Researchers soon identified TGF-β1 as a key growth inhibitory factor, but to understand the way it worked, they had to find the receptors to which it bound. The logic of how they went about their search is representative of typical biochemical approaches to identifying receptors (see Section 15.2). Investigators first reacted the purified growth factor with the radioisotope iodine-125 (125I) under conditions that caused the iodine to become covalently linked to exposed tyrosine residues, tagging them with a radioactive label. The 125I-labeled TGF-β protein was then incubated with cultured cells, and the incubation mixture was treated with a chemical agent that covalently cross-linked the labeled TGF-β to its receptors on the cell surface. Purification of the 125I-labeled TGF-β–receptor complexes revealed three different polypeptides with molecular weights of 55, 85, and 280 kDa, referred to as RI, RII, and RIII TGF-β receptors, respectively.
Figure 16-3 (steps 1 and 2) depicts the relationship and function of the three TGF-β receptor proteins. The most abundant, RIII, also called β-glycan, is a cell-surface proteoglycan. A proteoglycan consists of a protein bound to glycosaminoglycan (GAG) chains such as heparan sulfate and chondroitin sulfate (see Figure 20-32). RIII, a transmembrane protein, binds and concentrates TGF-β molecules near the cell surface, facilitating their binding to RII receptors. The RI and RII receptors are dimeric transmembrane proteins with serine/threonine kinases as part of their cytosolic domains. RII exhibits constitutive kinase activity; that is, it is active even when not bound to TGF-β. Binding of TGF-β to RII generates a new molecular surface at the TGF-β–RII interface that docks to RI, inducing the formation of complexes containing two copies each of RI and RII—an example of ligand-induced receptor hetero-oligomerization, which we will encounter often in this chapter. An RII subunit then phosphorylates serine and threonine residues in a highly conserved sequence of the RI subunit adjacent to the cytosolic face of the plasma membrane, thereby activating the RI kinase activity.