Figure 3.

Mutagenesis of an 11 amino acid domain in the type I receptor abolishes chimeric TGF-βR down-regulation and PAI-1 expression. (A) The amino acid sequence of the COOH-terminal 37–41 residues in the activin-like kinases ALK-1 to ALK-5 as well as the Xenopus BMP (X.BMP) and Drosophila thickveins (TKV) and saxophone (SAX) type I receptors is depicted. The conserved region from amino acid 482–492 is in bold. ALK-5 is the type I TGF-βR; ALK-1 is also referred to as TSR-1 or R3; ALK-2 binds activin and is also called ActR-1, Tsk7L, SkrR1, or R1; ALK-3 binds activin and BMP and has been referred to as Brk-1, BMPR-IA, or mTFRII; and ALK-4 can also bind activin or BMP and is called ActR-IB, R2, or SKR-2 (Kingsley, 1994; Miyazono et al., 1994; Massagué, 1996). (B) Down-regulation assays were performed on the parental A105 cells (▴) and clones expressing a chimeric type I receptor containing the depicted 11 amino acid changes in residues 482–492 (●; BOX-ANA) shown in A. The BOX-ANA results represent the mean ± SE of three clones analyzed three separate times in duplicate. Because this study was run in parallel with the truncations shown in Figure 2, the control A105 curve is the same. (C) Cells expressing the wild-type chimeric type I receptor (A105) or a chimeric type I receptor with the 11 amino acid substitution (BOX-ANA), both in the context of a wild-type type II chimeric receptor, were left untreated (−) or treated with 10 ng/ml GM-CSF (G) or TGF-β2 (β) for 4 h at 37°C to activate the chimeric or endogenous TGF-β receptors, respectively. Expression of endogenous PAI-1 was determined as described in MATERIALS AND METHODS.