Characterization of Mutated Transforming Growth Factor-.beta.s Which Possess Unique Biological Properties

Abstract

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation. On the basis of the crystal structure of TGF-beta 2, we have designed and synthesized two mutant TGF-beta s, TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73). Although both of these molecules inhibited the growth of Mv1Lu mink lung epithelial cells and LS1034 colorectal cancer cells, which are affected equally by TGF-beta 1 and TGF-beta 2, TGF-beta 1 (delta 69-73) was much less potent than TGF-beta 1 or TGF-beta 1 (71 Trp) at inhibiting the growth of LS513 colorectal cancer cells which are growth-inhibited by TGF-beta 1 but not TGF-beta 2. Both TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73) increased levels of mRNAs for fibronectin and plasminogen activator inhibitor with Mv1Lu cells, whereas only TGF-beta 1 (71 Trp) and not TGF-beta 1 (delta 69-73) up-regulated the mRNA level of carcinoembryonic antigen in LS513 cells. The expression level of carcinoembryonic antigen mRNA in LS1034 cells was not altered by either wild-type or mutant TGF-beta s. Receptor labeling experiments demonstrated that TGF-beta 1 (71 Trp) bound with high affinity to the cell-surface receptors of Mv1Lu, LS1034, and LS513 cells while TGF-beta 1 (delta 69-73) bound effectively to the receptors of Mv1Lu and LS1034 cells but much less to the receptors on LS513 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The crystal structure of TGF‐β3 and comparison to TGF‐β2: Implications for receptor binding

“…Residues 69-73 from the other subunit contribute to the electrostatic potential around N2. Qian et al (1994) showed that the deletion of these residues had a profound effect on the biological activities of TGF-0 1, indicating that this area might be involved in the recognition. It is possible that Asp 23, Gln 26, and Asp 27 are involved in the recognition as well, because they possess different electrostatic potentials in TGF-02 and TGF-03, and are located close to residues 69-73.…”

Binding Affinity of Transforming Growth Factor-β for Its Type II Receptor Is Determined by the C-terminal Region of the Molecule

“…The other domain (amino acids 69 -73) is important for receptor binding in the colorectal cancer cell line LS513 (33). Deletion of amino acids 69 -73 of TGF-␤1 dramatically decreased its binding to TGF-␤ receptors in this cell line, but not on other cells such as Mv1Lu cells and another colorectal cancer cell line, LS1034.…”

“…Construction of expression plasmids for TGF-␤2/␤1-(40 -82), TGF-␤2/ ␤1-(40 -47), TGF-␤1-(⌬69 -73), and TGF-␤1-(Trp 71 ) has been previously described (31)(32)(33). Expression constructs of TGF-␤1/␤2-(40 -112), TGF-␤1/␤2-(83-112), TGF-␤1/␤2-(91-96), TGF-␤2/␤1-(83-112), and TGF-␤2/ ␤3-(40 -82) were generated using a two-step overlapping polymerase chain reaction method (31).…”

“…Fig. 3A shows that the two TGF-␤1 mutants TGF-␤1-(⌬69 -73) and TGF-␤1-(Trp 71 ), which were designed to investigate the possibility that the exposed loop of TGF-␤ centered at amino acid 71 participates in TGF-␤ receptor binding (33), competed with biotinylated TGF-␤1 for binding to T␤RII, suggesting that this loop is not an important epitope. Fig.…”

“…In addition, distribution of charged residues on the protein surface varies considerably (30). In our previous studies, TGF-␤ mutants including isoform chimeras, amino acid substitutions, or deletions were used to identify the specific amino acids (positions 45 and 47) in TGF-␤2 responsible for its high affinity binding to ␣ 2 -macroglobulin (␣ 2 M) (31,32) and another region (amino acids 69 -73) in TGF-␤1 responsible for its binding to receptors on LS513 colon carcinoma cells (33). In this study, we have applied the same techniques and utilized recombinant soluble human T␤RII to delineate the specific region(s) in the TGF-␤ molecule responsible for its high affinity binding to T␤RII.…”