In the basal state, where the rate of exocytosis is relatively low, the vast majority of the GLUT4 glucose transporters reside in an as yet poorly characterized intracellular compartment (1,3,5). Stimulation of adipose cells with insulin leads to an increase in the rate of exocytosis of GLUT4-containing vesicles, resulting in a rapid shift in the steady state distribution of GLUT4 to the plasma membrane (1-3). After clearance of the hormone, the rate of GLUT4 exocytosis decreases and the steady state distribution shifts back to the intracellular compartment.The primary focus of recent investigations has been the identification and characterization of signaling molecules (e.g. p85/ p110 phosphatidylinositol 3-kinase) and other cellular components (e.g. soluble NSF attachment protein receptors (SNAREs)) possibly involved in the regulated exocytosis of GLUT4 (6 -11). However, little is known about the mechanism of GLUT4 endocytosis. Previous reports provided indirect evidence that GLUT4 might be internalized by a mechanism involving clathrin-mediated endocytosis. Potassium depletion, known to disrupt formation of clathrin-coated vesicles (12), results in a decreased internalization of GLUT4 and mannose-6-phosphate receptors in rat adipose cells (13). In 3T3-L1 adipocytes, GLUT4 has been shown to co-purify with clathrin-coated vesicles derived from the plasma membrane after treatment of the cells with the fungal toxin brefeldin A (14). Previous morphological analysis showed association of GLUT4 with clathrin-coated...

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The TGF-β isoforms, TGF-β1, -β2, and -β3, share greater than 70% sequence identity and are almost structurally identical. TGF-β2 differs from the others, however, in that it binds the TGF-β type II receptor (TβR-II) with much lower affinity than either TGF-β1 or -β3. It has been previously shown that three conserved interfacial residues, Arg25, Val92, Arg94, in TGF-β1 and -β3 are responsible for their high-affinity interaction with TβR-II. In this study, the role of each of these residues was examined by creating single, double, and triple substitutions resulting in both TGF-β3 loss-offunction and TGF-β2 gain-of-function variants. One-dimensional 1 H NMR spectra of the variants confirmed a lack of large structural perturbations. Affinities, kinetics, and thermodynamics for TβR-II binding were determined by surface plasmon resonance biosensor analysis. Double substitutions revealed that nearly all of the high-affinity binding is contributed by Arg25 and Arg94. Single site substitutions showed that Arg94 makes the greatest contribution. Substitution of Arg25 and Arg94 with alanine verified the requirement of the arginine guanidinium functional groups for the highly specific hydrogen-bonded ion pairs formed between Arg25 and Arg94 of TGF-β1 and -β3, and Glu119 and Asp32 of TβR-II. Further kinetic and thermodynamic analyses confirmed that Arg25 and Arg94 are primarily responsible for high-affinity binding and also revealed that noninterfacial longer range effects emanating from the TGF-β structural framework contribute slightly to TβR-II binding. Growth inhibition assays showed that binding changes generally correlate directly with changes in function; however, a role Val92 in this cellular context was uncovered.Transforming growth factor β (TGF-β) 1 isoforms are 25 kDa homodimeric polypeptide signaling ligands that control multiple cellular processes including proliferation, extracellular matrix deposition, and epithelial-mesenchymal transition (1). TGF-βs and other structurally related members of the TGF-β superfamily, such as activins, bone morphogenic proteins (BMPs), and growth and differentiation factors (GDFs), exert their biological effects by binding and bringing together two pairs of structurally similar, single-pass trans-membrane receptors, known as the type I and type II receptors, TβR-I and TβR-II (2). The ligand-mediated † This work was supported by the Genomics and Health Initiative, National Research Council, Canada, the National Institutes of Health (GM58670), and the Robert A. Welch Foundation (AQ1431 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; EDTA, ethylenediaminetetraacetic acid; FBS, fetal bovine serum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; TβR-I, transforming growth factor β type I receptor; TβR-II, transforming growth factor β type II receptor; TGF-βII, transforming growth factor β; TGF-β2-TM, TGF-β2-K25R/I92V/K94R variant. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 January 5. assembly of these tw...