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.
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.
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