Characterization of follistatin-type domains and their contribution to myostatin and activin a antagonism

Follistatin (FST)-type proteins are important antagonists of some members of the large TGF-β family of cytokines. These include myostatin, an important negative regulator of muscle growth, and the closely related activin A, which is involved in many physiological functions, including maintenance of a normal reproductive axis. FST-type proteins, including FST and FST-like 3 (FSTL3), differentially inhibit various TGF-β family ligands by binding each ligand with two FST-type molecules. In this study, we sought to examine features that are important for ligand antagonism by FST-type proteins. Previous work has shown that a modified construct consisting of the FST N-terminal domain (ND) followed by two repeating follistatin domains (FSD), herein called FST ND-FSD1-FSD1, exhibits strong specificity for myostatin over activin A. Using cell-based assays, we show that FST ND-FSD1-FSD1 is unique in its specificity for myostatin as compared with similar constructs containing domains from FSTL3 and that the ND is critical to its activity. Furthermore, we demonstrate that FSD3 of FST provides affinity to ligand inhibition and confers resistance to perturbations in the ND and FSD2, likely through the interaction of FSD3 of one FST molecule with the ND of the other FST molecule. Additionally, our data suggest that this contact provides cooperativity to ligand antagonism. Cross-linking studies show that this interaction also potentiates formation of 1:2 ligand-FST complexes, whereas lack of FSD3 allows formation of 1:1 complexes. Altogether, these studies support that domain differences generate FST-type molecules that are each uniquely suited ligand antagonists.     

The structure of the follistatin:activin complex reveals antagonism of both type I and type II receptor binding

TGF-beta ligands stimulate diverse cellular differentiation and growth responses by signaling through type I and II receptors. Ligand antagonists, such as follistatin, block signaling and are essential regulators of physiological responses. Here we report the structure of activin A, a TGF-beta ligand, bound to the high-affinity antagonist follistatin. Two follistatin molecules encircle activin, neutralizing the ligand by burying one-third of its residues and its receptor binding sites. Previous studies have suggested that type I receptor binding would not be blocked by follistatin, but the crystal structure reveals that the follistatin N-terminal domain has an unexpected fold that mimics a universal type I receptor motif and occupies this receptor binding site. The formation of follistatin:BMP:type I receptor complexes can be explained by the stoichiometric and geometric arrangement of the activin:follistatin complex. The mode of ligand binding by follistatin has important implications for its ability to neutralize homo- and heterodimeric ligands of this growth factor family.     

Heparin-mediated dimerization of follistatin

Heparin and heparan sulfate (HS) are highly sulfated polysaccharides covalently bound to cell surface proteins, which directly interact with many extracellular proteins, including the transforming growth factor-β (TGFβ) family ligand antagonist, follistatin 288 (FS288). Follistatin neutralizes the TGFβ ligands, myostatin and activin A, by forming a nearly irreversible non-signaling complex by surrounding the ligand and preventing interaction with TGFβ receptors. The FS288-ligand complex has higher affinity than unbound FS288 for heparin/HS, which accelerates ligand internalization and lysosomal degradation; however, limited information is available for how FS288 interactions with heparin affect ligand binding. Using surface plasmon resonance (SPR) we show that preincubation of FS288 with heparin/HS significantly decreased the association kinetics for both myostatin and activin A with seemingly no effect on the dissociation rate. This observation is dependent on the heparin/HS chain length where small chain lengths less than degree of polymerization 10 (dp10) did not alter association rates but chain lengths >dp10 decreased association rates. In an attempt to understand the mechanism for this observation, we uncovered that heparin induced dimerization of follistatin. Consistent with our SPR results, we found that dimerization only occurs with heparin molecules >dp10. Small-angle X-ray scattering of the FS288 heparin complex supports that FS288 adopts a dimeric configuration that is similar to the FS288 dimer in the ligand-bound state. These results indicate that heparin mediates dimerization of FS288 in a chain-length-dependent manner that reduces the ligand association rate, but not the dissociation rate or antagonistic activity of FS288.     

Ligand affinity for amino-terminal and juxtamembrane domains of the corticotropin releasing factor type I receptor: regulation by G-protein and nonpeptide antagonists

Peptide ligands bind the CRF(1) receptor by a two-domain mechanism: the ligand's carboxyl-terminal portion binds the receptor's extracellular N-terminal domain (N-domain) and the ligand's amino-terminal portion binds the receptor's juxtamembrane domain (J-domain). Little quantitative information is available regarding this mechanism. Specifically, the microaffinity of the two interactions and their contribution to overall ligand affinity are largely undetermined. Here we measured ligand interaction with N- and J-domains expressed independently, the former (residues 1-118) fused to the activin IIB receptor's membrane-spanning alpha-helix (CRF(1)-N) and the latter comprising residues 110-415 (CRF(1)-J). We also investigated the effect of nonpeptide antagonist and G-protein on ligand affinity for N- and J-domains. Peptide agonist affinity for CRF(1)-N was only 1.1-3.5-fold lower than affinity for the whole receptor (CRF(1)-R), suggesting the N-domain predominantly contributes to peptide agonist affinity. Agonist interaction with CRF(1)-J (potency for stimulating cAMP accumulation) was 12000-1500000-fold weaker than with CRF(1)-R, indicating very weak direct agonist interaction with the J-domain. Nonpeptide antagonist affinity for CRF(1)-J and CRF(1)-R was indistinguishable, indicating the compounds bind predominantly the J-domain. Agonist activation of CRF(1)-J was fully blocked by nonpeptide antagonist, suggesting antagonism results from inhibition of agonist-J-domain interaction. G-protein coupling with CRF(1)-R (forming RG) increased peptide agonist affinity 92-1300-fold, likely resulting from enhanced agonist interaction with the J-domain rather than the N-domain. Nonpeptide antagonists, which bind the J-domain, blocked peptide agonist binding to RG, and binding of peptide antagonists, predominantly to the N-domain, was unaffected by R-G coupling. These findings extend the two-domain model quantitatively and are consistent with a simple equilibrium model of the two-domain mechanism: (1) The N-domain binds peptide agonist with moderate-to-high microaffinity, substantially increasing the local concentration of agonist and so allowing weak agonist-J-domain interaction. (2) Agonist-J-domain interaction is allosterically enhanced by receptor-G-protein interaction and inhibited by nonpeptide antagonist.     

Structural study on ligand specificity of human vitamin B12 transporters

Studies comparing the binding of genuine cobalamin (vitamin B12) to that of its natural or synthetic analogues have long established increasing ligand specificity in the order haptocorrin, transcobalamin and intrinsic factor, the high-affinity binding proteins involved in cobalamin transport in mammals. In the present study, ligand specificity was investigated from a structural point of view, for which comparative models of intrinsic factor and haptocorrin are produced based on the crystal structure of the homologous transcobalamin and validated by results of published binding assays. Many interactions between cobalamin and its binding site in the interface of the two domains are conserved among the transporters. A structural comparison suggests that the determinant of specificity regarding cobalamin ligands with modified nucleotide moiety resides in the beta-hairpin motif beta3-turn-beta4 of the smaller C-terminal domain. In haptocorrin, it provides hydrophobic contacts to the benzimidazole moiety through the apolar regions of Arg357, Trp359 and Tyr362. Together, these large side chains may compensate for the missing nucleotide upon cobinamide binding. Intrinsic factor possesses only the tryptophan residue and transcobalamin only the tyrosine residue, consistent with their low affinity for cobinamide. Relative affinity constants for other analogues are rationalized similarly by analysis of steric and electrostatic interactions with the three transporters. The structures also indicate that the C-terminal domain is the first site of cobalamin-binding since part of the beta-hairpin motif is trapped between the nucleotide moiety and the N-terminal domain in the final holo-proteins.     

Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Ligand selectivity and affinity of chemokine receptor CXCR1. Role of N-terminal domain

Glu-Leu-Arg ("ELR") CXC chemokines interleukin-8 (IL-8) and melanoma growth stimulatory activity (MGSA) recruit neutrophils by binding and activating two receptors, CXCR1 and CXCR2. CXCR1 is specific, binding only IL-8 with nanomolar affinity, whereas CXCR2 is promiscuous, binding all ELRCXC chemokines with high affinity. Receptor signaling consists of two events: interactions between the ligand N-terminal loop (N-loop) and receptor N-terminal domain (N-domain) residues (site I), and between the ligand N-terminal ELR and the receptor juxtamembrane domain (J-domain) residues (site II). It is not known how these interactions mediate ligand affinity and selectivity, and whether binding at one site influences binding and function at the other. Sequence analysis and structure-function studies have suggested that the receptor N-domain plays an important role in ligand selectivity. Here, we report ligand-binding properties and structural characteristics of the CXCR1 N-domain in solution and in detergent micelles that mimic the native membrane environment. We find that IL-8 binds the N-domain with significantly higher affinity in micelles than in solution (approximately 1 microM versus approximately 20 microM) and that MGSA does not bind the N-domain in solution but does in micelles with appreciable affinity (approximately 3 microM). We find that the N-domain is structured in micelles and that the entire N-domain interacts with the micelle in an extended fashion. We conclude that the micellar environment constrains the N-domain, and this conformational restraint influences its ligand-binding properties. Most importantly, our data suggest that for both ligands, site I interaction provides similar affinity and that differential coupling between site I and II interactions is responsible for the observed differences in affinity.     

Structural determinants of binding and specificity in transforming growth factor-receptor interactions.

Transforming growth factor (TGF-beta) protein families are cytokines that occur as a large number of homologous proteins. Three major subgroups of these proteins with marked specificities for their receptors have been found-TGF-beta, activin/inhibin, and bone morphogenic protein. Although structural information is available for some members of the TGF-beta family of ligands and receptors, very little is known about the way these growth factors interact with the extracellular domains of their cell surface receptors, especially the type II receptor. In addition, the elements that are the determinants of binding and specificity of the ligands are poorly understood. The structure of the extracellular domain of the receptor is a three-finger fold similar to some toxin structures. Amino acid exchanges between multiply aligned homologous sequences of type II receptors point to a residue at the surface, specifically finger 1, as the determinant of ligand specificity and complex formation. The "knuckle" epitope of ligands was predicted to be the surface that interacts with the type II receptor. The residues on strands beta2, beta3, beta7, beta8 and the loop region joining beta2 and beta3 and joining beta7 and beta8 of the ligands were identified as determinants of binding and specificity. These results are supported by studies on the docking of the type II receptor to the ligand dimer-type I receptor complex.     

Glucagon-induced self-association of recombinant proteins in Escherichia coli and affinity purification using a fragment of glucagon receptor

The specific molecular interactions of alpha-helical peptide, human glucagon (i.e., intermolecular self-association and specific receptor-binding affinity) provided a rationale for using the glucagon as the fusion expression partner to achieve high productivity of foreign proteins both in vivo (in bacterial fusion-expression system) and in vitro (in affinity column chromatography). The fusion of glucagon peptide(s) effectively promoted homogeneous aggregate formation of recombinant proteins while avoiding intermolecular crosslinking by disulfide bridges. High sensitivity of the self-aggregation to sequence effects resulted from two distinct nonpolar domains of glucagon, determining specificity of molecular interaction and aggregate size of recombinant proteins. An N-terminal domain of glucagon molecule (Phe6-Tyr10-Tyr13) could be a certain hydrophobic moiety involved in intermolecular self-association (probably, via helix-helix docking), while a C-terminal domain (Phe22-Trp25-Leu26) seems to critically affect the oligomer size in the off-pathway aggregation of synthesized fusion proteins. An N-terminal extracellular domain of human glucagon receptor was recombinantly expressed in Escherichia coli, immobilized to a chromatography column, and efficiently renatured to a conformation that attains high specificity in interaction with N-terminus glucagon molecules of recombinant fusion proteins. Through column chromatography employing the receptor fragment as affinity ligand, the recombinant proteins were efficiently purified from total intracellular proteins, and the long-term ligand stability was evidently proven through multiple cyclic-purification experiments. Major scaffolds for using protein ligands are large-scale production in a low-cost expression system and long-term stable operation with selective-binding affinity. From this point of view, the extracellular fragment of human glucagon receptor used in this study seems to be a new potent ligand for fusion protein-based affinity chromatography.     

Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding

TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.     

Characterization of the Ligand Binding Functionality of the Extracellular Domain of Activin Receptor Type IIB

The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu⁷⁹ effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.     

Crystal structure of the SH3 domain of betaPIX in complex with a high affinity peptide from PAK2

The p21-activated kinases (PAKs) are important effector proteins of the small GTPases Cdc42 and Rac and control cytoskeletal rearrangements and cell proliferation. The direct interaction of PAKs with guanine nucleotide exchange factors from the PIX/Cool family, which is responsible for the localization of PAK kinases to focal complexes in the cell, is mediated by a 24-residue peptide segment in PAKs and an N-terminal src homology 3 (SH3) domain in PIX/Cool. The SH3-binding segment of PAK contains the atypical consensus-binding motif PxxxPR, which is required for unusually high affinity binding. In order to understand the structural basis for the high affinity and specificity of the PIX-PAK interaction, we solved crystal structures for the N-terminal SH3 domain of betaPIX and for the complex of the atypical binding segment of PAK2 with the N-terminal SH3 domain of betaPIX at 0.92 A and 1.3A resolution, respectively. The asymmetric unit of the crystal contains two SH3 domains and two peptide ligands. The bound peptide adopts a conformation that allows for intimate contacts with three grooves on the surface of the SH3 domain that lie between the n-Src and RT-loops. Most notably, the arginine residue of the PxxxPR motif forms a salt-bridge and is tightly coordinated by a number of residues in the SH3 domain. This arginine-specific interaction appears to be the key determinant for the high affinity binding of PAK peptides. Furthermore, C-terminal residues of the peptide engage in additional interactions with the surface of the RT-loop, which significantly increases binding specificity. Compared to a recent NMR structure of a similar complex, our crystal structure reveals an alternate binding mode. Finally, we compare our crystal structure with the recently published betaPIX/Cbl-b complex structure, and suggest the existence of a molecular switch.     

Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.     

Molecular dynamics directed rational design and fluorescence binding assay of phosphopeptide ligands for PLK polo-box domain

Polo-like kinase 1 (PLK1) plays a critical role during multiple stages of cell cycle progression and is involved in the development and metastasis of malignant tumours. The protein contains a regulatory polo-box domain (PBD) that can recognise and bind to a wide variety of phosphorylated substrates. Here, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was created based on the crystal structures of the domain in complex with its natural phosphopeptide ligands. With the profile we were able to explore the structural basis and energetic landscape of phosphopeptide binding to the domain. Moreover, in addition to domain peptide-binding pocket we also examined the intermolecular interaction between the N-terminal region of phosphopeptide and a newly discovered crystal packing site of the domain. All the harvested information was successfully integrated to guide the structural design and optimisation of phosphopeptide ligands with improved affinity and selectivity for the domain, which were then confirmed in vitro by fluorescence anisotropy assays. Structural analysis and energetic analysis revealed that the phosphopeptide ligand can be divided into three functionally independent sections: a N-terminal flexible tail, a C-terminal unbinding tail and a central binding region. The N-terminal tail possesses a high flexibility that can roll over on the surface of PBD crystal packing site, while the C-terminal tail points out of the peptide-binding pocket of the domain and no substantial interaction can be observed between them. In contrast, the central region binds tightly to the domain pocket and exhibits modest conformational change over different ligands.     

A point mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor increases its affinity for a family of beta receptor antagonists.

Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.     

Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway

The Slit-Robo (sr) GTPase-activating protein (GAPs) are important components in the intracellular pathway mediating Slit-Robo signaling in axon guidance and cell migration. We report the first crystal structure of the srGAP1 SH3 domain at 1.8-A resolution. The unusual side chain conformation of the conserved Phe-13 in the P1 pocket renders the ligand binding pocket shallow and narrow, which contributes toward the low binding affinity. Moreover, the opposing electrostatic charge and the hydrophobic properties of the P3 specificity pocket are consistent with the observed binding characteristics of the srGAP1 SH3 domain to its ligand. Surface plasmon resonance experiments indicate that the srGAP1 SH3 domain interacts with its natural ligand inaCtoN orientation. The srGAP1 SH3 domain can bind to both the CC2 and CC3 motifs in vitro. The N-terminal two acidic residues in the CC3 motif recognition site are necessary for srGAP1 SH3 domain binding. A longer CC3 peptide (CC3-FL) binds with greater affinity than its shorter counterpart, suggesting that the residues surrounding the proline-rich core are important for protein-peptide interactions. Our study reveals previously unknown properties of the srGAP-Robo interaction. Our data provide a structural basis for the srGAP-Robo interaction, consistent with the role of the Robo intracellular domain in interacting with other downstream signaling molecules and mediating versatile and dynamic responses to axon guidance and cell migration cues.     

Identification of ligand-selective peptidic ActRIIB-antagonists using phage display technology

ActRIIB (activin receptor type-2B) is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN), growth differentiation factor 11 (GDF11), and bone morphogenetic protein 9 (BMP9). Notably, the protein-protein interaction (PPI) between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9), AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity.     

Growth differentiation factor 9 (GDF9) suppresses follistatin and follistatin-like 3 production in human granulosa-lutein cells

Background

We have demonstrated that growth differentiation factor 9 (GDF9) enhances activin A-induced inhibin β _B-subunit mRNA levels in human granulosa-lutein (hGL) cells by regulating receptors and key intracellular components of the activin signaling pathway. However, we could not exclude its effects on follistatin (FST) and follistatin-like 3 (FSTL3), well recognized extracellular inhibitors of activin A.

Methodology

hGL cells from women undergoing in vitro fertilization (IVF) treatment were cultured with and without siRNA transfection of FST, FSTL3 or GDF9 and then treated with GDF9, activin A, FST, FSTL3 or combinations. FST, FSTL3 and inhibin β _B-subunit mRNA, and FST, FSTL3 and inhibin B protein levels were assessed with real-time RT-PCR and ELISA, respectively. Data were log transformed before ANOVA followed by Tukey''s test.

Principal Findings

GDF9 suppressed basal FST and FSTL3 mRNA and protein levels in a time- and dose-dependent manner and inhibited activin A-induced FST and FSTL3 mRNA and protein expression, effects attenuated by BMPR2 extracellular domain (BMPR2 ECD), a GDF9 antagonist. After GDF9 siRNA transfection, basal and activin A-induced FST and FSTL3 mRNA and protein levels increased, but changes were reversed by adding GDF9. Reduced endogenous FST or FSTL3 expression with corresponding siRNA transfection augmented activin A-induced inhibin β _B-subunit mRNA levels as well as inhibin B levels (P values all <0.05). Furthermore, the enhancing effects of GDF9 in activin A-induced inhibin β _B-subunit mRNA and inhibin B production were attenuated by adding FST.

Conclusion

GDF9 decreases basal and activin A-induced FST and FSTL3 expression, and this explains, in part, its enhancing effects on activin A-induced inhibin β _B-subunit mRNA expression and inhibin B production in hGL cells.     

A flexible activin explains the membrane-dependent cooperative assembly of TGF-beta family receptors

A new crystal structure of activin in complex with the extracellular domain of its type II receptor (ActRIIb-ECD) shows that the ligand exhibits an unexpected flexibility. The motion in the activin dimer disrupts its type I receptor interface, which may account for the disparity in its affinity for type I versus type II receptors. We have measured the affinities of activin and its antagonist inhibin for ActRIIb-ECD and found that the affinity of the 2-fold symmetric homodimer activin for ActRIIb-ECD depends on the availability of two spatially coupled ActRIIb-ECD molecules, whereas the affinity of the heterodimer inhibin does not. Our results indicate that activin's affinity for its two receptor types is greatly influenced by their membrane-restricted setting. We propose that activin affinity is modulated by the ligand flexibility and that cooperativity is achieved by binding to two ActRII chains that immobilize activin in a type I binding-competent orientation.