Analysis of the contribution of receptor subdomains to the cooperative binding and internalization of transforming growth factor-β (TGF-β) type I and type II receptors

The mechanistic basis underlying the striking cooperativity observed for the assembly of TGF-β family ligand/receptor complexes is not well understood. We report here an investigation in which we used a novel ligand sequestration assay, in combination with immunofluorescent light microscopy and flow cytometry analyses, to examine and quantify cooperative assembly of TGF-β ligand/receptor complexes on the cell surface, as well as ligand/receptor complex internalization. We analyzed the roles played by the ecto/transmembrane (ecto/TM) domains and endodomains of RI and RII TGF-β receptors in these processes by transfecting 293 or HeLa cells with different combinations of receptor mutants. We found that the ecto/TM domains of RII and RI cooperated together to promote the formation of cell surface receptor/ligand complexes. Furthermore, in agreement with the recently determined structure of the TGF-β3/RII ectodomain/RI ectodomain complex [J. Groppe, C.S. Hinck, P. Samavarchi-Tehrani, C. Zubieta, J.P. Schuermann, A.B. Taylor, P.M. Schwarz, J.L. Wrana, A.P. Hinck, Cooperative assembly of TGF-beta superfamily signaling complexes is mediated by two disparate mechanisms and distinct modes of receptor binding, Mol. Cell 29 (2008) 157–168], we observed that the N-terminus of the RII ectodomain was required for full assembly. With respect to endodomains, we found that the RI endodomain enhanced cooperative complex assembly at the cell surface, whereas both the RI and RII endodomains enhanced internalization. Finally, we observed that ligand/receptor internalization, but not complex assembly at the cell surface, was partly raft-dependent. In light of these results, currently proposed mechanisms of cooperative ligand/receptor assembly are discussed.