( he wells of the FlashPlate. Nevertheles...)Use of FlashPlate Technology for In Vitro Measurement of 125I-Labeled TGF-1 Binding on Chimeric Extracellular Domain of Type II Transforming Growth Factor Receptor

Moreover, we compared the affinity of hTRIIs-Fc (1370 + 363 pM) to the affinity of Cos-1 cells expressing either TRII alone (1123 + 413 pM), or both TRI and TRII (470 + 32 pM). In these experiments, the affinity of the chimeric soluble receptor for TGF-1 was threefold lower than the affinity of the heterodimer receptor complex (TRI and TRII) on the cell surface, but was very similar to that of TRII when expressed alone in the Cos-1 cell line.

One significant result is that the chimeric protein shows the same selectivity for different TGF- isoforms as the native protein. TGF-2 is not able to compete with the binding of ¹²⁵I-labeled TGF-1, although TGF-1 and TGF-3 both show an IC₅₀ value in the 3-4 nM range. Previous data have shown that TRII binds TGF-2 with low affinity, and co-expression of TRIII is required.

In conclusion, in vitro binding assays demonstrate that hTRIIs-Fc is able to bind TGF-1 with a similar affinity to that of wild type hTRII when overexpressed alone in Cos-1 cells, and can be used accordingly in Protein A FlashPlate microplates to search for TGF- agonist and antagonist compounds.

References

1. Massague, J. (1996) TGFbeta signaling: receptors, transducers, and Mad proteins. Cell 85, 947-950.

2. Komesli, S., Vivien D., & Dutartre, P. (1998) Chimeric extracellular domain of type II transforming growth factor (TGF)- receptor fused to the Fc region of human immunoglobulin as a TGF- antagonist. Eur. J. Bioch. 234, 505-513.

3. D. Vivien and J. L. Wrana (1995) Ligand-induced recruitment and phosphorylation of reduced TGF-beta type 1 receptor, Exp. Cell Res. 221, 60-65.

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