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  • Title: Reconstitution of a pentameric complex of dimeric transforming growth factor beta ligand and a type I, II, III receptor in baculoviral-infected insect cells.
    Author: Matsuzaki K, Kan M, McKeehan WL.
    Journal: In Vitro Cell Dev Biol Anim; 1996 Jun; 32(6):345-60. PubMed ID: 8842749.
    Abstract:
    Two transmembrane serine-threonine kinases (type I and II receptors), a membrane-anchored proteoglycan (type III), and a homodimeric ligand participate in the transforming growth factor beta type one (TGF beta 1) signal transduction complex. The expression of recombinant receptors in insect cells co-infected with up to three recombinant baculoviruses was employed to study interactions among the ectodomains of the three types of receptors and the TGF beta 1 ligand in absence of uncontrollable extrinsic factors in mammalian cells. Multi-subunit complexes were assembled in intact cells and purified on glutathione-conjugated beads for analysis by tagging one of the subunits with glutathione S-transferase (GST). Intrinsic ligand-independent interactions were observed among receptor subunits as follows: type III-III, type I-I, type III-I, and type II-I. The homeotypic complex of type II-II receptors and the heterotypic type III-II interaction was ligand dependent. The type I, but not the type III, subunit displaced about 50% of the type II component in either ligand-dependent homomeric type II-type II complexes or heteromeric type III-type II complexes to form type II-I or type III-II-I oligomers, respectively. The type II subunit displaced type I subunits in oligomers of the type I subunit. Specificity of type I receptors may result from differential affinity for the type II receptor rather than specificity for ligand. A monomeric subunit of the TGF beta 1 ligand bound concurrently to type III and type II or type III and type I receptors, but failed to concurrently bind to the type II and type I subunits. The binding of TGF beta 1 to the type I kinase subunit appears to require an intact disulfide-linked ligand dimer in the absence of a type III subunit. The combined results suggest a pentameric TGF beta signal transduction complex in which one unit each of the type III, type II, and type I components is assembled around the two subunits of the dimeric TGF beta ligand. An immobilized GST-tagged subunit of the receptor complex was utilized to assemble multi-subunit complexes in vitro and to study the phosphorylation events among subunits in the absence of extrinsic cell-derived kinases. The results revealed that (a) a low level of ligand-independent autophosphorylation occurs in the type I kinase; (b) a high level of autophosphorylation occurs in the type II kinase; (c) both the type III and type I subunits are trans-phosphorylated by the type II subunit; and (d) the presence of both type I and II kinases complexed with the type III subunit and dimeric TGF beta 1 ligand in a pentameric complex causes maximum phosphorylation of all three receptor subunits.
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