Fig. 1. TGFβ type II-B receptor is an alternatively spliced form of TβRII. (A) The amino acid sequence of TβRII-B compared with TβRII contains an insert of 26 amino acids after Ser31, replacing Val32 of TβRII. The insertion sequence of human TβRII-B is underlined. A potential N-linked glycosylation site (Asn48) and two Cys residues (Cys44, Cys47) are shown in shaded boxes. (B) Schematic outline of alternative splicing of the tβrII-intron 1 resulting in an additional exon, exon 1A.

Fig. 2. All three TGFβ isoforms bind TβRII-B. COS-7 cells transfected with TβRII or TβRII-B were affinity labelled with [¹²⁵I]TGF-β1 (lanes 1 and 2), [¹²⁵I]TGF-β2 (lanes 3 and 4) or [¹²⁵I]TGF-β3 (lanes 5 and 6), crosslinked and immunoprecipitated with α-CRII, an antibody raised against the C-terminus of both type II receptors. Unlike TβRII, TβRII-B binds the isoform TGF-β2, when expressed singly in COS-7 cells (lanes 4 and 3). In contrast, iodinated activin A (lane 7) or BMP-2 (lane 9) does not bind to TβRII-B, but do bind to their respective high-affinity receptors ActRII-B (lane 8) and ALK3 (lane 10).

Fig. 3. Binding and complex formation of TβRII-B upon co-expression with TβRI or TβRIII. (A) Receptor complexes containing TβRII and TβRI or TβRII-B and TβRI were detected after binding and crosslinking of [¹²⁵I]TGF-β1 (lanes 1 and 2), [¹²⁵I]TGF-β2 (lanes 3 and 4) and [¹²⁵I]TGF-β3 (lanes 5 and 6) by immunoprecipitation with α-CRII. Receptor combinations are indicated above each lane. (B) COS-7 cells were transiently transfected with plasmids encoding TβRII or TβRII-B alone, or cotransfected with TβRIII (indicated above each lane). After affinity labelling with [¹²⁵I]TGF-β1 (lanes 1 and 2) or [¹²⁵I]TGF-β2 (lanes 3–6) receptors were detected by immunoprecipitation with α-CRII. The positions of ligand-bound TβRII, TβRII-B and TβRIII are indicated. Both type II receptors interact with TβRIII in the presence of TGF-β1 (lanes 1 and 2) or TGF-β2 (lanes 5 and 6). TβRII can bind to TGF-β2 only if co-expressed with TβRIII (lane 5), but not without any associated receptor (lane 3). TGF-β2 binding to TβRII-B is not dependent on the formation of receptor complexes (lane 4). (C) Binding of [¹²⁵I]TGF-β2 to TGF-β receptors at the cell surface of Mv1Lu and R1b/L17 cells. Immunoprecipitations were performed using the TβRII-B-specific antibody, α-RII-B (lanes 1 and 4), the RII/RII-B antibody, α-CRII (lanes 2 and 5) and the type I receptor antibody, α-RI (lanes 3 and 6). R1b/L17 cells lack TβRI, whereas Mv1Lu-cells do not (lanes 3 and 6).

Fig. 4. Type II/II-B receptor hetero-oligomers are detected at the cell surface after ligand binding. COS-7 cells were cotransfected with HA-epitope-tagged TβRII and non-tagged TβRII-B. Binding and crosslinking were performed with [¹²⁵I]TGF-β1 (lanes 1–6 and 8) and [¹²⁵I]TGF-β2 (lane 7). The heteromeric complex of TβRII and TβRII-B was detected by sequential immunoprecipitations (IPs) using the human TβRII-B-specific antibody α-hRIIB in the first IP and the α-HA antibody in second IP (lanes 4, 7 and 8).

Fig. 5. Neither alternative disulfide bond formation nor N-glycosylation, but addition of N-terminal epitope tags, influences ligand binding to TβRII-B. (A) COS-7 cells were transiently transfected with the wild-type TβRII-B (lane 1) and mutant forms of TβRII-B, where Cys44 (lane 2), Cys47 (lane 3) or Cys44 and Cys47 (lane 4) or Asn48 (lane 5) were mutated to alanine. After binding and crosslinking with [¹²⁵I]TGF-β2, receptors were immunoprecipitated with α-CRII. The position of ligand-bound TβRII-B is indicated. All four mutants of TβRII-B are able to bind TGF-β2. (B) COS-7 cells were transfected with HA-tagged TβRII or TβRII-B. After metabolic labelling with [³⁵S]cysteine/methionine (lanes 1–3) or binding and crosslinking using [¹²⁵I]TGF-β1 (lanes 4–8), the receptors were immunoprecipitated using antibodies as indicated. Control immunoprecipitations were carried out with α-hRIIB (lane 5) and α-CRII (lanes 6 and 8).

Fig. 6. Restricted expression pattern of TβRII-B. (A) RT–PCR analysis of TβRII-B mRNA in different cell lines. The cDNAs were prepared from human osteosarcoma cells (U2OS), human fetal osteoblasts (hFOB), murine mesenchymal precursor cells MC3T3, C3H10T1/2 cells and C2C12 myoblasts, the human hepatoma cell line Hep3B, human neuroblastoma cells (IMR32), Mv1Lu cells and rat myoblasts (L6). PCR products were obtained using the primers P1 and P5 (odd lane numbers) or the TβRII-B-specific primers Pins combined with P5 (even lane numbers). Two PCR products using P1/P5 (for example, lane 1) as well as a single PCR product using Pins/P5 (for example, lane 2) indicate the presence of TβRII-B mRNA. A single PCR product using P1/P5 (lanes 13 and 15) and no PCR product using Pins/P5 (lanes 14 and 16) indicate the expression of only TβRII mRNA. C2C12 cells were analysed either undifferentiated (lanes 17 and 18) or after differentiation in low serum (LS; lanes 19 and 20) or in LS containing 40 nM BMP-2 (lanes 21 and 22). (B) Endogenous expression of TβRII and TβRII-B at the cell surface of different cell lines was detected by affinity labelling with [¹²⁵I]TGF-β1. Cell lysates were immunoprecipitated either with α-CRII (odd lane numbers), the antibody specific for the human TβRII-B, α-hRIIB (lanes 2, 4, 10 and 12) or α-RIIB, which recognizes also the murine TβRII-B (lanes 6, 8, 14 and 16). Hep3B, IMR32, Mv1Lu and L6 cells do not show any TβRII-B protein at the cell surface. (C) Upregulation of TGF-β receptors during differentiation of C2C12 cells. Cell surface expression of TGF-β type II receptors (lanes 1–3) and specifically TβRII-B (lanes 4–6) was determined after affinity labelling using iodinated TGF-β1 on C2C12 cells, which are either undifferentiated (lanes 1 and 4), differentiated into multinucleated myotubes (lanes 2 and 5) or differentiated into the osteoblast lineage by BMP-2 (lanes 3 and 6).

Fig. 7. TβRII-B transduces TGF-β2 signals via Smad2 independently of TβRIII. (A) U2OS cells (lanes 1–3) and L6 cells (lanes 4–6) were treated with 200 pM TGF-β1 (lanes 2 and 5) or 200 pM TGF-β2 (lanes 3 and 6). Total cell lysates were used for western blotting with PS2 antiserum (upper panel). Equal loading was confirmed using α-Smad2 (α-SED) antiserum (lower panel). (B) U2OS cells were transfected with the TGF-β-sensitive reporter plasmid p3TP-luc and pRL-TK for reference. After stimulation with 200 pM TGF-β1 or 200 pM TGF-β2, luciferase activity was measured. Data were normalized to pRL-TK activity to control for transfection efficiency. (C) L6 cells were transfected with the receptors indicated, p3TP-luc and pRL-TK, and then incubated with 200 pM (white), 500 pM TGF-β1 (grey), 200 pM TGF–β2 (dark grey) or 500 pM TGF-β2 (black). Data were normalized to pRL-TK activity and represent the mean of three independent experiments. (D) DR26 cells were transfected with p3TP-luc and pRL-TK together with TβRII-B or TβRII constructs. Luciferase activity was determined as described above.

Fig. 8. TβRII-B signals via TβRI (ALK5) to the reporter p3TP-luc. R1b/L17 cells were transiently transfected with ALK1–7 in the absence or presence of TβRII-B (as indicated). Reporter gene activity was measured on p3TP-luc after treatment with either TGF-β1 (black bars) or TGF-β2 (grey bars). Luciferase activity was determined as described.

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