THE UNGLYCOSYLATED EXTRACELLULAR DOMAIN OF TYPE-II RECEPTOR FOR TRANSFORMING GROWTH-FACTOR-BETA - A NOVEL ASSAY FOR CHARACTERIZING LIGAND AFFINITY AND SPECIFICITY

The activation of the human transforming growth factor (TGF-beta) syst em begins with the cytokine-induced association of the extracellular d omains of two structurally related receptor subunits. To study the pro tein-protein interactions between TGF-beta and the ligand-specific rec eptor subunit, the extracellular domain of the human TGF-beta receptor type II (T beta R-II) has been expressed as an intracellular protein in insect cells using the baculovirus expression system. The cDNA cons truct was engineered to encode amino acids 24-159 (the signal sequence 1-23 was lacking) preceded by one initiator methionine residue and si x histidine residues added at the carboxy terminus. The soluble recept or accumulated in the cytoplasm of infected cells and was purified by one-step nickel-chelate affinity chromatography. The purified protein was not glycosylated; it migrated as a single band of apparent mass 19 .5 kDa in SDS/polyacrylamide gels, and had a homogenous N-terminal seq uence. We have established a solid-phase binding assay using radioiodi nated TGF-beta 3 and capture antibodies to immobilize the soluble rece ptor. In this assay, the apparent dissociation constant of the TGF-bet a type-II receptor ectodomain for TGF-beta 3 was approximately 150 nM (this value is approximately 1000-fold higher than that of the cell-me mbrane receptor complex of living cells). The affinity of TGF-beta 3 f or the unglycosylated ectodomain of T beta R-II from insect cells was lower than the affinity for the recombinant glycosylated ectodomain T beta R-II from mouse cells. The novel assay has been used to character ize affinities and specificities of TGF-beta 3, TGF-beta 2, correspond ing mutants and hybrid proteins, as well as a related protein, BMP-2. The assay could also be used to search for inhibitors.