Identification of the high affinity binding site in transforming growth factor-beta involved in complex formation with alpha(2)-macroglobulin - Implications regarding the molecular mechanisms of complex formation between alpha(2)-macroglobulin and growth factors, cytokines, and hormones

The biological activities of transforming growth factor-beta isoforms (TGF- beta (1,2)) are known to be modulated by alpha (2)-macroglobulin (alpha M-2 ). alpha M-2 forms complexes with numerous growth factors, cytokines, and h ormones, including TGF-beta. Identification of the binding sites in TGF-bet a isoforms responsible for high affinity interaction with a2M many unravel the molecular basis of the complex formation. Here we demonstrate that amon g nine synthetic pentacosapeptides with overlapping amino acid sequences sp anning the entire TGF-beta (1), molecule, the peptide (residues 41-65) cont aining Trp-52 exhibited the most potent activity in inhibiting the formatio n of complexes between I-125-TGF-beta (1) and activated alpha M-2 (alpha M- 2*) as determined by nondenaturing polyacrylamide gel electrophoresis and b y plasma clearance in mice. TGF-beta (2) peptide containing the homologous sequence and Trp-52 was as active as the TGF-beta (1) peptide, whereas the corresponding TGF-beta (3) peptide lacking Trp-52, was inactive. The replac ement of the Trp-52 with alanine abolished the inhibitory activities of the se peptides. I-125-TGF-beta (3), which lacks Trp-52, bound to a2M* with an affinity lower than that of I-125-TGF-beta (1). Furthermore, unlabeled TGF- beta (3) and the mutant TGF-beta (1)W52A, in which Trp-52 was replaced with alanine, were less potent than unlabeled TGF-P, in blocking I-125-TGF-beta (1) binding to alpha M-2*.TGF-beta (1) and TGF-beta (2) peptides containin g Trp-52 were also effective in inhibiting I-125-nerve growth factor bindin g to a2M*. These results suggest that Trp-52 is involved in high affinity b inding of TGF-beta to alpha M-2*. They also imply that TGF-beta and other g rowth factors/cytokines/hormones may form complexes with a2M* via a common mechanism involving the interactions between topologically exposed Trp and/ or other hydrophobic residues and a hydrophobic region in alpha M-2*.