Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis

Transforming growth factor-β (TGF-β) internalization was studied by monitoring the uptake of125I-TGF-β1 in Mv1Lu cells, which endogenously express TGF-β receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 °C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with 125I- TGF-β1 at 37 °C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-β1 at 4 °C for 2 h prior to switching the cells to 37 °C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-β1 bound to the cell surface at 37 °C and by a reduction in the labeling intensities of RI and RII in125I-TGF-β1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-β1 at 4 °C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 °C, suggesting that prebinding promotes formation of stable RI·RII complexes that can signal independently of ligand

Predominant intracellular localization of the type I transforming growth factor-beta receptor and increased nuclear accumulation after growth arrest

NRC publication: Ye

Down-regulation of transforming growth factor-beta receptors: cooperativity between the types I, II, and III receptors and modulation at the cell surface

UI - 99458858NRC publication: Ye

Identification of a functional site on the type I TGF-b receptor by mutational analysis of its ectodomain

NRC publication: Ye

Analysis of the contribution of receptor subdomains to the cooperative binding and internalization of transforming growth factor-\u3b2 (TGF-\u3b2) type I and type II receptors

The mechanistic basis underlying the striking cooperativity observed for the assembly of TGF-\u3b2 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-\u3b2 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-\u3b2 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-\u3b23/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\u2013168], 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.Peer reviewed: YesNRC publication: Ye

Binding and functional profiling of antibody mutants guides selection of optimal candidates as antibody drug conjugates.

An increasingly appreciated conundrum in the discovery of antibody drug conjugates (ADCs) is that an antibody that was selected primarily for strong binding to its cancer target may not serve as an optimal ADC. In this study, we performed mechanistic cell-based experiments to determine the correlation between antibody affinity, avidity, internalization and ADC efficacy. We used structure-guided design to assemble a panel of antibody mutants with predicted Her2 affinities ranging from higher to lower relative to the parent antibody, Herceptin. These antibodies were ranked for binding via SPR and via flow-cytometry on high-Her2 SKOV3 cells and low-Her2 MCF7 cells, the latter acting as a surrogate for low-Her2 normal cells. A subpanel of variants, representative of different Her2-binding affinities (2 strong, 2 moderate and 3 weak), were further screened via high-content imaging for internalization efficacies in high versus low-Her2 cells. Finally, these antibodies were evaluated in ADC cytotoxicity screening assays (using DM1 and MMAE secondary antibodies) and as antibody-drug conjugates (DM1 and PNU159682). Our results identified specific but weak Her2-binding variants as optimal candidates for developing DM1 and PNU ADCs since they exhibited high potencies (low to sub-nM) in high-Her2 SKOV3 cells and low toxicities in low-Her2 cells. The 2 strong-affinity variants were highly potent in SKOV3 cells but also showed significant toxicities in low-Her2 cells and therefore are predicted to be toxic in normal tissues. Our findings show that pharmacological profiling of an antibody library in multiple binding and functional assays allows for selection of optimal ADCs