No abstract

Transforming growth factor (TGF)-bs are dimeric polypeptides that have vital roles in regulating cell growth and differentiation. They signal by assembling a receptor heterotetramer composed of two TbRI:TbRII heterodimers. To investigate whether the two heterodimers bind and signal autonomously, one of the TGF-b protomers was substituted to block receptor binding. The substituted dimer, TGF-b3 WD, bound the TbRII extracellular domain and recruited the TbRI with affinities indistinguishable from TGF-b3, but with one-half the stoichiometry. TGF-b3 WD was further shown to retain one-quarter to one-half the signalling activity of TGF-b3 in three established assays for TGF-b function. Single-molecule fluorescence imaging with GFP-tagged receptors demonstrated a measurable increase in the proportion of TbRI and TbRII dimers upon treatment with TGF-b3, but not with TGF-b3 WD. These results provide evidence that the two TbRI:TbRII heterodimers bind and signal in an autonomous manner. They further underscore how the TGF-bs diverged from the bone morphogenetic proteins, the ancestral ligands of the TGF-b superfamily that signal through a RI:RII:RII heterotrimer.

No abstract

Betaglycan is an accessory receptor of members of the transforming growth factor-β (TGF-β) superfamily, which regulates their actions through ligand-dependent interactions with type II receptors. A natural soluble form of betaglycan is found in serum and extracellular matrices. Soluble betaglycan, prepared as a recombinant protein using the baculoviral expression system, inhibits the actions of TGF-β. Because of its potential use as an anti-TGF-β therapeutic agent, we have purified and characterized baculoviral recombinant soluble betaglycan. Baculoviral soluble betaglycan is a homodimer formed by two 110kDa monomers associated by non-covalent interactions. This protein is devoid of glycosaminoglycan chains, although it contains the serine residues, which, in vertebrate cells, are modified by these carbohydrates. On the other hand, mannose-rich carbohydrates account for approximately 20kDa of the mass of the monomer. End-terminal sequence analysis of the soluble betaglycan showed that Gly24 is the first residue of the mature protein. Similarly to the natural soluble betaglycan, baculoviral soluble betaglycan has an equilibrium dissociation constant (Kd) of 3.5nM for TGF-β1. Ligand competition assays indicate that the relative affinities of recombinant soluble betaglycan for the TGF-β isoforms are TGF-β2>TGF-β3>TGF-β1. The anti-TGF-β potency of recombinant soluble betaglycan invitro is 10-fold higher for TGF-β2 than for TGF-β1. Compared with a commercial pan-specific anti-TGF-β neutralizing antibody, recombinant soluble betaglycan is more potent against TGF-β2 and similar against TGF-β1. These results indicate that baculoviral soluble betaglycan has the biochemical and functional properties that would make it a suitable agent for the treatment of the diseases in which excess TGF-β plays a central physiopathological role.

Our results for the first time indicate that TGFbeta blockade by systemic sBG administration can inhibit DU145 prostate xenograft growth and angiogenesis. The inhibition is likely in part mediated by the attenuation of TGFbeta-induced MMP-9 expression.

SummaryBetaglycan is a co-receptor for members of the TGF-β superfamily. Mutagenesis has identified two ligand binding regions, one at the membrane-distal and the other at the membrane-proximal half of the betaglycan ectodomain. Here we show that partial plasmin digestion of soluble betaglycan produces two proteolysis-resistant fragments of 45 and 55 kDa, consistent with the predicted secondary structure, which indicates an intervening non-structured linker region separating the highly structured N-and C-terminal domains. Amino terminal sequencing indicates that the 45 and 55 kDa fragments correspond, respectively, to the membrane-distal and -proximal regions. Plasmin treatment of membrane betaglycan results in the production of equivalent proteolysis-resistant fragments. The 45 and 55 kDa fragments, as well as their recombinant soluble counterparts, Sol Δ10 and Sol Δ11, bind TGF-β, nonetheless, compared to intact soluble betaglycan, have severely diminished ability to block TGF-β activity. Surface plasmon resonance (SPR) analysis indicates that soluble betaglycan has K d s in the low nanomolar range for the three TGF-β isoforms, while those for Sol Δ10 and Sol Δ11 are 1 -2 orders of magnitude higher. SPR analysis further shows that the K d s of Sol Δ11 are not changed in the presence of Sol Δ10, indicating that the high affinity of soluble betaglycan is a consequence of tethering of the domains together. Overall, these results, suggest that betaglycan ectodomain exhibits a bi-lobular structure in which each lobule folds independently, binds TGF-β through distinct non-overlapping interfaces, and that linker modification may be an approach to improve soluble betaglycan's TGF-β neutralizing activity.Address correspondence to Jose-Luis Montiel, PhD, Av. Universidad 1001, Colonia Chamilpa, Cuernavaca, Morelos, 62209, México. Phone and fax: +(52) 77 73297000 ext. 3371. jlmontiel@uaem.mx. Supporting InformationThis manuscript presents biochemical evidence that betaglycan ectodomain is composed of two independently folded TGF-β binding domains. Determination of their TGF-β affinity binding constants indicate both of these domains are required for the high affinity TGF-β binding and the complete TGF-β neutralizing activity of the full-length soluble betaglycan. Supporting information can be found at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptBetaglycan (BG) is a membrane proteoglycan that serves as a co-receptor for diverse members of the TGF-β superfamily of autocrine and paracrine factors. These factors are involved in diverse biological functions that include, among others, embryonic development, cell differentiation and proliferation, control of the immune response and wound repair (1). BG binds TGF-β superfamily factors with a characteristic selectivity: TGF-β2 > TGF-β1 > inhibin A, and establishes ligand-dependent complexes with several type II receptors (2). Th...

Transforming growth factor (TGF) β1, β2, and β3 (TGF-β1–TGF-β3, respectively) are small secreted signaling proteins that each signal through the TGF-β type I and type II receptors (TβRI and TβRII, respectively). However, TGF-β2, which is well-known to bind TβRII several hundred-fold more weakly than TGF-β1 and TGF-β3, has an additional requirement for betaglycan, a membrane-anchored nonsignaling receptor. Betaglycan has two domains that bind TGF-β2 at independent sites, but how it binds TGF-β2 to potentiate TβRII binding and how the complex with TGF-β, TβRII, and betaglycan undergoes the transition to the signaling complex with TGF-β, TβRII, and TβRI are not understood. To investigate the mechanism, the binding of the TGF-βs to the betaglycan extracellular domain, as well as its two independent binding domains, either directly or in combination with the TβRI and TβRII ectodomains, was studied using surface plasmon resonance, isothermal titration calorimetry, and size-exclusion chromatography. These studies show that betaglycan binds TGF-β homodimers with a 1:1 stoichiometry in a manner that allows one molecule of TβRII to bind. These studies further show that betaglycan modestly potentiates the binding of TβRII and must be displaced to allow TβRI to bind. These findings suggest that betaglycan functions to bind and concentrate TGF-β2 on the cell surface and thus promote the binding of TβRII by both membrane-localization effects and allostery. These studies further suggest that the transition to the signaling complex is mediated by the recruitment of TβRI, which simultaneously displaces betaglycan and stabilizes the bound TβRII by direct receptor–receptor contact.

The metalloproteinase BMP-1 (bone morphogenetic protein-1) plays a major role in the control of extracellular matrix (ECM) assembly and growth factor activation. Most of the growth factors activated by BMP-1 are members of the TGF-β superfamily known to regulate multiple biological processes including embryonic development, wound healing, inflammation and tumor progression. In this study, we used an iTRAQ (isobaric tags for relative and absolute quantification)-based quantitative proteomic approach to reveal the release of proteolytic fragments from the cell surface or the ECM by BMP-1. Thirty-eight extracellular proteins were found in significantly higher or lower amounts in the conditioned medium of HT1080 cells overexpressing BMP-1 and thus, could be considered as candidate substrates. Strikingly, three of these new candidates (betaglycan, CD109 and neuropilin-1) were TGF-β co-receptors, also acting as antagonists when released from the cell surface, and were chosen for further substrate validation. Betaglycan and CD109 proved to be directly cleaved by BMP-1 and the corresponding cleavage sites were extensively characterized using a new mass spectrometry approach. Furthermore, we could show that the ability of betaglycan and CD109 to interact with TGF-β was altered after cleavage by BMP-1, leading to increased and prolonged SMAD2 phosphorylation in BMP-1-overexpressing cells. Betaglycan processing was also observed in primary corneal keratocytes, indicating a general and novel mechanism by which BMP-1 directly affects signaling by controlling TGF-β co-receptor activity. The proteomic data have been submitted to ProteomeXchange with the identifier PXD000786 and doi: 10.6019/PXD000786 .